CA2379661A1 - Paracellular drug delivery system - Google Patents

Abstract

The present invention provides a paracellular drug delivery system, related to Claudin-6.
In another aspect of the present invention there is provided a composition comprising the Claudin-6-derived, specific peptides and peptide analogs to be generated or delivered for use within this system; the peptides may be conjugated to a drug to be used in targeted paracellular drug delivery. In another aspect of the present invention there is provided transgenic animals, wherein uses include, but are not limited to, models for studying human disease, paracellular drug delivery and tight junction permeability barrier biology in vivo.

Description

FIELD OF THE INVENTION
The present invention pertains to the field of drug delivery.
BACKGROUND OF THE INVENTION
The transdermal administration of drugs is becoming increasingly accepted as a preferred mode of drug delivery. The transdermal route of administration of therapeutically active drugs has been used to deliver drugs into the systemic circulation of mammals, including humans. However, despite the development of various means for the transdermal delivery of drugs, the skin of humans and other animals provides an excellent barrier to the penetration of chemical substances that are exogenously applied.
The epidermal permeability barrier in the skin resides in the extracellular, lipid-enriched membranes of the stratum corneum. The epidermal permeability barrier is crucial for the survival of organisms by retarding dehydration and inhibiting the invasion of microorganisms and noxious materials through the skin (Cartride, 2000; Rutter, 2000).
One highly pertinent example is the condition seen in premature human infants (babies born before 32 weeks of gestation) in which an aberrant epidermal permeability barrier has life threatening consequences (i.e., dehydration, loss of ability to maintain body temperature and toxicity related to resorption of external chemicals) (Harpin and Rutter, 1983; Kalia et al, 1998).
The stratum corneum is composed of vertically-stacked, polyhedral corneocytes surrounded by a matrix of lipid-enriched membranes encased by a chemically-resistant, yet flexible protein shell, the cornified envelope. The cornified envelope is a complex structure which is comprised of numerous different proteins expressed by progressively differentiating epidermal cells in the basal compartment. These proteins include, for example, involucrin, loricrin, small proline rich proteins (SPRRs), calcium binding 5100 :l0 proteins, cystatin A (keratolinin) repetin sciellin, NICE-1, and late envelope proteins (LEPs) as well as several others (Presland and Dale, 2000; Marenholz et al., 2001;
Marshall et al., 2001), and are cross linked by disulfide and Ne-('y-glutaminyl) lysine isodipeptide bonds, the formation of which is catalysed by transglutaminase (Hohl, 1990).
These proteins are sealed together via lipids in a bricks and mortar fashion to form the :15 cornified envelope (Nemes and Steinert, 1999; Steinert, 2000).
Epidermal permeability barrier formation occurs during terminal differentiation, as keratinocytes move upwards to the skin surface and undergo both morphological and biochemical changes. Concurrent with their drastic shape and adhesion changes, the 20 keratin component of their cytoskeleton becomes incorporated into the forming cornified envelope, a process requiring keratin filament bundling by filaggrin. The processing of profilaggrin to filaggrin normally occurs in the keratohyalin granules of the granular layer under the action of various processing enzymes. Once the filaggrin-mediated bundling of keratin intermediate filaments occurs, the development of the cornified envelope scaffold ;?5 continues via the activity of molecules such as involucrin and loricrin in addition to cross-linkers such as SPRRs which contribute to cornified envelope rigidity and resistance to mechanical stress (Steinert et. al, 1998).
Tight junction formation is a prerequisite for the sealing of proteins into cornified :30 envelopes as well as for the formation of the epidermal permeability barrier and the maintenance of barrier function (Mitic and Anderson, 1998). The molecular nature of tight junctions is becoming better understood with the recent cloning of a super-family of integral membrane proteins caked Claudins (Morita et al., 1999, Turksen and Troy, 2001).

The Claudin family consists of at least twenty highly conserved members with great diversity in tissue distribution (Morita et al, 1999; Tsukita et al, 2001).
Although their cell and tissue distribution during development (as well as in adult tissue) is not known, it appears that there is a need for more than one Claudin to make a tight junction (Tsukita et al, 2001). Together with the existence of a large number of Claudins, it appears that the overall levels and combinations of Claudin molecules in a given cell type and tissue must be very precisely regulated to provide the degree of sealing required for epidermal permeability barrier formation.
Although the epidermal permeability barrier serves a crucial role in survival, for the proper treatment of skin disorders and diseases, it is important that the pharmacologically active agent penetrate the stratum corneum and be made available at appropriate physiological concentrations at the site of action.
Transdermal delivery of drugs provides many advantages over conventional oral administration, in cases where drugs produce gastric problems or in cases where drugs are not well absorbed. Advantages include convenience, non-interrupted therapy, improved patient compliance, reversibility of treatment (by removal of the system from the skin), better control of regulating drug delivery, elimination of the "hepatic first pass" effect, a high degree of control over the blood concentration of any particular drug delivered and a consequent reduction of side effects. Although transdermal systems have many advantages, most drugs are not amenable to this mode of administration due to the barrier properties of the skin. Molecules moving from the environment into and through intact skin must first penetrate the stratum corneum and any material on its surface.
The molecule must then penetrate the viable epidermis, the papillary dermis, and then the capillary walls and finally into systemic circulation. Along the way, each of the above mentioned tissue barriers will exhibit a different resistance to penetration by the same molecule or drug. However, it is the stratum corneum that presents the greatest barrier to absorption of topical compositions or transdermally administered drugs because it is a complex structure of compact keratinized cell remnants separated by lipid domains.
Compared to the oral or gastric mucosa for example, the stratum corneum is much less permeable to a wide variety of compounds including many drugs.

Many drugs administered with a transdermal delivery system are given in conjunction with a permeability enhancer. To be considered useful, a permeation enhancer should have the ability to enhance the permeability of the skin for at least one and preferably a significant number of drugs. More importantly, it should be able to enhance the skin permeability such that the drug delivery rate from a reasonably sized system (preferably 5-60 cm) is at therapeutically effective levels. Additionally, the enhancer when applied to the skin surface should be non-toxic, non-irritating on prolonged exposure and under occlusion, and non-sensitizing on repeated exposure. Preferably, it should be odorless, physiologically inactive, and capable of delivering drugs without producing burning or tingling sensations. In addition to these permeation enhancer-skin interaction considerations, a permeation enhancer must also be evaluated with respect to possible interactions within the transdermal system itself. For example, the permeation enhancer must be compatible with the drug to be delivered, the adhesive, and the polymer matrix in which the drug is dispersed.
In an effort to increase skin permeability so that drugs can be delivered in therapeutically effective amounts, it has been proposed to pre-treat the skin with various chemicals or to concurrently deliver the drug in the presence of a permeation enhancer.
Various materials have been suggested for this, as described in U.S. Patent Nos. 3,472,931;
3,527,864;
3,896,238; 3,903,256; 3,952,099; 4,046,886; 4,130,643; 4,130,667; 4,299,826;

4,335,115;
4,343,798; 4,379,454; 4,405,616; 4,568,343; 4,746,515; 4,764,379; 4,788,062;
4,863,738;
4,865,848; 4,900,555; 5,053,227; 5,059,426; 5,378,730; WO 95/09006; and British Patent No. 1,011,949. Williams et al. (1992) Critical Review in Therapeutic Drug Carner Systems, pp. 305-353 provides a recent review of transdermal permeation enhancers.
These compounds and their specific activity as penetration enhancers, are more fully discussed in the text Transdermal Delivery of Drugs (1987) CRL Press.
The flux of a drug across the skin can be increased by changing different parameters including but not limited to a) the resistance (diffusion coefficient) and b) the driving force, which is dependent on the solubility of the drug in the stratum corneum and consequently the gradient for its diffusion across this barrier. Many enhancer compositions have been developed to change tine or more of these factors, and are known in the art. U.S.
Patent Nos. 4,006,218, 3,551,154 and 3,472,931, for example, respectively describe the use of dimethylsulfoxide (DMSD), dimethyl formamide (DM)~ and N,N-dimethylacetamide (DMA) to enhance the absorption of topically applied drugs through the stratum corneum. Combinations of enhancers consisting of diethylene glycol monoethyl or monomethyl ether with propylene glycol monolaurate and methyl laurate are disclosed in U.S. Patent No. 4,973,468 as enhancing the transdermal delivery of steroids such as progestrones and estrogens. A dual enhancer consisting of glycerol monolaurate and ethanol for the transdermal delivery of drugs is shown in U.S. Patent No.
4,820,720.
U.S. Patent No. 5,006,342 lists numerous enhancers for transdermal drug administration consisting of fatty acid esters or fatty alcohol ethers of C2 to C4 alkanediols, where each fatty acid/alcohol portion of the ester/ether is of about 8 to 22 carbon atoms. U.S. Patent No. 4,863,970 shows penetration enhancing compositions for topical application comprising an active permeant contained in a penetration enhancing vehicle containing specified amounts of one or more cell-envelope disordering compounds such as oleic acid, 1 S oleyl alcohol, and glycerol esters of oleic acid; a CZ or C3 alkanol and an inert diluent such as water.
Regardless of the advantages of transdermal drug delivery many of the enhancer systems currently in use, possess negative side effects such as toxicity, skin irritation and incompatibility with the drugs or other ingredients making up the transdermal drug composition. This incompatibility may result in drug instability and degradation when the enhancers and the drug are co-formulated into a pharmaceutically acceptable composition for use in warm-blooded mammals, including humans. As a consequence, the practitioner in the art is hampered by an inability to employ certain permeation enhancers for increasing the skin permeation of a drug. In some instances the permeation enhancer and the drug cannot be mixed and stored together without the drug becoming unstable over time and degrading to produce unwanted and potentially harmful by-products.
Associated with the formation of such drug degradation products is the risk of administering such products into the circulation of a warm-blooded mammal, including human patients, along with the active drug. The degradation products can have additional and uncharacterised effects on the patient, potentially including toxicity and reduced drug efficacy. Hence a drug with demonstrated efficacy in treating a particular affliction but with a low rate of skin permeation, which is unstable in a long-term formulation with permeation enhancing compositions, becomes ineffective for medical and clinical development. The end result is that its use in therapy will become greatly diminished, if not abolished.
Accordingly, in view of the foregoing, and because, upon storage, the permeation enhancer degrades the drug in question, or vice versa, one skilled in the art would be led away from using a method of permeation enhancement with particular drugs and with particular permeation enhancers, and vice 'versa. Methods to solve this problem have been tried and are known in the art. U.S. Pat. No. 5,156,846, discloses a percutaneous drug delivery system and method. This method involves pre-treating the skin with an enzyme preparation which serves as the permeation enhancer, occluding the area of the skin to which the skin permeation enhancing enzyme preparation is applied and applying a drug after rinsing the area. It is disclosed that the skin can again be occluded following application of the drug on the enzyme-pre-treated site.
U.S. Patent No. 5,254,342 discloses one potentially promising route to achieve selective delivery of a drug or protein transdermally, a carrier-mediated transport known as receptor-mediated transcytosis. This method described in Rodman et al. (1989) Current Opinion in Cell Biology 2:664-672, involves the use of epithelial or endothelial cell receptors as markers and receptar-building ligands as vehicles for the transcellular transport of drugs through the skin. This method has been used to transport honmones (King and Johnson (1985) Science 227:1583-1586), proteins (Ghitescu et al.
(1986) J. Cell Biol. 102:1304-1311), and immunoglobulins (Rodewald (1980) J. Cell Biol. 85:18-32;
Underdown (1989) Immunol. Invest. 18:287-297). Drug delivery via receptor-mediated transcytosis is highly specific because it enhances only the transport of molecules that are conjugated to receptor-binding ligands. However, receptor-mediated transcytosis has so far failed to demonstrate to be an effective means for increasing transepithelial or trasendothelial drug transport. One of the major drawbacks of drug delivery via transcytosis is that the rate of transport by this mechanism is usually very low due to the polarised distribution of receptors on the apical and basal plasma membranes of these cell types.
Finally, U.S. Patent No. 6,110,747 discloses a method to increase cell vasopermeability using modulating agents to enhance or inhibit occludin-mediated cell adhesion.
These modulating agents comprise at least one occludin cell adhesion recognition sequence or an antibody or fragment thereof that specifically binds the occludin cell adhesion recognition sequence to inhibit the functioning of occludin protein. The inhibition of occluding activity perturbs the tight junction permeability barrier facilitating the transport of molecules through the barrier. However, one disadvantage of this method is that these modulating agents can also stimulate the formation of tight junctions in, for example, epithelial cells which can indiscriminately inhibit paracellular drug transport across the TJ
permeability barrier.
:l0 In order to overcome the drawbacks and improve upon previous transdermal drug delivery systems, the present invention provides a transdermal delivery method that will allow for the following when used and applied as described herein. Firstly, the delivery of a variety of types of drugs including those that exhibit low permeation rates, are generally incompatible when combined with skin permeation enhancers or during long-term storage :l5 with these enhancers and are too large to be delivered effectively through transdermal systems presently known in the art. Secondly, the ability to maintain strict therapeutically effective control over the dosage of a delivered drug. Finally, to significantly increase skin penetration rates of all drugs delivered with the method described in this invention.
20 This invention involves the manipulation of a novel protein termed Claudin-6 to control the permeability barrier of tight junctions that reside in epithelial and endothelial cells.
Synthetic peptides corresponding to, either part of, or the entire first or second extracellular domains of Claudin-6 have been generated and shown to substantially reduce transepithelial electrical resistance (TER). The transient perturbation of the barrier ;?5 function of the tight junction by these peptides is contemplated as having use in medical therapeutics such as, for example, facilitating drug delivery across the epithelial, blood-brain and blood-retina barriers, increasing delivery of nutrients across the intestinal lumin, and decreasing disruption of tight junctions that occurs in many diseases including hepatitis, Celiac Spruce disease, Crohn's disease, and gastritis, as well as other disorders :30 where tight junction permeability barriers are affected.
This background information is provided for the purpose of making known information believed by the applicant to be of possible relevance to the present invention. No admission is necessarily intended, nor should be construed, that any of the preceding information constitutes prior art against the present invention.
SUMMARY OF THE INVENTION
The present invention provides a paracellular drug delivery system, related to Claudin-6.
In another aspect of the present invention there is provided a composition comprising the Claudin-6-derived, specific peptides and peptide analogs to be generated or delivered for use within this system; the peptides may be conjugated to a drug to be used in targeted paracellular drug delivery. In another aspect of the present invention there is provided :l0 transgenic animals, wherein uses include, but are not limited to, models for studying human disease, paracellular drug delivery and tight junction permeability barrier biology in vivo.
BRIEF DESCRIPTION OF THE FIGURES
:15 Figure 1 depicts the sequence of Claudin-6. The full length of the Claudin-6 Open Reading Frame (ORF) with both nucleic acid and amino acid sequences (Figure 1A) is 219 amino acids long with an estimated molecular weight of 23kb. This sequence has been submitted to Genbank (AF125305, AF125306), included is a copy of the submission. A
20 predicted schematic of the structure is represented by Figure 1B.
Figure 2 shows two representative schematic diagrams of the predicted molecular structure of Claudin-6. Analysis predicts that Claudin protein contains 4 trans-membrane domains (Figure 2A). However, other protein prediction programs predict that Claudin has 5 trans-'25 membrane domains (Figure 2B).

