Note: Descriptions are shown in the official language in which they were submitted.
CA 02379661 2002-03-28
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
CA 02379661 2002-03-28
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).
2
CA 02379661 2002-03-28
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.
3
CA 02379661 2002-03-28
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
4
CA 02379661 2002-03-28
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
5
CA 02379661 2002-03-28
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
6
CA 02379661 2002-03-28
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
7
CA 02379661 2002-03-28
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).
8
CA 02379661 2002-03-28
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
9
CA 02379661 2002-03-28
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.
CA 02379661 2002-03-28
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.
11
CA 02379661 2002-03-28
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).
12
CA 02379661 2002-03-28
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.
13
CA 02379661 2002-03-28
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
14
CA 02379661 2002-03-28
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
1S
CA 02379661 2002-03-28
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
16
CA 02379661 2002-03-28
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
17
CA 02379661 2002-03-28
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
18
CA 02379661 2002-03-28
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
19
CA 02379661 2002-03-28
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
CA 02379661 2002-03-28
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
21
CA 02379661 2002-03-28
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.
22
CA 02379661 2002-03-28
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
23
CA 02379661 2002-03-28
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.
24
CA 02379661 2002-03-28
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
CA 02379661 2002-03-28
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.
26
CA 02379661 2002-03-28
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
27
CA 02379661 2002-03-28
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.
28
CA 02379661 2002-03-28
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-
29
CA 02379661 2002-03-28
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
CA 02379661 2002-03-28
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;
31