Figure 3 shows a comparison of the homology between mouse Claudin-1 through to Claudin-7 using the Blast program, www.ncbi.nlm.nih.govBLAST/, for sequence analysis.
Figure 4 illustrates a comparison of two Claudin proteins, mouse Claudin-6 and human Claudin-6, to mouse Occludin. Sequence analysis with the Blast program as above shows that there is no relationship between Claudin and Occludin proteins.
Figure 5 shows a comparison of mouse Scullin and mouse Claudin-6. Search and sequence alignment done using the Clustal program (www2.ebi.ac.ulc/clustalw) indicates that Scullin and Claudin-6 are the same, at the amino acid and nucleic acid level.
Figure 6 depicts representative sequence alignments between human Claudin-6 and mouse Claudin-6. Using Claudin sequences, for comparison known human sequences were screened and it was found that Claudin maps to two regions of chromosome 16. Based on comparison at the amino acid level it appears that one of them is human Claudin-6 and the other is similar to Claudin-9.
Figure 7 illustrates the organisation of human Claudin-6 and Claudin-9.
Figure 8, a Northern blot, shows the tissue distribution of mouse Claudin-6 and Claudin-9.
Figure 9 depicts the tissue specific targeting of Claudin-6 and it.~ effects using transgenic mouse technology. PCR results for seven founder mice shown in Figure 9B are generated using the cassette depicted in Figure 9A. Two major phenotypes are shown in Figure 9C, both having poor permeability formation, Figure 9D.
Figure 10 is an illustration of data showing that animals expressing high levels of the Claudin-6 transgene tend to become dehydrated.
Figure 11 illustrates a comparison of phenotypes between wild type mice and transgenic animals that express lower levels of Claudin-6. Animals expressing lower levels of Claudin-6 in the skin survive and have distinct phenotypic traits including: a wavy hair pattern (Figure 11A); curly whiskers (Figure 11C); and a delay of 4-6 days in the opening of the eyes (Figure 11E). Wild type controls are shown in Figure 11B and Figure 11 D as comparisons. Additionally, keratin expression analysis of trangenic animals expressing Claudin-6, indicates that there is an increase in keratin 1 expression while the expression of filagrin and loricrin is less uniform and disrupted than in wild type mice.
Figure 12 depicts the composition of hair fibres in wild type and Claudin-6 transgenic mice. The proportion of the four types of hair fibres in Claudin-6 mice is drastically-different than those of wild type mice. The greater percentage of zigzag fibres to guard hairs is what contributes to the curly look of the coat in transgenic animals.
Figure 13 shows a photograph of the occurrence of prostate tumors in Claudin-6 transgenic mice. In a small yet significant subset of the population (~5%), Claudin-6 mice develop large prostate tumors after 6 to 8 months.
Figure 14 shows that application of TPA (a tumor promoter) to Claudin-6 mice results in papillorna formation in these transgenic animals.
Figure 15 exhibits schematics of three tail truncations of Claudin-6 proteins produced in transgenic mice in order to investigate the role of the tail region of Claudin protein. Wild type, normal, Claudin-6 sequence is shown in Figure 15A. Truncations were made at position 206, 194 and 186, as represented by Figure 15B, Figure 15C and Figure 15D, respectively.
Figure 16 illustrates transgenic mice with Claudin-6-FLAG truncated at c~194.
Truncation in position 194 generates mice with no hair fibers, they are totally and completely bald. This truncation seems to have no effect on whisker morphology.
Figure 17 depicts the loop deletion that is being made to investigate its role in the functioning of Claudin-6 in vivo. A 13 amino acid portion of the second loop has been removed and is currently being injected for the production of transgenic mice.

Figure 18 displays the promoter used to target Claudin-6 to the intestine, Intestinal Fatty Acid Binding Promoter (1FABP) (Figure 18A). This promoter is active only in intestinal epithelial cells. A series of injections with this construct has given us 7 DNA positive animals as shown through PCR analysis (Figure 18B). Indirect immunofluoresence studies using monoclonal antibodies against the FLAG tag of the construct indicate that the lines generated are indeed expressing the transgene in intestine Figure 18C, Figwe 18D.
Figure 19 shows that Claudin-6 can also be targeted to heart by using the a-MHC
promoter (Figure 19A). Three lines have been generated that are positive in PCR screens (Figure 19B). The PCR positive transgenic animals labelled with anti-FLAG
antibodies indicate that Claudin-6 was targeted to the myocyte cell membrane by the aMHC
promoter Figure 19C and Figure 19D. In addition, these transgenic animals are smaller than their normal littermates Figure 19E, with smaller hearts Figure 19F.
Figure 20 depicts a comparison of heart specific cytoskeletal markers for aMHC
transgenic and wild type animals.
Figure 21 shows that a very small percentage of transgenic animals develop grossly enlarged kidneys within 2-3 weeks, Figure 21A-C.
Figure 22 illustrates that Claudin-6 can also be targeted to lung tissue using the human SPC promoter, Figure 22A. Two positive transgenic animals have been generated, Figure 22B.
Figure 23 illustrates the expression characteristics of Claudin-6. A schematic diagram of the predicted Claudin-6 protein is represented by Figwe 23A. RT-PCR and immunohistochemistry on wild type tissue sections show the tissue distribution of Claudin-6 (Figures 23B and C, respectively). PCR results for transgenic mice shown in Figure 23E are generated using the cassette depicted in Figure 23D.
Comparative RT-PCR
and immunohistochemistry findings are shown in Figures 23F-I.
Figure 24 lists the specific primers used in RT-PCR analyses.

Figure 25 shows the Claudin profile of Inv-Claudin-6 transgenic epidermis as resolved by PCR (Figure 25A) with band intensities in RNA samples shown in Figure 25B.
Immunohistochemical anaysis of Claudin-1 expression in backskin samples is shown in Figure 25C.
Figure 26 depicts the skin abnormalities in transgenic mice overexpressing Claudin-6 (Figure 26A) as also shown in histopathology findings (Figures 26B-G).
:l0 Figure 27 illustrates the abnormal epidermal barrier function exhibited in Inv-Claudin-6 transgenic mice as demonstrated using a ~3-gal assay on transgenic and wild type animals (Figure 27A), by trans-epidermal water loss (TEWL) measurements (Figure 27B), and comparative conufied envelope observations (Figure 27C).
Figure 28 shows the expression of epidermal keratin and terminal differentiation markers in the skin of transgenic and normal animals as evaulated by immunofluorescence.
Figure 29 a Western blot shows the expression of the structural proteins in the skin.
Fillaggrin, lorcicrin, transglutaminase-3 and involucrin are shown in Figures 29A and B
and the expression levels of repetin and several SPRRs are shown in Figure 29C.
DETAILED DESCRIPTION OF THE INVENTION
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Generally, the nomenclature used herein and the laboratory procedures in spectroscopy, drug discovery, cell culture, molecular genetics, diagnostics, amino acid and nucleic acid chemistry, described below are those well known and commonly employed in the art. Standard techniques are typically used for signal detection, recombinant nucleic acid methods, polynucleotide synthesis, and microbial culture and transformation (e.g., electroporation, lipofection).

The techniques and procedures are generally performed according to conventional methods in the art and various general references (see generally, Sambroak et al.
Molecular Cloning: A Laboratory Manual, 2d ed. (1989) Cold Spring Harbcn Laboratory Press, Cold Spring Harbor, N.Y., and Lakowicz, J. R. Principles of Fluorescence Spectroscopy, New York: Plenum Press (1983) for fluorescence techniques) which are provided throughout this document. Standard techniques are used for chemical syntheses, chemical analyses, and biological assays. As employed throughout the disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings:
As used herein, the term "drug" is to be construed in its broadest sense to mean any material which is intended to produce some biological, beneficial, therapeutic, or other intended effect, such as permeation enhancement, for example, an the organism to which it is applied.
As used herein, the term "transdermal" refers to the use of skin, mucosa, and/or other body surfaces as a portal for the administration of drugs by topical application of the drug thereto.
As used herein, the term "therapeutically effective" amount or rate refers to the amount or rate of drug needed to effect the desired therapeutic result.
As used herein, the phrase "sustained time period" intends at least about 12 hours and will typically intend a period in the range of about one to about seven days.
As used herein, the term "individual" intends a living mammal and includes, without limitation, humans and other primates, livestock and sports animals such as cattle, pigs and horses, pets such as cats and dogs and mice.
As used herein, the phrase "predetermined area of skin" intends a defined area of intact unbroken skin or mucosal tissue. That area will usually be in the range of about 5 cm2 to about 100 cm2.

As used herein, the tam "permeation enhancer" intends an agent or a mixture of agents which, alone or in combination, acts to increase the permeability of the skin to a drug.
As used herein, the term "permeation enhancement" intends an increase in the permeability of skin to a drug in the presence of a permeation enhancer as compared to permeability of skin to the drug in the absence of a permeation enhancer.
As used herein, the term "permeation-enhancing" intends an amount or rate of a permeation enhancer which provides permeation enhancement throughout a substantial portion of the administration period.
Other chemistry terms herein are used according to conventional usage in the art, as exemplified by The McGraw-Hill Dictionary of Chemical Terns (ed. Parker, S.,1985), McGraw-Hill, San Francisco).
The ParaceDular Drug Delivery System In general, the Claudin-derived peptides and analogs thereof and compositions containing them as described herein may be used for modulating the permeability barrier of a cell or cells in vivo or in vitro by disrupting the formation of tight junctions within a cell, cells or tissue. Nucleic acid sequences may be used to generate and thereby deliver such peptides to the epithelial cells of interest. Certain methods involving the disruption of cell permeability barriers as described herein have the advantage over prior techniques in that they permit passage of molecules that are large and/ or charged across barriers of Claudin expressing cells. It has been found, within the context of the present invention, that tight junctions of epithelial cells can be disrupted by linear peptides containing the Claudin-derived amino acid sequences ---- (SEQ 1D No.l - 20).
The specific and related polypeptide analogs, derivatives or fragments of Claudin-6 can be used according to the present invention to modulate the permeability of the TJ
of epidermal cells of mammals. Manipulation of the TJ in this manner increases the permeability of the epidermal cells without irreversibly compromising the epithelial cell barrier's integrity and function. Ultimately this allows the passage of large macromolecules >1 kDa through the barrier, as well as provide a safe and specific mode of drug delivery, particularly, paracellular drug delivery. The Claudin-6-derived polypeptides of the present invention exhibit a time- and dose-dependent effect on the passage of large macromolecules through the barrier.
In one embodiment of the present invention the Claudin-6-derived, specific peptides) are employed in drug delivery, for example paracellular drug delivery. In a related embodiment the present invention provides fusion proteins comprising Claudin-6 analogs, derivatives or fragments thereof" fused to a second protein or peptide. The second protein or peptide can be a drug and/or a targeting protein that wilt facilitate drug targeting to specific tissues or organs. Such fusion proteins can be used in tissue specific drug delivery, to for example, epidermal, cardiac, intestinal or pulmanary tissue.
In addition, these peptides or fusion proteins may be used to facilitate drug delivery through, for example, colon epithelium, lung epithelium, cornea, and the endothelium of the blood brain barrier.
Within further aspects, the present invention provides cell permeability modulating agents that comprise a cyclic or linear peptide as described above. Within specific embodiments, such modulating agents may be linked to one or more of the following, a targeting agent, a drug, a solid support or support molecule, or a detectable marker. Within further specific embodiments, cell permeability modulating agents are provided that comprise a Claudin polypeptide sequence and derivatives of the foregoing polypeptide sequences having one or more C-terminal, N-terminal and/or side chain modifications. Such modifications will depend on the degree of regulation required to enhance or inhibit cellular permeability barrier(s). In a further embodiment of the present invention the Claudin-derived, specific peptide analogs are employed in the development of drugs, including but not limited to inhibiting peptides, that cleave the loops of endogenous Claudin to inactivate this protein, thus disrupting the ability of the tight junction to close, increasing permeability.
In addition, any of the above cell permeability modulating agents may further comprise one or more of: {a) a cell permeability recognition sequence that is bound by a TJ molecule or protein other than a Claudin, wherein said cell permeability recognition sequence is separated by a linker; and/or (b) an antibody or antigen-binding fragment thereof that specifically binds to a cell permeability recognition sequence bound by a TJ
molecule or protein other than a Claudin.
The specific and related polypeptide analogs, derivatives or fragments of Claudin-6 can be used according to the present invention to determine effective pharmaceutical compounds that can decrease the level of permeability or entry of molecules, bacteria or viruses, for example HIV, transported through the TJ of epidermal cells of mammals.
Similarly, it is known that many bacterially-derived molecules (including peptides) can bind to TJ
proteins and disrupt cellular permeability barriers) during the course of an infection.
Thus, anti-bacterial drugs can be developed and screened for using the Claudin-derived peptide analogs and method of the invention.
In another embodiment of the present invention the Claudin-derived, specific peptides) are used in the design of anti-inflammatory drugs. For example, these drugs are useful for treatment of psoriasis, male infertility and CNS degeneration. These potential drugs all enhance the normal function of Claudin, which is to limit the transport of molecules, particularly large molecules from passing through the permeability barrier formed by the TJ.
Therefore, in the case of paracellular drug transport, an endogenous "shutoff ' mechanism for drug transport is available through the enhancement of Claudin protein function. In the case of anti-viral or anti-bacterial treatments, drugs found to bind to the site where viral or bacterial-derived molecules or peptides bind Claudin to disrupt the permeability barrier and which do not themselves disrupt the morphology of the TJ by so binding may be used to inhibit the binding of viral and/or bacterial proteins to Claudin and thus prevent a disruption in the permeability barrier formed by the TJ of a cell. In addition, the utility of these drugs designed by this method can be studied according to the present invention.
The regulation of the TJ permeability barrier protein Claudin through the use of the Claudin-derived peptide analogs, derivatives or fragments thereof of this invention can be applied to tightly regulate the process of paracellular drug transport. In this embodiment Claudin-derived peptides that bind to Claudin protein to open the permeability barrier are applied along with the drug to be transported in such a manner as to allow for the drug to be delivered in a highly controlled manner. By adjusting the level of Claudin-derived peptide analogs that open the permeability barrier, the time of drug transport, targeting and dosage of drug delivered can be controlled and monitored. Such a method is useful in the treatment of a wide variety of diseases, and in conjunction with a wide variety of drugs.
Further, the methods of this invention are easily adaptable to treatment protocols where multiple drug therapy is required, reducing the number of different drugs a patient must take into one treatment protocol, for example.
Transdermal delivery of drugs is a convenient and non-invasive method that can be used to maintain relatively constant blood levels of a drug. In general, to facilitate drug delivery via the skin, it is necessary to perturb adhesion between the epithelial cells (keratinocytes) and the endothelial cells of the microvasculature. Using currently available techniques, only small, uncharged molecules may be delivered across skin in vivo. The methods described herein are not subject to the same degree of limitation.
Accordingly, a wide variety of drugs may be transported across the epithelial and endothelial cell layers of skin, for systemic or topical administration. Such drugs may be delivered to melanomas or may enter the blood stream of the mammal for delivery to other sites within the body.
Claudin-6 Analogs, Derivatives or Fragments Thereof In one aspect, the present invention provides novel polypeptide analogs, derivatives or fragments thereof of Claudin-6 protein, a protein that resides in the tight junction (TJ) of epidermal cells in mammals that functions to regulate cellular permeability barriers. The present invention also provides a novel method of paracellular drug delivery that utilises properties of polypeptides derived from Claudin-6. In one aspect of the present invention there is provided specific polypeptides related to the extracellular domain of Claudin-6.
Within other embodiments, such compounds may be linear peptides comprising a polypeptide sequence of Claudin-6 or a variant thereof. Such peptides are preferably 5-50 amino acid residues in length, preferably 5-20 amino acid residues, and more preferably 5-10 amino acid residues.
Antibodies to Claudin-Derived Peptides and Peptide Analogs Polyclonal and monoclonal antibodies may be raised against a Claudin-derived peptide sequence using conventional techniques. See, e.g., Harlow and Lane, Antibodies: A
Laboratory Manual, Cold Spring Harbor laboratory, 1988. 1n one such technique, an immunogen comprising the Claudin-6 sequence is initially injected into any of a wide variety of mammals (e.g., mice, rats, rabbits, sheep or goats). The smaller immunogens (i.e., less than about 20 amino acids) should be joined to a carrier protein, such as bovine serum albumin or keyhole limpet hemocyanin. Following one or more injections, the animals are bled periodically. Polyclonal antibodies specific for the Claudin-6 sequence may then be purified from such antisera by, for example, affinity chromatography using the modulating agent or antigenic portion thereof coupled to a suitable solid suppotrt.
Monoclonal antibodies specific for portions of the Claudin-6 sequence may be prepared, for example, using the technique of Kohler and Milstein, Eur. J. Immunol.
6:511-519, 1976, and improvements thereto. Briefly, these methods involve the preparation of immortal cell lines capable of producing antibodies having the desired specificity from spleen cells obtained from an animal immunized as described above. The spleen cells are immortalized by, for example, fusion with a myeloma cell fusion partner, preferably one that is syngeneic with the immunized animal. Single colonies are selected and their culture supernatants tested for binding activity against the modulating agent or antigenic portion thereof. Hybridomas having high reactivity and specificity are preferred.
Monoclonal antibodies may be isolated from the supernatants of growing hybridoma colonies, with or without the use of various techniques known in the art to enhance the yield. Contaminants may be removed from the antibodies by conventional techniques, such as chromatography, gel filtration, precipitation, and extraction. Antibodies having the desired activity may generally be identified using immunofluorescence analyses of tissue sections, cells or other samples where the target Claudin protein is localized.
Within certain embodiments, the use of antigen-binding fragments of antibodies may be preferred. Such fragments include Fab fragments, which may be prepared using standard techniques. Briefly, immunoglobulins may be purified from rabbit serum by affinity chromatography on Protein A bead columns (Harlow and Lane, Antibodies: A
Laboratory Manual, Cold Spring Harbor Laboratory,1988; see especially page 309) and digested by papain to yield Fab and Fc fragments. The Fab and Fc fragments may be separated by affinity chromatography on protein A bead columns (Harlow and Lane, 1988, pages 628-29).
Specific Applications of the Paracellular Drug Delivery System To enhance the delivery of a drug through the skin, a Claudin-derived peptide analog as described herein and a drug are contacted with the skin surface. Preferred Claudin-derived peptide analogs, derivatives or fragments thereof for use within such methods include the amino acid sequence corresponding to SEQ 117 NO: and SEQ ID NO: . Preferred antibody modulating agents include Fab fragments directed against either SEQ ID NO: or SEQ ID
NO:. Alternatively, a separate modulator of non-Claudin-mediated cell permeability may be administered in conjunction with the Cluaudin-derived peptide(s), either within the same pharmaceutical composition or separately. Contact may be achieved by direct application of the peptides, generally within a composition formulated as a cream or gel, or using any of a variety of skin contact devices for transdermal application (such as those described in US Patent No. 5,613,958 and US Patent No. 5,505,956). A skin patch provides a convenient method of administration (particularly for slow-release formulations). Such patches may contain a reservoir of modulating agent and drug separated from the skin by a membrane through which the drug diffuses. Within other patch designs, the Claudin-derived peptides) or analogs thereof and drug may be dissolved or suspended in a polymer or adhesive matrix that is then placed in direct contact with the patient's skin. The Claudin-derived peptides) or analogs thereof and drug may then diffuse from the matrix into the skin. The Ctaudin-derived peptides) or analogs thereof and drugs) may be contained within the same composition or skin patch, or may be separately administered, although administration at the same time and site is preferred.
In general, the amount of the Claudin-derived peptides) or analogs thereof administered via the skin varies with the nature of the condition to be treated or prevented, but may vary as described above. Such levels may be achieved by appropriate adjustments to the device used, or by applying a cream formulated as described above. Transfer of the drug across the skin and to the target tissue may be predicted based on in vitro studies using, for example, a Franz cell apparatus, and evaluated in vivo by appropriate means that will be apparent to those of ordinary skill in the art. As an example, monitoring of the serum level of the administered drug over time provides an easy measure of the drug transfer across the skin.
Transdermal drug delivery as described herein is particularly useful in situations in which a constant rate of drug delivery is desired, to avoid fluctuating blood levels of a drug. For example, morphine is an analgesic commonly used immediately following surgery.
When given intermittently in a parenteral form (intramuscular, intravenous), the patient usually feels sleepy during the first hour, is well during the next 2 hours and is in pain during the last hour because the blood level goes up quickly after the injection and goes down below the desirable level before the 4 hour interval prescribed for re-injection is reached.
Transdermal administration as described herein permits the maintenance of constant levels for long periods of time (e.g., days), which allows adequate pain control and mental alertness at the same time. Insulin provides another such example. Many diabetic patients need to maintain a constant baseline level of insulin that is different from their needs at the time of meals. The baseline level may be maintained using transdermal administration of insulin, as described herein. Antibiotics may also be administered at a constant rate, maintaining adequate bactericidal blood levels, while avoiding the high levels that are often responsible for the toxicity (e.g., levels of gentamycin that are too high typically result in renal toxicity) .
Drug delivery by the methods of the present invention also provide a more convenient method of drug administration. For example, it is often particularly difficult to administer parenteral drugs to newborns and infants because of the difficulty associated with finding veins of acceptable caliber to catheterize. However, newborns and infants often have a relatively large skin surface as compared to adults. Transdermal drug delivery permits easier management of such patients and allows certain types of care that can presently be given only in hospitals to be given at home. Other patients who typically have similar difficulties with venous catheterization are patients undergoing chemotherapy or patients on dialysis. In addition, for patients undergoing prolonged therapy, transdermal administration as described herein is more convenient than parenteral administration.
Transdermal administration as described herein also allows the gastrointestinal tract to be bypassed in situations where parenteral uses would not be practical. For example, there is a growing need for methods suitable for administration of therapeutic small peptides and proteins, which are typically digested within the gastrointestinal tract. The methods described herein permit administration of such compounds and allow easy administration over long periods of time. Patients who have problems with absorption through their gastrointestinal tract because of prolonged ileus or specific gastrointestinal diseases limiting drug absorption may also benefit from drugs formulated for transdermal application as described herein.
The present invention also provides methods for enhancing drug delivery to the central nervous system of a mammal. The bloodlbrain barrier is largely impermeable to most neuroactive agents, and delivery of drugs to the brain of a mammal often requires invasive procedures. Using one or more Claudin-derived peptides or analogs thereof as described herein, however, delivery may be by, for example, systemic administration of a modulating agent-drug-targeting agent combination, injection of a Claudin-derived peptide or analog thereof (alone or in combination with a drug and/or targeting agent) into the carotid artery or application of a skin patch comprising a Claudin-derived peptide or analog thereof to the head of the patient. Certain preferred Claudin-derived peptides or analogs thereof for use within such methods are SEQ ID NO: and SEQ 1D NO: . Preferred antibody modulating agents include Fab fragments directs against either SEQ >D NO: or SEQ )D
NO: .
In general, the amount of Claudin-derived peptide or analog thereof administered varies as described above, and with the method of administration and the nature of the condition to be treated or prevented. Transfer of the drug to the central nervous system may be evaluated by appropriate means that will be apparent to those of ordinary skill in the art, such as magnetic resonance imaging (MR>) or PET scan (positron emitted tomography).
Within further aspects, Claudin-derived peptides or analogs thereof as described herein may be used for modulating the immune system of a mammal in any of several ways.
Claudin-derived peptide or analog thereof may generally be used to modulate specific steps within cellular interactions during an immune response or during the dissemination of malignant lymphocytes. For example, a Claudin-derived peptide or analog thereof as described herein may be used to treat diseases associated with excessive generation of otherwise normal T cells. Accordingly, Claudin-derived peptide or analog thereof may be used to treat certain types of diabetes and rheumatoid arthritis.
In addition, one or more Claudin-derived peptides or analogs thereof may also be administered to patients afflicted with certain skin disorders (such as cutaneous lymphomas), acute B cell leukemia and excessive immune reactions involving the humoral immune system and generation of immunoglobulins, such as allergic responses and antibody-mediated graft rejection.
Methods for Modulating the Permeability of a Cell Using the Claudin-Derived Peptide Analogs of the Invention Methods for enhancing paracellular drug transpoR using the Claudin-derived peptide analogs of the invention are presented in the following embodiments. One method for modulating cell permeability comprising contacting a Claudin-expressing cell with a cell permeability modulating agent or the pharmaceutical composition as described above.
Such a method increases cell vasopermeability in a mammal cell or cells wherein the modulating agent inhibits Claudin protein function.
In yet another emodiment, the present invention provides methods for enhancing the delivery of a drug through the skin of a mammal, comprising contacting epithelial cells of a mammal with a cell permeability modulating agent as provided above and a drug, wherein the modulating agent inhibits Claudin-mediated activity, and wherein the step of contacting is performed under conditions and for a time sufficient to allow passage of the drug across the epithelial cells.
In another embodiment the present invention further provides methods for enhancing the delivery of a drug to a tumor in a mammal, comprising administering to a mammal a cell permeability modulating agent as provided above and a drug, wherein the modulating agent inhibits Claudin protein function.

In yet another embodiment, the present invention provides methods for enhancing drug delivery to the central nervous system of a' mammal, comprising administering to a mammal a cell permeability modulating agent as provided above, wherein the modulating agent inhibits Claudin.
In another embodiment, methods are provided for modulating the immune system of a mammal, comprising administering to a mammal a permeability modulating agent as described above, wherein the modulating agent inhibits Claudin-mediated function.
In a further embodiment, the present invention further provides methods for identifying an agent capable of modulating Claudin-mediated cell permeability. One such method x0 comprises the steps of (a) culturing cells that express a Claudin in the presence and absence of a candidate agent, under conditions and for a time sufficient to allow cell TJ
regulation to occur; and (b) visually evaluating the extent of cell permeability among the cells.
In another embodiment, such methods may comprise the steps of: (a) culturing human :l5 aortic endothelial cells in the presence and absence of a candidate agent, under conditions and for a time sufficient to allow cell TJ regulation to occur; and (b) comparing the level of cell surface Claudin for cells cultured in the presence of candidate agent to the level for cells cultured in the absence of candidate agent.
In another embodiment, the present invention further provides methods for detecting the '20 presence of Claudin-expressing cells in a sample, comprising: (al contacting a sample with an antibody that binds to a Claudin under conditions and for a time sufficient to allow formation of an antibody-Claudin complex; and (b) detecting the level of antibody-Claudin complex, and therefrom detecting the presence of Claudin-expressing cells in the sample.
Kits for Use in Enhancing Paracellular Drug Transport In a further embodiment, the present invention provides kits for detecting the presence of Claudin-expressing cells in a sample, comprising: {a) an antibody that binds to a modulating agent comprising the sequence XXX-XXX; and (b) a detection reagent.
In yet another embodiment, the present invention further provides kits for enhancing transdennal drug delivery, comprising: (a) a skin patch; and (b) a cell permeability modulating agent, wherein said modulating agent comprises an isolated Claudin peptide sequence or fragment thereof, and wherein the modulating agent inhibits Claudin-mediated cell permeability.
Characterisation of Genetic and Protein Sequences of ScuIlin/Claudin-6 While the physiological significance of the tight junction is well recognized, the molecular components) involved in the formation of a functional tight junction barrier are not yet established. Several cytoplasmic peripheral membrane proteins, including ZO-1, Z02, cingulin, 7H6, and rob 13 (Anderson, J.M et al. (1988) J. Cell Biol. 106: 1141-1149;
Anderson, J.M et al. (1989) ,l. Cell Biol. 109:1047-1056; Citi, S. et al.
(1988) Nature 333:
272-276; Citi, S. et al (1989) J. Cell Sci. 93: 107-122; Gumbiner, B et al.
(1991) Proc.
Natl. Acaa~ Sci. USA. 88: 3460-3464; Stevenson, B.R. et al. (1986) J. Cell Biol. 103: 755-766; Zahraoui, A. et al. (1994) J Cell Bial. 124: 101-115; Zhong, Y. et al.
(1993) J. Cell Biol. 120: 477-483; Zhong, Y. et al. (1994) Exp Cell Res. 214:614-20) and an integral membrane protein, occludin, have been found to localize at the tight junction (Furuse, M.
et al. (1993) J. Cell Biol. 123: 1777-1788). Occludin was shown to localise to functional fibrils by immunogold labelling of freeze-fracture replicas of tight junctions (Fujimoto, K.
(I995) J. Cell Sci. 108: 3443-3449). The cytoplasmic tail of occludin is necessary for its localisation to cell-cell contacts, perhaps via binding to ZO-1 and ZO-2 (Furuse, M. et al.
(1994) J. Cell BioL 127:1617-1626). Although initially it was predicted that occludin by itself was responsible for tight junction formation, a number of biochemical studies and more recently gene targeting by homologous recombination in embryonic stem cells have demonstrated that occludin-knockout cells are still capable of making tight junctions.
These observations indicated that occludin is dispensable in tight junction formation and suggested that there are probably still unidentified molecules that are involved in assembly of the tight junction and in a manner not yet clear.

As part of an effort to isolate novel genes regulated during epithelial development, a new gene, termed Scullin (Claudin-6), has been identified (Figurel). Based on s~uence analysis and in vitro transfection studies, this newly identified putative integral membrane protein is a likely candidate for participation in formation of the functional intercellular seal of the tight junction. The primary amino acid sequence of mouse Claudin-6 predicts an integral membrane protein with four or five membrane-spanning regions, two extracellular loops, and a unique cytoplasmic tail(Figures 2 and 23A). Both extracellular domains of Scullin/Claudin-6 consist solely of uncharged residues with the exception of one or two charged residues adjacent to the transmernbrane regions. A
comparison of the homology between the Scullin protein of the invention and the Claudin gene family (Claudins 1 to 7), suggests the Scullin has remarkable similarities with a number of Claudin proteins, particularly Claudin-6 (Figures 3 and 4). In addition a comparison of mouse Scullin, Claudin-6 and occludin proteins shows that one, occludin is not related to Scullin (Figure 4); and two, that mouse Scullin and Claudin-6 are identical at both the amino acid and nucleic acid level (Figure 5).
Mouse Scullin sequences were used to screen known human gene sequences and it was determined that the Scullin gene is very similar to, and can be mapped to, two regions of human chromosome 16 (Figure 7). Further, based on the amino acid sequence of these proteins encoded by these two genes, one protein appears to be human Scullin/Claudin-6 and the other human Claudin-9 (Figures 6 and 7). Interestingly, these two genes are organised in a fashion reminiscent of the Dlx genes (a member of the Hox gene family), i.e., head to head. There is a 1.4 kb intergenic region between these two genes. Using the sequence information, we isolat~i all three sequences by PCR and verified them by sequencing. We predict that the :intergenic region between the two genes must be important in the regulation of these genes. A close inspection of the intergenic region indicates that there are a number of sites for known regulatory molecules including, but not limited to, AP-2, Hox, and LEF-1.
The tissue distribution of mouse Scullin/Claudin-6 and Claudin-9 mRNA is illustrated in Figures 8 and 23B. Northern blot analysis indicates high levels of Claudin-6 mRNA in brain, kidney, liver, lung and stomach, whereas Claudin- 9 levels are highest in kidney and are found to a lesser extent in brain and liver {Figure 8). Similarly, RT-PCR
analysis using specific primers (Figure 24) indicates newborn mouse kidney and liver expressed high levels of Claudin-6 mRNA. Skin, tail, stomach, tongue, lung, brain, calvaria, spleen, thymus and heart expressed low to medium levels and the mRNA was undetectable in intestine (Figure 23B). Immunohistochemistry (Turksen and Aubin, 1991) on wild type sections of intestine, lung and kidney supported these RT-PCR findings (Figure 23C).
Claudin-6 then seems a likely candidate to target in order to manipulate the TJ
permeability barrier in a wide variety of tissues. Interestingly, in preliminary studies, Claudin-6 expression in the newborn mouse epidermis was low and present primarily in the differentiating cells, whereas, the levels of Claudin-9 mRNA was, to a greater extent, found in the skin of normal animals. Finally, based on organisational observations, these two genes might be regulated and expressed in a co-ordinated pair fashion.
Preparation of Synthetic Peptides Derived from the Claaxdin External Loop 1 and 2 Several synthetic peptides corresponding to each of the putative extracellular domains of mouse Scullin/Claudin-6 were prepared in order to demonstrate the nonpolar nature of the extracellular domains of Scullin/Claudin-C> and to demonstrate the conservation of their sequences between species (human, mouse). These studies demonstrated that Scullin/Claudin-6 have important functional roles and form the actual contact seal of the tight junction.
In one embodiment of the present invention, synthetic peptides are prepared which correspond to the sequences within the extacellular domains of Scullin. The predicted topology of Scullin/Claudin-6, based on its amino acid sequence, consists of two extracellular domains. The synthetic peptides of the present invention correspond to part of the entire first or second extracellular domains. Methods for generating the candidate synthetic peptides are well known to workers skilled in the art. For example, the peptides of the present invention can be synthesised by commercial facilities such as the Peptide Facility Sigma/Genosys (Texas, USA) using an automated synthesiser. In a prefenred embodiment the peptides of the present invention are prepared as 10 mM stock solutions in cell culture media are 5-15 amino acids in length, soluble in cell culture media and stable.

A "variant" of a Scullin/Claudin- 6-derived peptide refers to a molecule substantially similar to either the peptide, or a fragment thereof, which possesses biological activity that is substantially similar to a biological activity of the Scullin/Claudin-6-derived peptide. A
molecule is said to be "substantially similar" or "substantially identical" to another molecule if both molecules have substantially similar structures or if both molecules possess a similar biological activity.
Peptide Synthesis Peptide modulating agents (and peptide portions of modulating agents) as described herein may be synthesized by methods well known in the art, including chemical synthesis and recombinant DNA methods. For modulating agents up to about 50 residues in length, chemical synthesis may be performed using standard solution or solid phase peptide synthesis techniques, in which a peptide linkage occurs through the direct condensation of the alpha-amino group of one amino acid with the alpha-carboxy group of the other amino acid with the elimination of a water molecule. Peptide bond synthesis by direct condensation, as formulated above, requires suppression of the reactive character of the amino group of the first and of the carboxyl group of the second amino acid.
The masking substituents must permit their ready removal, without inducing breakdown of the labile peptide molecule.
In solution phase synthesis, a wide variety of coupling methods and protecting groups may be used (see Gross and Meienhofer, eds., "The Peptides: Analysis, Synthesis, Biology,"
Vol. 1-4 (Academic Press, 1979); Bodansky and Bodansky, "The Practice of Peptide Synthesis," 2d ed. (Springer Verlag, 1994)). In addition, intermediate purification and linear scale up are possible. Those of ordinary skill in the art wilt appreciate that solution synthesis requires consideration of main chain and side chain protecting groups and activation method. In addition, careful segment selection is necessary to minimize racemization during segment condensation. Solubility considerations are also a factor.
Solid phase peptide synthesis uses an insoluble polymer for support during organic synthesis. The polymer-supported peptide chain permits the use of simple washing and filtration steps instead of laborious purifications at intermediate steps.
Solid-phase peptide synthesis may generally be performed according to the method of Merrifield et al., J. Am.
Chem. Soc. 85:2149,1963, which involves assembling a linear peptide chain on a resin support using protected amino acids. Solid phase peptide synthesis typically utilizes either the Boc or Fmoc strategy. The Boc strategy uses a 1% cross-linked polystyrene resin. The standard protecting group for .alpha.-amino functions is the tart-butyloxycarbonyl (Boc) group. This group can be removed with dilute solutions of strong acids such as 25%
trifluoroaeetic acid (TFA). The next Boc-amino acid is typically coupled to the amino acyl resin using dicyclohexylcarbodiimide (DCC). Following completion of the assembly, the '10 peptide-resin is treated with anhydrous I-iF to cleave the benzyl ester link and liberate the free peptide. Side-chain functional groups are usually blocked during synthesis by benzyl-derived blocking groups, which are also cleaved by HF. The free peptide is then extracted from the resin with a suitable solvent, purified and characterized. Newly synthesized peptides can be purified, for example, by gel filtration, HPLC, partition chromatography and/or ion-exchange chromatogaphy, and may be characterized by, for example, mass spectrometry or amino acid sequence analysis. In the Boc strategy, C-terminal amidated peptides can be obtained using benzhydrylamine or methylbenzhydrylamine resins, which yield peptide amides directly upon cleavage with HF.
In the procedures discussed above, the selectivity of the side-chain blocking groups and of the peptide-resin link depends upon the differences in the rate of acidolytic cleavage.
Orthoganol systems have been introduced in which the side-chain blocking groups and the peptide-resin link are completely stable to the reagent used to remove the alpha-protecting group at each step of the synthesis. The most common of these methods involves the 9-fluorenylmethyloxycarbonyl (Fmoc) approach. Within this method, the side-chain protecting groups and the peptide-resin link are completely stable to the secondary amines used for cleaving the N-.alpha.-Fmoc group. The side-chain protection and the peptide-resin link are cleaved by mild acidolysis. The repeated contact with base makes the Merrifield resin unsuitable for Fmoc chemistry, and p-alkoxybenzyl esters linked to the resin are generally used. Deprotection and cleavage are generally accomplished using TFA.

Those of ordinary skill in the art will recognize that, in solid phase synthesis, deprotection and coupling reactions must go to completion and the side-chain blocking groups must be stable throughout the entire synthesis. In addition, solid phase synthesis is generally most suitable when peptides are to be made on a small scale.
N-acetylation of the N-terminal residue can be accomplished by reacting the final peptide with acetic anhydride before cleavage from the resin. C-amidation may be accomplished using an appropriate resin such as methylbenzhydrylamine resin using the Boc technology.
For longer modulating agents, recombinant methods are preferred for synthesis.
Within such methods, all or part of a modulating agent can be synthesized in living cells, using any of a variety of expression vectors known to those of ordinary skill in the art to be appropriate for the particular host cell. Suitable host cells may include bacteria, yeast cells, mammalian cells, insect cells, plant cells, algae and other animal cells (e.g., hybridoma, CHO, myeloma). The DNA sequences expressed in this manner may encode portions of an endogenous Claudin or other TJ molecule. Such sequences may be prepared based on known cDNA or genomic sequences (see Blaschuk et al., J. Mol. Biol. 211:679-682, 1990), or from sequences isolated by screening an appropriate library with probes designed based on known Claudin sequences. Such screens may generally be performed as described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor laboratories, Cold Spring Harbor, N.Y., 1989 (and references cited therein).
Polymerise chain reaction (PCR) may also be employed, using oligonucleotide primers in methods well known in the art, to isolate nucleic acid molecules encoding all or a portion of an endogenous adhesion molecule. To generate a nucleic acid molecule encoding a desired modulating agent, an endogenous Claudin sequence may be modified using well known techniques. For example, portions encoding one or more Claudin sequences may be joined, with or without separation by nucleic acid regions encoding linkers, as discussed above. Alternatively, portions of the desired nucleic acid sequences may be synthesized using well known techniques, and then ligated together to form a sequence encoding the Claudin-derived peptide or protein.
Methods for forming amide bonds are well known in the irt and are based on well established principles of chemical reactivity. Within one such method, carbodiimide-mediated lactam formation can be accomplished by reaction of the carboxylic acid with DCC, DIC, EDAC or DCCI, resulting in the formation of an O-acylurea that can be reacted immediately with the free amino group to complete the cyclization. The formation of the inactive N-acylurea, resulting from O-->N migration, can be circumvented by converting the O-acylurea to an active ester by reaction with an N-hydroxy compound such as 1-hydroxybenzotriazole, 1-hydroxysuccinimide, 1-hydroxynorbornene carboxamide or ethyl 2-hydroximino-2-cyanoacetate. In addition to minimizing O-~N migration, these additives also serve as catalysts during cyclization and assist in lowering racemization.
Alternatively, cyclization can be performed using the azide method, in which a reactive azide intermediate is generated from an alkyl ester via a hydrazide.
Iiydrazinolysis of the terminal ester necessitates the use of a t-butyl group for the protection of side chain carboxyl functions in the acylating component. This limitation can be overcome by using diphenylphosphoryl acid (DPPA), which furnishes an azide directly upon reaction with a carboxyl group. The slow reactivity of azides and the formation of isocyanates by their disproportionation restrict the usefulness of this method. The mixed anhydride method of lactam formation is widely used because of the facile removal of reaction by-products. The anhydride is formed upon reaction of the carboxylate anion with an alkyl chloroformate or pivaloyl chloride. The attack of the amino component is then guided to the carbonyl carbon of the acylating component by the electron donating effect of the alkoxy group or by the steric bulk of the pivaloyl chloride t-butyl group, which obstructs attack on the wrong carbonyl group. Mixed anhydrides with phosphoric acid derivatives have also been successfully used. Alternatively, cyclization can be accomplished using activated esters.
The presence of electron withdrawing substituents on the alkoxy carbon of esters increases their susceptibility to aminolysis. The high reactivity of esters of p-nitrophenol, N-hydroxy compounds and polyhalogenated phenols has made these "active esters" useful in the synthesis of amide bonds. The last few years have witnessed the development of benzotriazolyloxytris-(dimethylamino)phosphonium hexafluorophosphonate (BOP) and its congeners as advantageous coupling reagents. Their performance is generally superior to that of the well established carbodiimide amide bond formation reactions.
Within a further embodiment, a thioether linkage may be formed between the side chain of a thiol-containing residue and an appropriately derivatized alpha-amino acid.
By way of example, a lysine side chain can be coupled to bromoacetic acid through the carbodiimide coupling method (DCC, EDAC) and then reacted with the side chain of any of the thiol containing residues mentioned above to form a thioether linkage. In order to form dithioethers, any two thiol containing side-chains can be reacted with dibromoethane and diisopropylamine in DMF.
Methods to Chemically Modify a Candidate ScullinlClaudin-6-Derived Peptide Analog, Derivative or Fragment Thereof to Improve its Biological Activity Modification of the structure of the subject polypeptides can be for such purposes as enhancing therapeutic or prophylactic efficacy, stability (e.g., ex vivo shelf life and resistance to proteolytic degradation do vivo), or post-translational modifications (e.g., to alter the phosphorylation pattern of protein). Such modified peptides, when designed to retain at least one activity of the naturally occurring form of the protein, or to produce specific antagonists thereof, are considered functional equivalents of the polypeptides described in more detail herein. Such modified peptides can be produced, for instance, by amino acid substitution, deletion, or addition.
For example, it is reasonable to expect that an isolated replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar replacement of an amino acid with a structurally related amino acid (i.e.
isosteric and/or isoelectric mutations) will not have a major effect on the biological activity of the resulting molecule. Conservative replacements are those that take place within a family of amino acids that are related in their side chains. Genetically encoded amino acids are can be divided into four families:
(1) acidic=aspartate, glutamate;
(2) basic=lysine, arginine, histidine;
(3) nonpolar=alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar=glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine.
In similar fashion, the amino acid repertoire can be grouped as (1) acidic=aspartate, glutamate;

(2) basic=lysine, arginine, histidine;
(3) aliphatic=glycine, alanine, valine, leucine, isoleucine, serine, threonine, with serine and threonine optionally be grouped separately as aliphatic-hydroxyl;
(4) aromatic=phenylalanine, tyrosine, tryptophan;
(5) amide=asparagine, glutamine; and (6) sulphur-containing=cysteine and methionine. (See, for example, Biochemistry, 2nd ed., Ed. by L. Stryer, W H Freeman and Co.: 1981).
Whether a change in the amino acid sequence of a peptide results in a functional homologue (e.g. functional in the sense that the resulting polypeptide mimics or antagonises the wild-type form) can be readily determined by assessing the ability of the variant peptide to produce a response in cells in a fashion similar to the wild-type protein, or competitively inhibit such a response. Polypeptides in which more than one replacement has taken place can readily be tested in the same manner.
Generally, those skilled in the art will recognise that peptides as described herein may be modified by a variety of chemical techniques to produce compounds having essentially the same activity as the unmodified peptide, and optionally having other desirable properties.
For example, carboxylic acid groups of the peptide, whether carboxyl-terminal or sidechain, may be provided in the form of a salt of a pharmaceutically-acceptable cation or esterified to form a Cl-C16 ester, or converted to an amide of formula NRIR2 wherein RI
and R2, are each independently H or C1-Gi6 alkyl, or combined to form a heterocyclic ring, such as 5- or 6-rnembered. Amino groups of the peptide, whether amino-terminal or sidechain, may be in the form of a pharmaceutically-acceptable acid addition salt, such as the HCI, HBr, acetic, benzoic, toluene sulphonic, malefic, tartaric and other organic salts, or may be modified to C~-C16 alkyl or dialkyl amino or further converted to an amide.
Hydroxyl groups of the peptide sidechain may be converted to Cl-Ci6 alkoxy or to a Cl-C16 ester using well-recognised techniques. Phenyl and phenolic rings of the peptide sidechain may be substituted with one or more halogen atoms, such as fluorine, chlorine, bromine or iodine, or with Cl-C16 alkyl, Cl-C~6 alkoxy, carboxylic acids and esters thereof, or amides of such carboxylic acids. Methylene groups of the peptide sidechains can be extended to homologous CZ -C4 alkylenes. Thiols can be protected with any one of a number of well-recognised protecting groups, such as acetamide groups.

Those skilled in the art will also recognise methods for introducing cyclic structures into the peptides of this invention to select and provide conformational constraints to the structure that result in enhanced binding and/or stability. For example, a carboxyl-terminal or amino-terminal cysteine residue can be added to the peptide, so that when oxidised the peptide will contain a disulphide bond, thereby generating a cyclic peptide.
Other peptide cyclising methods include the formation of thioethers and carboxyl- and amino-terminal amides and esters.
Peptidomimetic and organomimetic embodiments are also hereby explicitly declared to be within the scope of the present invention, whereby the three-dimensional arrangement of the chemical constituents of such peptido-and organomimetics mimic the three-dimensional arrangement of the peptide backbone and component amino acid sidechains in the peptide, resulting in such peptido- and organomimetics of the peptides of this invention having substantial biological activity. It is implied that a phannacophore exists for each of the described activities of the Scullin-derived peptides. A pharmaeophore is an idealised, three-dimensional definition of the structural requirements for biological activity. Peptido-and organomimetics can be designed to fit each pharmacophore with current computer modelling software (computer aided drug design). The degree of overlap between the specific activities of pharmacophores remains to be determined.
In addition to peptides consisting only of naturally occun~ing amino acids, peptidomimetics or peptide analogues are also provided. Peptide analogues are commonly used in the pharmaceutical industry as non-peptide drugs with properties analogous to those of the template peptide. These types of non-peptide compound are ternned "peptide mimetics" or "peptidomime'cs" (Luthman, et al., A Textbook of Drug Design and Development, 14:386-406, 2nd Ed., Harwood Academic Publishers (1996); Grante (1994) Angew.
Chem.
Int. Ed. Engl. 33:1699-1720; Fauchere (1986) Adv Drug Res. 15:29; Veber and Freidinger (1985) TINS, p.392; and Evans, et al. (1987) J. Mead Chem. 30:1229, which are incorporated herein by reference;). Peptide mimetics that are structurally similar to therapeutically useful peptides may be used to produce an equivalent or enhanced therapeutic or prophylactic effect. Generally, pepddomimetics are structurally similar to a paradigm polypeptide (i.e., a polypeptide that has a biological or pharmacological activity), such as naturally-occurring receptor-binding polypeptide, but have one or more peptide linkages optionally replaced by a linkage selected from the group consisting of: --CH2NH----CHZS--, --CH2--CH2 --, --CH=CH-- (cis and traps), --COCHZ, --, --CH(OH)CH2 --, and -CH2S0--, by methods known in the art and further described in the following references:
Spatula, A. F. in Chemistry and Biochemistry of Amino Acids, Peptides, and Proteins, B.
Weinstein, eds., Marcel Dekker, New York, p. 267 (1983); Spatula, A. F., Vega Data (March 1983), Vol. 1, Issue 3, Peptide Backbone Modifications (general review); Morley (1980) Trends Pharm. Sci. pp. 463-468, (general review); Hudson, et al. (1979) Int. J.
Pent. Prox Res.,14:177-185 (--CHiNH--, CH2CH2 --); Spatula, et al. (1986) Life Sci., 38:1243-1249 (--CH2--S); Hann (1982) G'hem. Soc. Perkin Traps. I, 307-314 (--CH=CH--, cis and traps); Almquist, et al. (1980) J. Mead Chem., 23:1392-1398, (--COCH2 --);
Jennings-White, et a~ (1982) Tetrahedron Letx 23:2533, (--COCH2 --); Szelke, et al.
(1982)European Appln. EP 45665 (--CH(OH)CHZ --); Holladay, et al. (1983) Tetrahedron Letx, 24:4401-4404 (--C(OH)CH2 --); and Hruby (1982) Life Sci., 31:189-199 (--); each of which is incorporated herein by reference. A particularly preferred non-peptide linkage is -CH2 NH--. Such peptide mimetics may have significant advantages over polypeptide embodiments, including, for example: more economical production, greater chemical stability, enhanced pharmacological properties (half-life, absorption, potency, efficacy, etc.), altered specificity (e.g., a broad-spectrum of biological activities), reduced antigenicity, and others.
Systematic substitution of one or more amino acids of a consensus sequence with a D-amino acid of the same type (e.g., D-lysine in place of L-lysine) may be used to generate more stable peptides. In addition, constrained peptides comprising a consensus sequence or a substantially identical consensus sequence variation may be generated by methods known in the art (Rizo, et al. (1992) Ann. Rev. Biochem., 61:3$7, incorporated herein by reference); for example, by adding internal cysteine residues capable of forming intramolecular disulphide bridges which cycles the peptide.
Synthetic or non-naturally occurring amino acids refer to amino acids which do not natwally occur in vivo but which, nevertheless, can be incorporated into the peptide structures described herein. Preferred synthetic amino acids are the D-a,-amino acids of naturally occurring L-a-amino acid as well as non-naturally occurring D- and L-a,-amino acids represented by the formula HzIVCHRSCOOH where R5 is 1) a lower alkyl group, 2) a cycloalkyl group of from 3 to 7 carbon atoms, 3) a heterocycle of from 3 to 7 carbon atoms and 1 to 2 heteroatoms selected from the group consisting of oxygen, sulphur, and nitrogen, 4) an aromatic residue of from 6 to 10 carbon atoms optionally having from 1 to 3 substituents on the aromatic nucleus selected from the group consisting of hydroxyl, lower alkoxy, amino, and carboxyl, S) -alkylene-Y where alkylene is an alkylene group of from 1 to 7 carbon atoms and Y is selected from the group consisting of (a) hydroxy, (b) amino, (c) cycloalkyl and cycloalkenyl of from 3 to 7 carbon atoms, (d) aryl of from 6 to 10 carbon atoms optionally having from 1 to 3 substituents on the aromatic nucleus selected from the group consisting of hydroxyl, lower alkoxy, amino and carboxyl, (e) heterocyclic of from 3 to 7 carbon atoms and 1 to 2 heteroatoms selected from the group consisting of oxygen, sulphur, and nitrogen, (f) --C(O)R2 where R2 is selected from the group consisting of hydrogen, hydroxy, lower alkyl, lower alkoxy, and -NR3R4 where R3 and R4 are independently selected from the group consisting of hydrogen and lower alkyl, (g) --S(O)"R6 where n is an integer from 1 to 2 and R6 is lower alkyl and with the proviso that Rs does not define a side chain of a naturally occurring amino acid.
Other preferred synthetic amino acids include amino acids wherein the amino group is separated from the carboxyl group by more than one carbon atom such as /3-alanine, Y-aminobutyric acid, and the like.
"Detectable label" refers to materials, which when covalently attached to the peptides and peptide mimetics of this invention, permit detection of the peptide and peptide mimetics in vivo in the patient to whom the peptide or peptide mimetic has been administered. Suitable detectable labels are well known in the art and include, by way of example, radioisotopes, fluorescent labels (e.g., fluorescein), and the like. The particular detectable label employed is not critical and is selected relative to the amount of label to be employed as well as the toxicity of the label at the amount of label employed. Selection of the label relative to such factors is well within the skill of the art.
Covalent attachment of the detectable label to the peptide or peptide mimetic is accomplished by conventional methods well known in the art. For example, when the lasI
radioisotope is employed as the detectable label, covalent attachment of '2s1 to the peptide or the peptide mimetic can be achieved by incorporating the amino acid tyrosine into the peptide or peptide mimetic and then iodinating the peptide (see, e.g., Weaner, et al., Synthesis and Applications of Isotopically Labelled Compounds, pp. 137-140 (1994)). If tyrosine is not present in the peptide or peptide mimetic, incorporation of tyrosine to the N
or C terminus of the peptide or peptide mimetic can be achieved by well-known chemistry.
Likewise, 32P can be incorporated onto the peptide or peptide mimetic as a phosphate moiety through, for example, a hydroxyl group on the peptide or peptide mimetic using conventional chemistry.
Chimeric Peptides Containing Claudin-Derived Peptide Analogs, Derivatives or ~agments Thereof.
In another embodiment the use of recombinant DNA techniques may be used to form chimeric peptides in which a heterologous peptide is attached to the Scullin/Claudin-6-derived peptide. Such chimeric peptides will be useful in targeting and improving the stability of Scullin/Claudin-6-derived peptides for use as modulators of TJ permeability barriers.
Tests to Determine Biological Activity of Candidate Claudin-derived Peptides 1n one aspect of the present invention there is provided methods for testing the ability of the candidate Claudin-derived peptides to alter the tight junction (TJ) barrier function.
1. Calcium Switch Assay Expression and localisation of Caludin-6 in epidermal progenitor cells (EPCs) correlates with the development of transepithelial electrical resistance (TER) which is reduced in the presence of active synthetic peptides of the present invention, which correspond to either or both extracellular domains of Caludin-6.
It is known that epithelial EPCs form monolayers that have a very high TER of 8,000/cm2 and are impermeable to macromolecules with a molecular weight of 40 kD or greater. The induction of synchronised intercellular junction formation by a calcium switch and Claudin-6 localisation at cell boundaries, were correlated with the formation of tight junctions as monitored by measurements of TER. The expression levels of Claudin-6 during tight junction formation are roughly correlated with the increase in TER and Claudin-6 expression levels plateau as TER reaches maximal steady state levels. The establishment of the time course of Claudin-6 localisation and expression is consistent with the hypothesis that Claudin-6 participates in the formation of the tight junction.
To perform this assay EPCs are Cultured in normal growth medium until confluency is reached. The normal growth medium is exchanged with a low calcium medium and incubated for 18 hours. After this incubation the EPC cell cultures are replenished with the normal growth medium and the formation of tight junctions monitored by the 'l0 generation of transepithelial electrical resistance (TER), measured by a NOVA
transepidermal apparatus. TER is an indicator of epithelial permeability barrier disruption.
TER is calculated from the measured voltage and normalised by the area of the monolayer.
The background TER of blank Transwell filters is subtracted from the TER of cell monolayers.
LS
The synthetic, Claudin-derived candidate peptides were assayed for their ability to affect tight junctions as assessed by measurements of TER. Active peptides were those in which the treatment of EPC monolayers with the candidate peptide caused a substantial reduction of TER (e.g. from 6,000/cm2 to ~900/cm2). In each case the result of the assay in the '~0 presence of the candidate peptide was compared to the assay performed using DMSO and a scrambled control peptide containing a scrambled amino acid sequence from the same extracellular domains. This permited the determination of whether the candidate peptide was effective and specific in its ability to modulate TER.
?5 From the results of this assay using various candidate peptides, it was possible to establish that peptides of the present invention specifically reduce TER in mouse epithelial EPC cell monolayers.
Paracellular Tracer Flux Assay 30 Once it is determined whether the candidate Claudin-derived peptides reduce TER; it is necessary to distinguish between those that cause an increase in paracellular tight junction permeability and those that cause an increase in transcellular plasma membrane permeability to ions. To distinguish between the two possibilities, the flux of membrane-impermeant paracellular tracer molecules across the epithelial cell monolayers is assayed.
Various paracellular tracers can be used in the assay, including, but not limited to, neutral dextran, with a molecular weight of 3 kDa, conjugated with Texas red (Molecular Probes, Eugene, OR), and neutral dextran, with a molecular weight of 40 kDa, conjugated with Texas red (Molecular Probes). Newly formed EPC monolayers are treated with 5 ~,M of the candidate peptide for 36 hours. At the end of the 36 hour treatment (when control TER
has developed to 2,500/cm2), the paracellular tracer flux assays is performed.
As before, treatment of monolayers with effective peptides will result in reduction in TER. In the same monolayers, effective peptides are expected to cause a significant increase in the flux of paracellular tracers. Active peptides of the present invention will demonstrate an increase in paracellular permeability of the tight junction (e.g. the flux of dextran 3kDa) which is associated with the decrease in TER previously observed.
Paracellular tracers flux assays can be performed on 6.5-mm Transwells (in 6-well cell culture dishes). At the beginning of the paracellular flux assay, both sides of the bathing wells of Transwell filters are replaced with fresh medium without peptides.
The tracers are added to a final concentration of approximately 25 wg/100 p,1 for dextran (molecular weight 3 kDa) or approximately 50 pg/100 p1 for dextran (molecular weight 40 kDa) in the apical bathing wells containing 100 u1 of medium. The basal bathing well has no added tracers and contains 700 p1 of the same flux assay medium as in the apical compartment.
All flux assays are be performed at 25°C with gentle agitation. Cell monolayers are allowed to equilibrate for 30 minutes after the addition of tracers. For dextran (3 kD and 40 kD), the concentration is calculated from the amount of fluorescence emission at 610 nm (excitation at 587 nm) using a titration curve of known concentrations of the same tracers.
It is possible that the peptide will only alter the rates of movement of these relatively small paracellular tracers through the tight junction. To be effective in peptide induced drug delivery, we net to know whether changes to the permeability barrier will be effective in movement of macromolecules. 7.'o examine whether the functional tight junction barrier to macromolecules is disrupted by the peptides, the paracellular flux of neutral dextran with a molecular weight of 40kDa, to which EPC epithelial cell monolayers should be completely impermeable, will be measured. We anticipate that treatment of EPC cell monolayers with effective peptides, but not control peptide, will open the paracellular barrier to dextran 40K.
In general, there should be a close correlation between tracer fluxes and the magnitude of the drop in TER, i.e., effective peptides reduce TER and make tight junctions permeable to macromolecules. This correlation suggests that the decrease in TER caused by the candidate peptides) is predominantly, if not exclusively, due to an increase in paracellular permeability.
2. TER Recovery It is likely that the candidate Claudin-derived peptides found to be active, decrease TER
and Claudin-6 levels by specifically promoting Claudin-6 turnover and localisation rather than by non-specific toxicity. To be therapeutically useful the Claudin-derived peptides of the present invention should allow EPCs to remain healthy and capable of reforming the tight junction permeability barrier after the removal of the peptide. To assay for this, the previously treated EPC monolayers were tested for their ability to recover TER
after the removal of the candidate peptide.
This assay is performed after newly formed monolayers are treated with 5 p,M
candidate peptide for 24 h and the decrease in TER is measured. The candidate peptide-containing medium is then removed and replaced with fresh growth medium free of the candidate peptide. In the case of the therapeutically useful Claudin-derived peptides, after candidate peptide removal the TER slowly increases to the initial pre-treatment value.
Cells that are treated with the effective peptides of the present invention should also continue to exclude the vital dye, trypan blue, indicating that they remain intact and alive.
The reversibility of the effect of the peptide on protein transport suggests that the peptide only transiently alters the ability of EPCs to form functional tight junctions. Furthermore, the correlation of TER recovery with Claudin-6 reappearance at the tight junction provides strong evidence for a role of Claudin-6 in the formation of the tight junction permeability barrier.

3. Electrical Resistance Assay II
Yet another assay evaluates the effect of Claudin-derived peptide analogs or modulating agents on the electrical resistance across a monolayer of cells. For example, Madin Darby canine kidney (MDCK) cells can be exposed to the modulating agent dissolved in medium (e.g., at a final concentration of 0.5 rng/ml for a period of 24 hours). The effect on electrical resistance can be measured using standard techniques. This assay evaluates the effect of a modulating agent on tight junction formation in epithelial cells.
In general, the presence of 500 p,g/ml of modulating agent should result in a statistically significant increase or decrease in electrical resistance after 24 hours.
Interpretation of Assay Results The decrease in TER observed after treatment with active candidate peptides was attributed to a disruption of the tight junction permeability barrier when it was found to be associated with an increase in paracellular flux of membrane-impenneant tracers. These results demonstrate that extracellular domain peptides of Claudin-6 are acting specifically to perturb the permeability barrier function of the tight junction. The correlation of the physiological effects of the peptide with selective reduction of Claudin-6 provides evidence for a role for Claudin-6 in the formation of a functional tight junction seal.
The results of the TER recovery assay were used to establish that the effect of the peptides of the present invention is not a result of general cell toxicity or perturbation of the plasma membrane.
The data from the assays using candidate peptides demonstrate that the Claudin-derived peptides of the present invention permit transient disruption of TER and permeability in epidermal cells. A worker skilled in the art would easily recognise from these results that the peptides of the present invention will be useful far controlled delivery of drugs.
Preparation of Compositions and Therapeutic Formulations A preferred embodiment of the present invention is the use Claudin-derived peptides, in the preparation of pharmaceutical compositions, used for paracellular drug delivery, which comprise a biologically active Claudin-derived peptide or peptides, and a pharmaceutically acceptable diluent or excipient. A related embodiment is the compositions as above which additionally comprise a therapeutic compound, which may be chosen from the group comprising: antibiotics, anti-inflarnmatories, antidepressants, etc.
In another embodiment of the present invention there is provided pharmaceutical compositions comprising a cell permeability modulating agent, i.e. a Claudin-derived peptide analog, derivative or fragment thereof as described above, in combination with a pharmaceutically acceptable carrier. Such compositions may further comprise a drug. In addition, or alternatively, such compositions may further comprise one or more of: (a) a peptide comprising a cell permeability recognition sequence that is bound by a TJ
molecule or protein other than a Claudin; and/or (b) an antibody or antigen-binding fragment thereof that specifically binds to a cell permeability recognition sequence bound by a TJ molecule or protein other than a Claudin.
The pharmaceutical compositions of the present invention may be administered orally, topically, parenterally, by inhalation or spray or rectally in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles. The term parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intrasternal injection or infusion techniques. One or more protease inhibitor may be present in association with one or more non-toxic pharmaceutically acceptable carriers and/or diluents and/or adjuvants and, if desired, other active ingredients. The pharmaceutical compositions containing one or more protease inhibitor may be in a form suitable for oral use, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsion hard or soft capsules, or syrups or elixirs.
Compositions intended for oral use may be prepared according to any known to the art for the manufacture of pharmaceutical compositions and such compositions may contain one or more agents selected from the group consisting of sweetening agents, flavouring agents, colouring agents and preserving agents in order to provide pharmaceutically elegant and palatable preparations. Tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients, which are suitable for the manufacture of tablets.
These excipients may be for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate: granulating and disintegrating agents for example, corn starch, or alginic acid: binding agents, for example starch, gelatin or acacia, and lubricating agents, for example magnesium stearate, stearic acid or talc. The tablets may be uncoated or they may be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monosterate or glyceryl distearate may be employed.
Pharmaceutical compositions for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example peanut oil, liquid paraffin or olive oil.
Aqueous suspensions contain active materials in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents, for example sodium carboxymethylcellulose, methyl cellulose, hydropropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia: dispersing or wetting agents may be a naturally-occurring phosphatide, for example, lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethyene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example hepta-decaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate. The aqueous suspensions may also contain one or more preservatives, for example ethyl, or n-propylp-hydroxy-benzoate, one or more colouring agents, one or more flavouring agents or one or more sweetening agents, such as sucrose or saccharin.
Oily suspensions may be formulated by suspending the active ingredients in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those set forth above, and flavouring agents may be added to provide palatable oral preparations.
These compositions may be preserved by the addition of an anti-oxidant such as ascorbic acid.
Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above.
Additional excipients, for example sweetening, flavouring and colouring agents, may .also be present.
Pharmaceutical compositions of the invention may also be in the form of oil-in-water emulsions. The oil phase may be a vegetable oil, for example olive oil or arachis oil, or a mineral oil, for example liquid paraffin or mixtures of these. Suitable emulsifying agents may be naturally-occurring gums, for example gum acacia or gum tragacanth, naturally-occurring phosphatides, for example soy bean, lecithin, and esters or partial esters derived from fatty acids and hexitol, anhydrides, for example sorbitan monoleate, and condensation products of the said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monoleate. The emulsions may also contain sweetening and flavouring agents.
Syrups and elixirs may be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol or sucrose. Such formulations may also contain a demulcent, preservative and flavouring and colouring agents. The pharmaceutical compositions may be in the form of a sterile injectable aqueous or oleaginous suspension. This suspension may be formulation according to known art using those suitable dispersing or wetting agents and suspending agents that have been mentioned above. 'The sterile injectable preparation may also be sterile injectable solution or suspension in a non-toxic parentally acceptable diluent or solvent, for example as a solution in 1,3-butanediol.
Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, lactated Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oiI may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.

Further, there are many clinical situations where it is difficult to maintain compliance. For example, patients with mental problems (e.g., patients with Alzheimer's disease or psychosis) are easier to manage if a constant delivery rate of drug is provided without having to rely on their ability to take their medication at specific times of the day. Also patients who simply forget to take their drugs as prescribed are less likely to do so if they merely have to put on a skin patch periodically (e.g., every 3 days). Patients with diseases that are without symptoms, like patients with hypertension, are especially at risk of forgetting to take their medication as prescribed.
:l0 For patients taking multiple drugs, devices for transdermal application such as skin patches may be formulated with combinations of drugs that are frequently used together. For example, many heart failure patients are given digoxin in combination with furosemide.
The combination of both drugs into a single skin patch facilitates administration, reduces :LS the risk of errors (taking the correct pills at the appropriate time is often confusing to older people), reduces the psychological strain of taking "so many pills," reduces skipped dosage because of irregular activities and improves compliance.
The methods described herein are particularly applicable to humans, but also have a ~0 variety of veterinary uses, such as the administration of growth factors or hormones (e.g., for fertility control) to an animal.
A pharmaceutical composition may also, or alternatively, contain one or more drugs, which may be linked to a modulating agent or may be free within the composition.
~5 Virtually any drug may be administered in combination with a modulating agent or Claudin-derived peptide analog, derivative or fragment thereof as described herein, for a variety of purposes as described below. As noted above, a wide variety of drugs may be administered according to the methods provided herein. Some examples of drug categories that may be administered transdermally include anti-inflammatory drugs (e.g., :30 in arthritis and in other condition) such as all NSAID, indomethacin, prednisone, etc.;
analgesics (especially when oral absorption is not possible, such as after surgery, and when parenteral administration is not convenient or desirable), including morphine, codeine, Demerol, acetaminophen and combinations of these (e.g., codeine plus acetaminophen);

antibiotics such as Vancomycin (which is not absorbed by the GI tract and is fr~uently given intravenously) or a combination of INH and Rifarnpicin (e.g., for tuberculosis);
anticoagulants such as heparin (which is not well absorbed by the GI tract and is generally given parenterally, resulting in fluctuation in the blood levels with an increased risk of bleeding at high levels and risks of inefficacy at lower levels) and Warfarin (which is absorbed by the GI tract but cannot be administered immediately after abdominal surgery because of the normal ileus following the procedure); antidepressants (e.g., in situations where compliance is an issue as in Alzheimer's disease or when maintaining stable blood levels results in a significant reduction of anti-cholinergic side effects and better tolerance by patients), such as amitriptylin, imipramin, prozac, etc.; antihypertensive drugs (e.g., to improve compliance and r~uce side effects associated with fluctuating blood levels), such as diuretics and beta-blockers (which can be administered by the same patch;
e.g., furosemide and propanolol); antipsychotics (e.g., to facilitate compliance and make it easier for case giver and family members to make sure that the drug is received), such as haloperidol and chlorpromazine; and anxiolytics or sedatives (e.g., to avoid the reduction of alertness related to high blood levels after oral administration and allow a continual benefit throughout the day by maintaining therapeutic levels constant).
Numerous other drugs may be administered as described herein, including naturally occurring and synthetic hormones, growth factors, proteins and peptides. For example, insulin and human growth hormone, growth factors like erythropoietin, interleukins and inteferons may be delivered via the skin.
For imaging purposes, any of a variety of diagnostic agents may be incorporated into a pharmaceutical composition, either linked to a modulating agent or free within the composition. Diagnostic agents include any substance administered to illuminate a physiological function within a patient, while leaving other physiological functions generally unaffected. Diagnostic agents include metals, radioactive isotopes and radioopaque agents (e.g., gallium, technetium, indium, strontium, iodine, barium, bromine and phosphorus-containing compounds), radiolucent agents, contrast agents, dyes (e.g., fluorescent dyes and chromophores) and enzymes that catalyze a colorimetric or fluorometric reaction. In general, such agents may be attached using a variety of techniques as described above, and may be present in any orientation.

The compositions described herein may be administered as part of a sustained release formulation (i.e., a formulation such as a capsule or sponge that effects a slow release of modulating agent following administration). Such formulations may generally be prepared using well known technology and administered by, for example, oral, rectal or subcutaneous implantation, or by implantation at the desired target site.
Sustained-release formulations may contain a modulating agent dispersed in a carrier matrix and/or contained within a reservoir surrounded by a rate controlling membrane (see, e.g., European Patent Application 710,491 A). Carriers for use within such formulations are biocompatible, and may also be biodegradable; preferably the formulation provides a relatively constant level of modulating agent release. The amount of modulating agent contained within a sustained release formulation depends upon the site of implantation, the rate and expected duration of release and the nature of the condition to be treated or prevented.
Pharmaceutical compositions of the present invention may be administered in a manner appropriate to the disease to be treated (or prevented). Appropriate dosages and a suitable duration and frequency of administration will be determined by such factors as the condition of the patient, the type and severity of the patient's disease and the method of administration. In general, an appropriate dosage and treatment regimen provides the modulating agents) in an amount sufficient to provide therapeutic and/or prophylactic benefit. Within particularly preferred embodiments of the invention, a modulating agent or pharmaceutical composition as described herein may be administered at a dosage ranging from 0.001 to SO mg/kg body weight, preferably from 0.1 to 20 mg/kg, on a regimen of single or multiple daily doses. For topical administration, a cream typically comprises an amount of modulating agent ranging from 0.00001% to i%, preferably from 0.0001% to 0.2% and more preferably from 0.01% to 0.1%. Fluid compositions typically contain an amount of modulating agent ranging from 10 ng/mI to 5 mg/ml, preferably from 10 ~g to 2 mg/ml. Appropriate dosages may generally be determined using experimental models and/or clinical trials. In general, the use of the minimum dosage that is sufficient to provide effective therapy is preferred. Patients may generally be monitored for therapeutic effectiveness using assays suitable for the condition being treated or prevented, which will be familiar to those of ordinary skill in the art.

Kits for administering a drug via the skin of a mammal are also provided within the present invention. Such kits generally comprise a device for transdermal application (e.g., a skin patch) in combination with, or impregnated with, one or more modulating agents. A
drug may additionally be included within such kits.
Within a related aspect, the use of modulating agents as described herein to increase skin permeability may also facilitate sampling of the blood compartment by passive diffusion, permitting detection and/or measurement of the levels of specific molecules circulating in the blood. For example, application of one or more modulating agents to the skin, via a skin patch as described herein, permits the patch to function like a sponge to accumulate a small quantity of fluid containing a representative sample of the serum. The patch is then removed after a specified amount of time and analyzed by suitable techniques for the compound of interest (e.g., a medication, hormone, growth factor, metabolite or marker).
Alternatively, a patch may be impregnated with reagents to permit a color change if a specific substance (e.g. an enzyme) is detected. Substances that can be detected in this manner include, but are not limited to, illegal drugs such as cocaine, HIV
enzymes, glucose and PSA. This technology is of particular benefit for home testing kits.
There are a variety of assay formats known to those of ordinary skill in the art for using an antibody to detect a target molecule in a sample. See, e.g., Harlow and Lane, Antibodies:
A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. For example, the assay may be performed in a Western blot format, wherein a protein preparation from the biological sample is submitted to gel electrophoresis, transferred to a suitable membrane and allowed :25 to react with the antibody. The presence of the antibody on the membrane may then be detected using a suitable detection reagent, as described below.
In another embodiment, the assay involves the use of antibody immobilized on a solid support to bind to the target Claudin, or a proteolytic fragment thereof, and remove it from the remainder of the sample. The bound Claudin may then be detected using a second antibody or reagent that contains a reporter group. Alternatively, a competitive assay may be utilized, in which the Claudin is labelled with a reporter group and allowed to bind to the immobilized antibody after incubation of the antibody with the sample. The extent to which components of the sample inhibit the binding of a labelled Claudin to the antibody is indicative of the reactivity of the sample with the immobilized antibody, and as a result, indicative of the level of the Claudin in the sample.
The solid support may be any material known to those of ordinary skill in the art to which the antibody may be attached, such as a test well in a microtiter plate, a nitrocellulose filter or another suitable membrane. Alternatively, the support may be a bead or disc, such as glass, fiberglass, latex or a plastic such as polystyrene or polyvinylchloride. The antibody may be immobilized on the solid support using a variety of techniques known to those in the art, which are amply described in the patent and scientific literature.
In certain embodiments, the assay for detection of Claudin in a sample is a two-antibody sandwich assay. This assay may be performed by contacting an antibody that has been immobilized on a solid support, commonly the well of a microtiter plate, with the biological sample, such that the Claudin within the sample is allowed to bind to the immobilized antibody (a 30 minute incubation time at room temperature is generally sufficient). Unbound sample is then removed from the immobilized Claudin-antibody complexes and a second antibody (containing a reporter group such as an enzyme, dye, radionuclide, luminescent group, fluorescent group or biotin) capable of binding to a different site on the Claudin is added. The amount of second antibody that remains bound to the solid support is then determined using a method appropriate for the specific reporter group. The method employed for detecting the reporter group depends upon the nature of the reporter group. For radioactive groups, scintillation counting; or autoradiographic methods are generally appropriate. Spectroscopic methods may be used to detect dyes, luminescent groups and fluorescent groups. Biotin may be detected using avidin, coupled to a different reporter group (commonly a radioactive or fluorescent group or an enzyme).
Enzyme reporter groups may generally be detected by the addition of substrate (generally for a specific period of time), followed by spectroscopic or other analysis of the reaction products. Standards and standard additions may be used to determine the level of Claudin in a sample, using well known techniques.
The present invention also provides kits for use in such immunoassays. Such kits generally comprise one or more antibodies, as described above. In addition, one or more additional compartments or containers of a kit generally enclose elements, such as reagents, buffers and/or wash solutions, to be used in the immunoassay.
Within further aspects, modulating agents or antibodies (or fragments thereof) may be used to facilitate cell identification and sorting in vitro or imaging in vivo, pen~nitting the selection of cells expressing claudin (or different claudin levels).
Preferably, the modulating agents) or antibodies for use in such methods are linked to a detectable marker. Suitable markers are well known in the art and include radionuclides, luminescent groups, fluorescent groups, enzymes, dyes, constant immunoglobulin domains and biotin.
Within one preferred embodiment, a modulating agent linked to a fluorescent marker, such as fluorescein, is contacted with the cells" which are then analyzed by fluorescence activated cell sorting (FAGS).
Antibodies or fragments thereof may also be used within screens of combinatorial or other nonpeptide-based libraries to identify other compounds capable of modulating claudin-mediated cell permeability. Such screens may generally be performed using an ELISA or other method well known to those of ordinary skill in the art that detect compounds with a shape and structure similar to that of the modulating agent. In general, such screens may involve contacting an expression library producing test compounds with an antibody, and detecting the Level of antibody bound to the candidate compounds. Compounds for which the antibody has a higher affinity may be further characterized as described herein, to evaluate the ability to modulate Claudin-mediated cell permeability through cellular tight junction permeability barriers.
Nucleic Acid Sequence Based Methods The sequences of the invention can be introduced using molecular biology techniques.
Examples of this methodology are demonstrated in transgenic animals as described in the Examples Section. The sequences can be full-length or fragments thereof, such as deletion mutants. These fragments can be designed by systematic deletion of the full length from either the COOH or N-terminal ends, or by other designs known in the art, followed by testing in the systems described herein.

In a specific embodiment of the present invention there is provided a class of Claudin-6 derived peptides that are deletion peptides. These deletion peptides include those peptide having an N-terminal or C-terminal deletion, or a deletion of any region between the N-terminus or C-terminus of the Claudin-6 protein having the following amino acid sequence:
NH2-Met-Ala-Ser-Thr-Gly-Leu-Gln-Ile-Leu-Gly-Ile-Val-Leu-Thr-Leu-Leu-Gly-Trp-Val-Asn-Ala-Leu-Val-Ser-Cys-Ala-Leu-Pro-Met-Trp-Lys-Val-Thr-Ala-Phe-Ile-Gly-Asn-Ser-Ile-Val-Val-Ala-Gln-Met-Val-Trp-Glu-Gly-Leu-Trp-Met-Ser-Cys-Val-Val-Gln-Ser-Thr-Gly-Gln-Met-Gln-Cys-Lys-Val=Tyr-Asp-Ser-Leu-Leu-Ala-Leu-Pro-Gln-Asp-Leu-Gln-Ala-Ala-Arg-Ala-Leu-Cys-Val-Val-Thr-Leu-Leu-Ile-Val-Leu-Leu-Gly-Leu-Leu-Val-Tyr-Leu-Ala-Gly-Ala-Lys-Cys-Thr-Thr-Cys-Val-Glu-Asp-Arg-Asn-Ser-Lys-Ser-Arg-Leu-Val-Leu-Ile-Ser-Gly-Ile-Ile-Phe-Val-Ile-Ser-Gly-Val-Leu-Thr-Leu-Ile-Por-Val-Cys-Trp-Thr-Ala-His-Ser-lle-Ile-Gln-Asp-Phe-Tyr-Asn-Pro-Leu-Val-Ala-Asp-Ala-Gln-Lys-Arg-Glu-Leu-Gly-Ala-Ser-Leu-Tyr-Leu-Cily-Trp-Ala-Ala-Ser-Gly-Leu-Leu-Leu-Leu-Gly-Gly-Gly-Leu-Leu-Cys-Cys-Ala-Cys-Ser-Ser-Gly-Gly-Thr-Gln-Gly-Pro-Arg-His-Tyr-Met-Ala-Cys-Tyr-Ser-Thr-Ser-Val-Pro-His-Ser-Arg-Gly-Pro-Ser-Glu-Tyr-Pro-Thr-Lys-Asn-Tyr-Val-COOH
It would be readily apparent to a worker skilled in the art that a deletion could comprise a deletion of one or more amino acid residues from the amino acid sequence of the naturally occurring Claudin-6.
Exemplary N-terminal deletion peptides include:
NH2-Ala-Ser-Thr-Gly-Leu-Gln-Ile-Leu-Gly-Ile-Val-Leu-Thr-I,eu-Leu-Gly-Trp-Val-Asn-Ala-Leu-Val-Ser-Cys-Ala-Leu-Pro-Met-Trp-Lys-Val-Thr-Ala-Phe-lle-Gly-Asn-Ser-Ile-Val-Val-Ala-Gln-Met-Val-Trp-(ilu-Gly-Leu-Trp-Met-Ser-Cys-Val-Val-Gln-Ser-Thr-Gly-[G1n61 -Va1219]-COOH
NH2-Ser-Thr-Gly-Leu-Gln-Ile-Leu-Gly-lle-Val-Leu-Thr-Leu-Leu-Gly-Trp-Val-Asn-Ala-Leu-Val-Ser-Cys-Ala-Leu-Pro-Met-Trp-Lys-Val-Thr-Ala-Phe-De-Gly-Asn-Ser-lle-Val-Val-Ala-Gln-Met-Val-Trp-Glu-Gly-Leu-'Crp-Met-Ser-Cys-Val-Val-Gln-Ser-Thr-Gly-[G1n61 -Va1219]-COOH

NH2-Thr-Gly-Leu-Gln-Ile-Leu-Gly-Ile-Val-Leu-Thr-Leu-Leu-Gly-Trp-Val-Asn-Ala-Leu-Val-Ser-Cys-Ala-Leu-Pro-Met-T'rp-Lys-Val-T'hr-Ala-Phe-Ile-Gly-Asn-Ser-Ile-Val-Val-Ala-Gln-Met-Val-Trp-Glu-Gly-heu-Trp-Met-Ser-Cys-Val-Val-Ciln-Ser-Thr-Gly-[Gln6i -Val2i9]-COOH
Exemplary C-terminal deletion peptides are:
NH2-[Alai-Leu~~3]--Leu-Leu-Leu-Gly-Gly-Gly-Leu-Leu-Cys-Cys-Ala-Cys-Ser-Ser-Gly-Gly-T'hr-Gln-Gly-Pro-Arg-His-Tyr-Met-Ala-Cys-Tyr-Ser-Thr-Ser-Val-Pro-His-Ser-Arg-Gly-Pro-Ser-Glu-Tyr-Pro-Thr-Lys-Asn-Tyr-COOH
NHZ-[Alal-Leu173]--Leu-Leu-Leu-Gly-Gly-Gly-Leu-Leu-Cys-Cys-Ala-Cys-Ser-Ser-Gly-Gly-Thr-Gln-Gly-Pro-Arg-His-Tyr-Met-Ala-Cys-Tyr-Ser-Thr-Ser-Val-Pro-His-Ser-Arg-Gly-Pro-Ser-Glu-Tyr-Pro-Thr-Lys-Asn-COOH
NH2-[Alal-Leul~3]-Leu-Leu-Leu-Gly-Gly-Gly-Leu-Leu-Cys-Cys-Ala-Cys-Ser-Ser-Gly-Gly-Thr-Gln-Gly-Pro-Arg-His-Tyr-Met-Ala-Cys-Tyr-Ser-Thr-Ser-Val-Pro-His-Ser-Arg-Gly-Pro-Ser-Glu-Tyr-Pro-Thr-Lys-COON
:20 Production of Transgenic Animals Transgenic animals have been generated using the method as described in Example IV, which have an altered Scullin/Claudin-6 gene (Figures 9A and 23D). Generally, alterations to the naturally occurring gene can be modifications, deletions and substitutions.
Modifications and deletions render the naturally occurring gene non-functional, producing a "knockout" animal. Substitution of the naturally occurnng gene for a gene from a second species results in an animal that produces the gene product of the second species.
Substitution of the naturally occurring gene for a gene having a mutation, results in an animal that produces the mutated gene product. These transgenic animals are critical for antagonist or agonist studies, the creation of animal models of human diseases, and for ventual treatment of disorders or diseases associated with Claudin-6-mediated res nses. A transgenic animal carrying a "knockout" of Claudin-b is useful for the .r~. . .__...«..~~.,.._.~._.._ _ _.. .. .. _. _.

establishment of a non-human model for diseases involving Claudin-6 equivalents in a human.
Further, a transgenic mouse carrying the disrupted Claudin-6 gene can be generated by homologous recombination of a target DNA construct with the endogenous gene in the chromosome. The DNA construct can be prepared from a genomic clone (cDNA) of Claudin-6 isolated from a genomic DNA library.
The term "transgene" is used herein to describe genetic material that has been or is about 'LO to be artificially inserted into the genome of a mammal, particularly a mammalian cell of a living animal.
A "knock-out" of a gene means an alteration in the sequence of the gene that results in a decrease of function of the target gene, preferably such that target gene expression is L5 undetectable or insignificant. A knock-out of an endogenous Claudin-6 gene means that function of the Claudin-6 gene has been substantially decreased so that expression is not detectable or only present at insignificant levels. "Knock-out" transgenics can be transgenic animals having a heterozygous knock-out of the Claudin-6 gene or a homozygous knock-out of the Claudin-6 gene. "Knock-outs" also include conditional ~0 knock-outs, where alteration of the target gene can occur upon, for example, exposure of the animal to a substance that promotes target gene alteration, introduction of an enzyme that promotes recombination at the target gene site (e.g., Cre in the Cre-lox system), or other method for directing the target gene alteration postnatally.
'~5 A "knock-in" of a target gene means an alteration in a host cell genome that results in altered expression (e.g., increased (including ectopic)) of the target gene, e.g., by introduction of an additional copy of the target gene, or by operatively inserting a regulatory sequence that provides for enhanced expression of an endogenous copy of the target gene. Such transgenics can be heterozygous knock-in for the Claudin-6 gene or :30 homozygous for the knock-in of the Claudin-6 gene. "Knock-ins" also encompass conditional knock-ins.

The term "animal" is used herein to include all vertebrate animals, except humans. It also includes an individual animal in all stages of development, including embryonic and fetal stages. A "transgenic animal" is any animal containing one or more cells bearing genetic information altered or received, directly ar indirectly, by deliberate genetic manipulation at a subcellular level, such as by targeted recombination or microinjection or infection with recombinant virus. The term "transgenic animal" is not intended to encompass classical cross-breeding or in vitro fertilization, but rather is meant to encompass animals in which one or more cells are altered by, or receive, a recombinant DNA molecule. This recombinant DNA molecule may be specifically targeted to a defined genetic locus, may be randomly integrated within a chromosome, or it may be extxachromosomally replicating DNA. The term "germ cell line transgenic animal" refers to a transgenic animal in which the genetic alteration or genetic information was introduced into a germ line cell, thereby conferring the ability to transfer the genetic information to offspring. If such offspring in fact possess some or all of that alteration or genetic information, they are transgenic animals as well.
The alteration or genetic information may be foreign to the species of animal to which the recipient belongs, or foreign only to the particular individual recipient, or may be genetic information already possessed by the recipient. In the last case, the altered or introduced gene may be expressed differently than the native gene, or not expressed at all.
The altered Claudin-6 gene generally should not fully encode the same Claudin-6 as native to the host animal, and its expression product should be altered to a minor or great degree, or absent altogether. However, it is conceivable that a more modestly modified Claudin-6 gene will fall within the scope of the present invention.
The genes used for altering a target gene may be obtained by, a wide variety of techniques that include, but are not limited to, isolation from genomic sources, preparation of cDNAs from isolated mRNA templates, direct synthesis, or a combination thereof.
Any technique known in the art can be used to introduce the transgene into animals to produce the founder lines of transgenic animals. Such techniques include, but are not limited to pronuclear microinjection (Gordon et al., 19$0, Prcx. Natl. Acad.
Sci. USA 77:

7380-7384; Gordon & Ruddle, 1981, Science 214: 1244-1246; U.S. Pat. No.
4,873,191 (Oct. 10, 1989) T. E. Wagner and P. C. Hoppe); retrovirus mediated gene transfer into germ lines (Van der Putten et al., 1985, Proc. Natl. Acad. Sci. USA 82: 6148-152); gene targeting in embryonic stem cells (Thompson et al., 1989, Cell 56: 313-321);
electroporation of embryos (Lo, 1983, Mol. Cell. Biol. 3: 1803-1814); and sperm-mediated gene transfer (Lavitrano et al., 1989, Cell 57: 717-723); etc. For a review of such techniques, see Gordon, 1989, Transgenic Animals, Intl. Rev. Cytol. 115: 171-229. Once the founder animals are produced, they can be bred, inbred, crossbred or outbred to produce colonies.
'l0 The present invention provides fur transgenic animals that carry the transgene in all their cells, as well as animals which carry the transgene in some, but not all cells, i.e., mosaic animals. The transgene can be integrated as a single transgene or in tandem, e.g., head to head tandems, or head to tail or tail to tail.

In one example, the target cell for transgene introduction is the embryonal stem cell (ES).
ES cells may be obtained from pre-implantation embryos cultured in vitro [M.
J. Evans et al., Nature 292: 154-156 (1981); M. O. Bradley et al., Nature 309: 255-258 (1984);
Gossler et al. Proc. Natl. Acad. Sci. USA 83: 9065-9069 (1986); Robertson et al., Nature 20 322, 445-448 (1986); S. A. Wood et al. Proc. Natl. Acad. Sci. USA 90: 4582-(1993)). Transgenes can be efficiently introduced into the ES cells by standard techniques such as DNA transfection or by retrovirusmediated transduction. The resultant transformed ES cells can thereafter be combined with blastocysts from a non-human animal.
The introduced ES cells thereafter colonize the embryo and contribute to the germ line of the 25 resulting chimeric animal (R. Jaenisch, Science 240: 1468-1474 (1988)).
In order to analyse the role of the tight functional protein Claudin-6, transgenic animals that overexpress this protein were generated in which the Claudin-6 gene was targeted to various :30 epithelial and endothelial tissues that are known to have tight junctions as discussed below. In addition, the interactions of Claudin-6 protein in the cytoplasm were explored by generating transgenic animals that express this protein ectopically.
Further, in order to analyse the role of this TJ protein in the epidermis transgenic animals were generated by using a skin specific promoter, involucrin (Turksen et al., 1992, 1993). The involucrin promoter is a differentiation marker in the epidermis and therefore, faithfully targets transgenes, in this case the Claudin-6 transgene, to the suprabasal layer of the epidermis.
Three other tissues were targeted in order to determine the extent to which Claudin-6 overexpression effects the function of the TJ permeability barrier in these tissues. The Claudin-6 transgene was targeted to intestinal, cardiac, and pulmonary tissues using the Intestinal Fatty Acid Binding Promoter (IFABP), alpha-MHC promoter and the SPC
promoter, respectively. To generate transgenic animals, a 660bp cDNA of Scullin/Claudin-6 was subcloned into a promoter cassette and the fragment containing the promoter and the transgene was isolated and purified using standard microbiological techniques. The purified fragment was then injected into mouse eggs using standard implantation techniques known in the art for generation of transgenic mice as detailed in Example IV. This is a clear indication that by using this method any tissue, organ or cell type, such as skin, heart, intestine, lung, brain, kidney, liver etc. as well as any permeability barners formed in such tissues, for example the blood/brain barrier, an epithelial barrier, or the blood-retina barriers, can be directly targeted so that the TJ
permeability barrier can be easily manipulated, particularly in mammals. Such targeting methods will result in tissue specific regulation and/or modulation of Claudin-mediated responses, particularly those that control the formation and functioning of tissue permeability barriers. In addition, drags can be directly targeted to an organ, tissue or cell of interest to help prevent, protect against or treat a particular disease state.
The invention may be better understood by reference to the following examples which are intended for purposes of illustration only and are not to be construed as in any way limiting the scope of the present invention, which is defined in the claims appended hereto.
EXAMPLES
EXAMPLE I. Preparation of Claudin-Derived Peptide Analogs, Derivatives or Fragments Thereof for use in the Manipulation of Tight Junction Permeability Barriers) Peptides of mouse Claudin-6 (corresponding to amino acids [aa] _ ) and (SEQ 1D
Nos. ) are part of or the entire first and second putative extracellular domains, are part of or the entire transmembrane domain of TM l, TM2, TM3, or TM4, or are part of or the entire cytoplasmic domain of the amino terminal, intracellular loop or carboxylic tail of mouse Claudin-6, respectively. In addition, several different peptide forms of the first or second extracellular, transmembrane TM 1- TM4 or the cytoplasmic damains are synthesized as outline above. A scrambled peptide (SEQ ID No ), composed of a scrambled sequence of the same residues as SEQ ID No., can be synthesized. Peptides are prepared as 10 mM
stock solutions in DMSO and is added to both sides of the Transwell bathing wells. As well peptide sequences combining 1) one or more parts of the extracellular domain plus one or more parts of the 4 transmembrane domains and two, one or more parts of the cytoplasmic domain plus one or more parts of the 4 transmembrane domains can be synthsized in order to test their ability to disrupt the TJ protein Claudin and thus increase the permeability a cell or tissue to a drug for transport. All peptides can be synthesized as outlined above by the Peptide Facility at Sigrna/Synthesis (Texas, USA).
Example II. Sequence Analysis of Scullin/Claudin-6 Based on sequence analysis and in vitro transfection studies, Scullin/Claudin-6 a newly ;Z0 identified putative integral TJ membrane protein is a candidate for participation in formation of the functional intercellular seal of the tight junction. The full length of the Claudin-6 Open Reading Frame (ORF) with both nucleic acid and amino acid sequences (Figure 1A) is 219 amino acids long with an estimated molecular weight of 23kb. This sequence has been submitted to Genbank (AF125305, AF125306), included is a copy of the submission. A predicted schematic of the structure is represented by Figure 1B. The primary amino acid sequence of mouse Claudin-6 predicts four or five membrane-spanning regions and two extracellular loops (Figure 2). Both extracellular domains of Scullin/Claudin-6 consist solely of uncharged residues with the exception of one or two charged residues adjacent to the transmembrane regians.
A comparison of the homology between the Scullin protein of the invention and the Claudin gene family (Claudins 1 to 7), demonstrates the Scullin has remarkable similarities with a number of Claudin proteins, particularly Claudin-6 (Figures 3 and 4). In addition a comparison of mouse Scullin, Claudin-6 and occludin proteins shows that one, occludin is not related to Scullin (Figure 4); and two, that mouse Scullin and Claudin-6 are identical at both the amino acid and nucleic acid level (Figure 5).
Mouse Scullin sequences were used to screen known human gene sequences and it was determined that the Scullin gene is very similar to, and can be mapped to, two regions of human chromosome 16 (Figure 7). Further, based on the amino acid sequence of these proteins encoded by these two genes and on a search and sequence alignment done using the Clustal program (www2.ebi.ac.uk/clustalw) indicates one protein appears to be human Scullin/Claudin-6 and the other human Claudin-9 (Figures 6 and 7).
Interestingly, these two genes are organised in a fashion reminiscent of the Dlx genes (a member of the Hox gene family), i.e., head to head. There is a 1.4 kb intergenic region between these two genes. Using the sequence information for Scullin/Claudin-6 and Claudin-9 garnered using the sequence analysis program Blast (www.ncbi.nlm.nih.gov/BLAST~, these two sequences were isolated by PCR and verified by sequencing. The intergenic region between Claudin-6 and Claudin-9 may be important in the regulation of these two genes. A close inspection of the intergenic region indicates that there are a number of sites for known regulatory molecules including, but not limited to, AP-2, Hox, and LEF-1.
Northern blots illustrated in Figure 8, show the tissue distribution of mouse Claudin-6 and Claudin-9 mRNA levels. High levels of Claudin-6 mRNA are found in brain, kidney, liver, lung and stomach, whereas Claudin- 9 levels are highest in kidney and are found to a lesser extent in brain and liver. Claudin-6 then seems a likely candidate to target in order to maipulate the TJ permeability barrier in a wide variety of tissues.
Interestingly, the level of Claudin-9 mRNA is, to a greater extent, found in the skin of normal animals than is Claudin-6. Finally, based on organisational observations, these two genes might be regulated and expressed in a co-ordinated pair fashion.
Example III. Demonstration of Claudin-6's Role in Regulating the Tight Junction Permeability Barrier Using a Cell Model to Assess Permeability The epidermal progenitor cells (EPC) of the differentiating ES cell model were grown on Transwell filters (Costar Corp., Cambridge, MA) in 85% DMEM (high glucose, 1 g/liter glucose) supplemented with 5% FCS and maintained at 37°C and 5% COZ.
For the calcium !> switch assay (Troy and Turksen, 1999), EPC cells are allowed to grow in normal growth medium until confluent and were subsequently changed to low calcium medium for 18 h: At the end of the low calcium (<66 plvl) incubation, EPC cell cultures were replenished with normal calcium medium, and the formation of tight junctions monitored by the generation of transepithelial electrical resistance (TER), measured by a NOVA transepidermal apparatus.

11) First, measurements of TER can be performed to indicate the ability of the Claudin-derived peptide analogs ability to disrupt the epithelial permeabilty barrier. TER is calculated from the measured voltage and normalized by the area of the monolayer of EPC's. The background TER of blank Transwell filters was subtracted from the TER of cell monolayers.
A second 15 assay will measure the flux of paracellular tracer compounds across the EPC
monolayer in the presence or absence of one or more Claudin-derived peptide analogs.
The assays are performed on 6.5-mm Transwells (in 6-well cell culture dishes).
Two different paracellular tracers, neutral dextran (mol wt 3 kDa) conjugated with Texas red (Molecular 20 Probes, Eugene, OR), and neutral dextran (mol wt 40 kDa) conjugated with Texas red (Molecular Probes), are used in this assay. At the beginning of the flux assay, both sides of the bathing wells of Transwell filters were replaced with fresh medium without peptides. The tracers were added to a final concentration of 25 pg/100 w1 for dextran (mol wt 3 kDa) or 50 ~,g/100 p1 for dextran (mol wt 40 kDa) in the apical bathing wells containing 100 w1 of 2.5 medium. The basal bathing well had no added tracers and contained 700 ~,1 of the same flux assay medium as in the apical compartment. All flux assays are performed at 25°C with gentle agitation. Cell monolayers were allowed to equilibrate for 30 min after the addition of tracers. For dextran (3 kD and 40 kD), the concentration was calculated from the amount of fluorescence emission at 610 nm (excitation at 587 nm) using a tiuration curve of known 30 concentrations of the same tracers. Therefore, an increase in flux of the tracer across the EPC

monolayer is indicative of a particular peptide's ability to increase the disruption of the permeability barrier and thus is an excellent candidate for use in increasing paracellular drug transport.
Example IV. Establishment of Transgenic Mouse Models In order to demonstrate the paracellular drug delivery system of this invention, transgenic animals that overexpress this protein were generated in which the Claudin-6 gene was targeted to various epithelial and endothelial tissues that are known to have tight junctions. In 10~ addition, the interactions of Claudin-6 protein in the cytoplasm were demonstrated by generating transgenic animals that express this protein ectopically.
Further, transgenic animalswere generated by using a skin specific promoter, involucrin (Turksen et al., 1992, 1993) to demonstrate the effect of Claudin-b on epidermal la differentiation and tissue function. The involucrin promoter is a differentiation marker in the epidermis and therefore, faithfully targets transgenes, in this case the Claudin-6 transgene, to the suprabasal layer of the epidermis (Carroll et al, 1993, 1995) where involucrin is normally expressed (Rice and Green, 1977). Three other tissues were also targeted in order to demonstrate how Claudin-6 overexpression effects the function of the TJ
permeability barrier 20 in these tissues. The Claudin-6 transgene was targeted to intestinal, cardiac, and pulmonary tissues using the Intestinal Fatty Acid Binding Promoter (IFABP), alpha-MHC
promoter and the SPC promoter, respectively.
Generation of TYr~nsgenic Animals 25 To generate transgenic animals, a b60bp cDNA of Scullin/Claudin-6 was subcloned into an involucrin promoter cassette (Figures 9A and 23D). The fragment containing the promoter and the Claudin-6 transgene was isolated and purified using standard microbiological techniques. The purified fragment was then injected into mouse eggs using standard implantation techniques known in the art for generation of transgenic mice (Hogen et al., 30 1994; Turksen et ad.,1992, 1993).

Phenotypes of Transgenic Animals Using this technique, 7 founder mice that have the Claudin-6 transgene targeted to the suprabasal level of the epidermis were generated based on PCR screening (Figure 9B).
'i Two major phenotypes were observed in these aansgenic animals, severe/lethal and less severe/viable. Transgenic animals that have very high expression of Claudin-6 protein in the epidermis are very severely affected. At birth, they move very sluggishly and their skin appears shiny. Within 1- 3 days of birth the skin of these animals becomes dry and flaky 1U (Figure 9C). Due to the severe increase in permeability and because of the disrupted permeability barrier formation due to increased Claudin protein expression, these animals dehydrate and die within 3 days of their birth. This is consistent with defective permeability barrier formation in these mice. In addition, to assess the epidermal permeability barrier, the X-gal staining, skin permeability assay, (Hardman et al, 1993) was used and confirmed that to these animals have a poor permeability formation (Figure 9D). Further confirmation that transgenic mice that express high levels of Claudin-6 protein tend to become dehydrated is shown in Figure 10, wherein measurements of the hydration state of transgenic animals was much less than that found for control animals. Other methods may be used for measuring the hydration state of animals and include, but are not limited to, those of Goffin et al., 1999 Clin.
20 Exp. Dennatol. 24(4):308-311; andTagami et al., 1994 Derm. Venereol. Suppl.
185:29-33.

Less Severe Phenotypes Figure 11 illustrates a comparison of phenotypes between wild type mice and transgenic animals that express lower levels of Claudin-6. Animals expressing lower levels of Claudin-6 2!i in the skin survive and have distinct phenotypic traits including: a wavy hair pattern (Figure 11A); curly whiskers (Figure 11C); and a delay of 4-6 days in the opening of the eyes (Figure 11E). Wild type controls are shown in Figure 11B and Figure 11 D as comparisons.
Additionally, keratin expression analysis of trangenic animals expressing Claudin-6, indicates that there is an increase in keratin 1 expression while the expression of filagrin and loricrin is 30 less uniform and disrupted than in wild type mice. The composition of the hair fibres is also a distinguishing phenotypic characteristic of transgenic animals that express lower levels of Claudin-6 protein in the epidermis. Figure 12 depicts the composition of hair fibres in wild type and Claudin-6 transgenic mice. The proportion of the four types of hair fibres in Claudin-6 mice is drastically different than those of wild type mice. The greater percentage of zigzag fibres (61%) to guard hairs (39% total) in transgenic mice compared to 44% and 56%, respectively in control animals, is what contributes to the curly look of the coat in transgenic animals. The occurrence of prostate tumors in Claudin-6 transgenic mice is illustrated in Figure 13 A and B. In a small yet significant subset of the population (~5%), mice expressing-Claudin-6 develop large prostate tumors after 6 to 8 months. Transgenic animals are also exhibit an increased susceptibility to tumor induction when exposed to tumor promoting substances when compared to control animals. For example, Figure 14 shows that application of TPA (a tumor promoter) to Claudin-6 mice results in papilloma formation in these transgenic animals.
1 S In order to gain insight into the function of Claudin-6 protein's role in the formation of TJ in the epidermis various truncations where made in the Claudin-6 gene as illustrated in Figure 15. Truncation of Claudin-6 in transgenic mice where targeted to the tail region of Claudin-6 protein. Wild type, normal, Claudin-6 sequence is shown in Figure 15A.
Truncations were made at position 206, 194 and 186, as represented by Figure 15B, Figure 15C
and Figure 15D, respectively. Founder mice for the truncation in position number 194 have been generated.
Figure 16 illustrates transgenic mice with Claudin-6-FLAG truncated at c~194.
Truncation in position 194 generates mice with no hair fibers, they are totally and completely bald.
However, this truncation seems to have no effect on whisker morphology. In addition, animals with this truncation are visibly smaller than corresponding age matched controls.
The effect of deleting amino acids from the second extracellular loop of Claudin-6 protein is currently being investigated. Figure 17 depicts the loop deletion that has been made to investigate its role in the functioning of Claudin-6 in vivo. A 13 amino acid portion of the second loop has been removed and is currently being injected for the production of transgenic mice.

Tissue Specific Targeting of Claudin-6 Protein Tissue specific targeting has been demonstrated with the transgenic mouse models of this invention. This is a particularly powerful advantage in that by using this technique a variety of organs, tissues and cells may be targeted directly at the site of permeability barrier formation. Therefore, a particular permeability barrier including but not limited to, for example the blood brain barrier, can be modulated to allow for paracelluar drug transport across the barrier. By using tissue specific promoters it has been shown that Claudin-6 can be targeted to different tissues. In addition to the direct targeting to epidermal tissue as in the above example, other tissues have been targeting using this method including but not limited to intestinal, cardiac, and pulmonary tissues. Similarly, Claudin-6 has been targeted to the vascular endothelium and to neurons using the vasular endothelium promoter, VE-CAD
(Huber et al. 1999) and the neuruofilament-heavy chain promoter, resp~tively.
Direct evidence for tissue specific targeting is displayed with discussion of the following figures.
Figure 18A displays the promoter used to target Claudin-6 to the intestine, Intestinal Fatty Acid Binding Promoter (1FABP). This promoter is active only in intestinal epithelial cells. A
series of injections with this construct has produced 7 DNA positive animals as shown through PCR analysis (Figure 18B). Indirect immunofluoresence studies using monoclonal antibodies against the commercially available FLAG tag of the construct indicate that the transgenic lines generated are indeed expressing the transgene in intestine Figure 18C, Figure 18D.
Figure 19 shows that Claudin-6 can also be targeted to heart by using the a-MHC promoter (Figure 19A). Three tines have been generated that are positive in PCR screens (Figure 19B).
2~ The PCR positive transgenic animals labelled with anti-FLAG antibodies indicate that Claudin-6 was targeted to the myocyte cell membrane by the ocMHC promoter Figure 19C and Figure 19D. In addition, these transgenic animals are smaller than their normal littermates (and stay smaller throughout their lifespan) Figure 19E, with smaller hearts Figurl9F.
Severely phenotypic animals generally die 3 days following birth. These transgenic mice are 3(1 an excellent model system to study defects in heart development resulting in poor function and heart failure. Changes in transgenic heart tissue will assist in the study to determine some of the crucial players that are involved in functional and malfunctional hearts. Similarly, using such a method to target other organs such as, but not limited to, brain, kidney, intestine and liver may also be used as in vivo model systems to study diseases and disorders associated ~~ with the functioning of these organs.
Due to the ectopic expression of Claudin-6 in myocytes (cardiocytes) there may be a subsequent disruption of the cytoskeletal balance (architecture) in myocytes.
Such a disruption will consequently produce a weak phenotype that may be an excellent model 1(1 system to study heart function in vivo. A comparison of heart spe<;ific cytoskeletal markers that are known to contribute to the mechanical functioning of the heart have been analyzed for aMHC transgenic and wild type animals is illustrated in Figure 20. By utilizing PCR
techniques familiar to a person of skill in the art it has been determined that aMHC
transgenic animals possess all the same heart specific cytoskeletal markers as wild type mice.
lei However, myosin light chain-lA (MLC-lA) and atrial natriuretic factor (ANF) are dramatically down regulated in transgenic cardiac tissue by overexpression of the Claudin-6 transgene. Thus, the cytoarchitecture in transgenic hearts is affected by the overexpression of Claudin-6 in myocytes. Interestingly, a very small percentage of aMHC
transgenic animals develop grossly enlarged kidneys within 2-3 weeks, Figure 21A-C. The cause of this is 20 phenotype is not yet known however, an in depth analysis is being undertaken in order to better understand the alterations in kidney growth, structure and physiology of this particular phenotype. Lastly, Claudin-6 has been successfully targeted to lung tissue using the human SPC promoter, Figure 22A. Although only two DNA positive transgenic animals have been generated the screening for other potential founders is currently underway.
2.'i Example V. Demonstration of Claudin-6's Role in Epithelial Differentiation and Epidermal Permeability Barrier Formation Analysis of Inv-Claudin-6 Expression in Transgenic Mice Seventeen transgenic mice were generated using the same technique as described above.
Among them seven were mosaic and survived while the remaining founders died within 48 hours. Lines were established from mosaic founders exhibiting similar phenotypes and transgenic mice were identified using PCR (Figures 23E and 24). To ensure amplification of mRNA of transgenic, but not non-transgenic skin, RT-PCR was conducted with primers spanning the junction of the Inv exon and Glaudin-6 sequences (Figure 23F).
Conversely, with Claudin-6 forward and reverse primers, RT-PCR was perfornied and a 660bp band diagnostic of mouse Claudin-6 was amplified both endogenously and exogenously (Figure 1(1 23G). It was estimated that expression of the transgene was ~$-fold over endogenous Claudin-6 expression. Protein analysis also revealed a significant increase in the expression of Claudin-6 in the transgenic epidermis (Figure 23H). Overexpression of Claudin-6 was further confirmed by indirect immunofluoresence on frozen sections of transgenic and wild type backskin using polyclonal antibodies specific for Claudin-6. As expected, the Claudin-6 1-'i protein in wild type and transgenic epidermis was restricted to the upper spinous and granular layers, where it localizes to cell-cell junctions (Figure 23I), with transgenic mice exhibiting appreciably higher levels in relative terms in agreement with the PCR results.
The Claudin Profile of Inv-Claudin-6 Transgenic Epidermis 20 The effect of the perturbation of one Claudin on the expression and homeostasis of the other Claudins was examined. Using primers specific for known mouse Claudins (Figure 24), pilot PCR runs at 25, 30 and 35 cycles was done to find the linear range of signal detection (Figure 25A) and the band intensities quantified (Figure 25B) in RNA samples from transgenic and wildtype mouse epidermis. Claudin-6 expression is clearly increased in the transgenic mice 2'.i indicating the substantial level of overexpression and ease of detection of the transgene. On the other hand, Claudin-1 expression is only slightly decreased in the transgenic epidermis:
~2-fold at 30 cycles and marginally at 35 cycles. This is supported by the immunohistochemical analysis (Turksen and Aubin, 1991) of Claudin-1 expression in backskin samples indicating no obvious differences between the normal and transgenic 31) samples (Figure 25C).

RT-PCR results further indicated tlhat other Claudins were decreased to varying degrees in response to the overexpression of Claudin-fi, i.e., decreases were seen in Claudin-3 (~2.6-fold at 35 cycles), Claudin-4 (~2.4-fold at 30 cycles), Claudin-7 (~1.7-fold at 35 cycles), Claudin-8 (~2.5-fold at 30 cycles), Claudin-10 (~2.6-fold at 35 cycles), Claudin-11 (~2-fold at 30 cycles) and Claudin-14 (~2-fold at 35 cycles). Claudin-9 remained unchanged as no detectable signal was evident in either wild type or transgenic samples (not shown). There was no Claudin-2 expression detectable by RT-PCR or immunohistochemistry analysis (Figure 25C).
As antibodies for other specific Claudins become available, they will also be assessed and compared to the RT-PCR results. A control with no reverse transcriptase shows that there was no DNA contamination in the RNA samples used and a control with primers for GAPDH
indicate that signal differences observed are truly due to Claudin expression rather than sample inconsistencies.
Skin Abnormalities in Transgenic Mice Overexpressing Claudin-6 Transgenic newborn mice were immediately identifiable by their smaller size (~30% by weight) as well as the very distinct appearance of their skin, which was red, shiny and sticky to the touch. These neonates lost sufficient body moisture to cause the skin to become dried up and cracked (Figure 26A), and the dehydration resulted in death within 24 to 48 hours, suggesting an impairment of the skin's barrier function (see below).
Histopathology showed the transgenic epidermis to be thicker and disorganised, with improper packing of cells and with basal cells lacking their usual uniformly cuboidal shape (Figure 26B-G).
In addition, there were areas of the epidermis that had few or no visible keratohyalin granular cells (Figure 26B vs. C) and the stratum corneum (SC) was moderately thicker and frequently fragmented (Figure 26D vs. E). Collectively, these findings suggested that certain steps of epidermal terminal differentiation might be retarded or disrupted in the transgenic mice. Strikingly, there was a marked decrease in subcutaneous fat pads (Figure 26F vs. G) such that the skin of transgenic animals was notably thinner than that of control animals in spite of the thicker epidermis.
3(1 Abnormal Epidermal Barrier Function Exhibited in Inv-Claudin-6 Transgenic Mice To determine whether the observed dehydration was due to defective epidermal barrier function, permeability barrier formation was compared using a (3-gal assay (Hardman et. al, 1993) on transgenic and wild type animals at embryonic age 16.5 and 18.5 (E16.5 and E18.5) as well as newborns (Figure 27A). Typically, the mouse EPB forms 2-3 days prior to birth corresponding to the first detection of the SC in the mouse at E17.5 (Hardman et. al, 1993;
Aszterbaum et. al, 1992; Williams et.al, 1998; Elias and Feingold, 2001).
Since an intact EPB blocks the penetration of (3-gal through the skin, this assay is a reliable means to assess EPB formation. As expected, (3-gal penetrated the skin of both transgenic and wild type 1(1 animals at E16.5. By E18.5, however, there was no penetration of the dye in wild type mice whereas transgenic embryos still did not possess an EPB, a situation that continued to exist even after birth.
To compliment the (3-gal assay, the amount of water that was lost through the skin's surface at birth (traps-epidermal water loss: TEWL) was also measured using a dermal phase meter (DPM). In representative experiments, significantly higher DPM values (reproducibly in the range of 440-470) were obtained from transgenic samples compared to wild type values (reproducibly in the range of 118-1.22), indicating a ~3-fold increase in water loss through the skin in transgenic newborns (Figure 27B). To assess further whether the water loss was sufficient to cause the neonatal lethality observed, the weight of newborn transgenic and wild type mice was tabulated for a period of six hours after birth. Transgenic mice lost up to 5% of their birth 'weight due to fluid evaporation attributed to a compromised EPB
(not shown). It has been noticed that such a rapid rate of dehydration exists in premature babies, which results in hypovolaemic shock due to severe dehydration leading to death as observed in Inv-Claudin-2ri 6 transgenic mice. These results confirm that the EPB of transgenic animals were poorly formed, indicating that the aberrant expression of Claudin-6 affects barrier formation and causes neonatal lethality.

Defective Cornified Envelopes (CEs) in Inv-Claudin-6 Epidermis The CEs of Inv-Claudin-6 and wild type skin were investigated to determine if there was a disruption in their formation and morphology. CEs were isolated by boiling the epidermis in the presence of an ionic detergent and a reducing agent (Hohl et al, 1991).
The CEs of wild ~~ type mice were abundant, uniformly rigid and polygonal (Figure 27D and F), but within the transgenic samples there were fewer CEs and they were generally more fragile in appearance (Figure 27C and E). In addition, transgenic CEs were morphologically less uniform and mostly rounded in shape reflecting their weakened nature (Figure 27E). The defect in the shape of the CEs, with many rounded rather than polygonal in appearance, may also explain 1(1 the thicker less compact nature of the overall SC observed histologically (Figure 26D).
Overexpression of Claudin-6 in the Suprabasal Layer of the Epidermis Affects the Epidermal Differentiation Program In general, keratin and intermediate filament associated protein expression are very reliable 1 ~ biochemical indicators of whether the program of epidermal differentiation is altered (Fuchs and Byrne, 1994; Turksen and Troy, 1998). The expression of epidermal keratin and terminal differentiation markers in the skin of transgenic and normal animals was evaluated by immunofluorescence (Figure 28). Basal cell specific keratins KS/K14 were present at similar levels in transgenic and normal skin, indicating that the basal cells continue to express the 2() major structural proteins characteristic of this layer despite their abnormal morphology and arrangement. On the other hand, the suprabasal differentiation marker Kl, which is generally restricted to the spinous and granular layers of the epidermis, showed an increased expression in the transgenic skin.
25 Skin samples were further assayed for the expression of K6/K16, keratins normally associated with the outer root sheath of hair follicles and not seen in the interfollicular epidermis unless there is hyperproliferation of the stratified epithelia such as under wound healing conditions (McGowan and Coulombe, 1998a). Patchy K6lKlb expression was seen in the lower strata primarily in the suprabasal layers of the epidermis of Claudin-6 transgenic mice. However, 3() K17 (a marker of early epithelial differentiation expressed normally in some basal cells of newborn epidermis and upregulated during hyperproliferation [Mc~Gowan and Coulombe, 1998b]) was similar in wild type and transgenic samples (not shown).
Immunohistochemistry with antibodies against histone-3, a proliferation marker, showed no obvious differences between wild type and transgenic samples, suggesting that there is not an increase in proliferation rate in transgenic epidermis (not shown).
The expression of the structural proteins filaggrin, loricrin, transglutaminase-3 and involucrin were evaluated by immunofluorescence (Figure 28) and western blotting (Figure 29A). All four of these markers were abnormally expressed with a noticeably less compact distribution in transgenic as compared to wild type epidermis, again indicating a dysfunction in differentiation. Most interestingly, filaggrin expression was seen to extend into the suprabasal layers of the epidermis of transgenic mice. The markedly increased filaggrin expression suggests a dysfunction in the processing of profilaggrin. Western blotting showed that the processing of profilaggrin to filaggrin was enhanced as evidenced by the increased proportion of smaller (processed filaggrin) versus larger sized (processing profilaggrin) bands in transgenic versus wild type samples. In fact, fully processed filaggrin was shown to be increased ~ 13-fold in the transgenic samples (Figure 29A). Alterations in filaggrin processing are also supported by histological observations indicating the discontinuous and disrupted pattern of keratohyalin granules in the transgenic epidermis. Since filaggrin expression changes in accordance with the enhancement or disappearances of granular cells, the abnormal distribution of granular cells supports the hypothesis that the terminal epidermal differentiation program was not progressing normally in the transgenic animals.
In addition to filaggrin, the expression of loricrin, transglutaminase-3 and involucrin was increased, though more modestly, in the transgenic compared to wild type samples (~ 1.5-fold, Figure 29A). RT-PCR analysis revealed no major detectable alterations in the expression of these markers.
The discontinuous keratohyalin granules, fragile CEs and abnormal histological observations led to the question of whether the regulators of the formation of the CEs were defective. The expression levels of repetin and several SPRRs (namely SPRR1A, 1B, 2A, 2B, 2C, 2D, 2G
and 3), molecules known to be involved in the cross-linking of CE molecules (Figure 29C), were studied. SPRR2B, 2C and 3 (SPRRs known not to be expressed in newborn epidermis) as well as SPRR1B were not detected suggesting that the overexpression of Claudin-6 did not 'l cause an alteration in the expression of these SPRRs. On the other hand, repetin and SPRRlA
and 2A expression was decreased while the expression of SPRR2D and 2G was increased in the transgenic epidermis. Disruption in the known regulators of repetin, SPRR1A and 2A
were looked at for clues to the mechanism of deregulation of these cross-linking proteins.
Since a KI,F4 binding site exists on the SPRR2A promoter (Sark et. al, 1998;
Fischer et. al, 1U 1996), KLF4 was a potential candidate. Analysis of KLF4 expression in transgenic epidermis by RT-PCR indicated that its levels were decreased ~4-fold as compared to the wild type (Figure 29C). The downregulation of Klf4, given its transactivator role in SPRR expression, may be responsible for the cascade of changes that lead to disruption of the CE cross-linking process and hence the perturbation of epidermal differentiation as well as the processing of 15 late markers that contribute to EPB formation.
From the foregoing, it will be evident that although specific embodiments of the invention have been described herein for the purpose of illustrating the invention, it will be obvious that the same may be varied in many ways. Such variations are not to be regarded as a departure from the scope of the invention, and all such modifications as would be obvious to one skilled 2f3 in the art are intended to be included within the scope of the following claims.

Claims (6)

1. A composition comprising one or more Claudin-derived peptide analogs, derivatives or fragments thereof, a drug to be delivered transdermally and an acceptable pharmaceutical carrier.

2. A method for identifying a compound capable of modulating Claudin-mediated cell permeability, comprising:
(a) contacting a test compound with an antibody or antigen-binding fragment thereof that binds to a modulating agent comprising the sequence of SEQ ID NO:1, wherein the agent comprises no more than 50 consecutive amino acid residues present within an Claudin molecule; and (b) detecting the level of antibody that binds to the test compound, and therefrom identifying a compound capable of modulating Claudin-mediated cell permeability.

3. A method according to claim 2, wherein the agent is a linear peptide.
3. A method according to claim 3, wherein the agent comprises 5-16 consecutive amino acid residues present within a Claudin.

4. A method according to claim 2, wherein the step of detecting is performed using an ELISA.

5. A method according to claim 2, wherein the test compound is produced by an expression library.

6. A transgenic mouse that overexpresses a Claudin protein for use as an animal model.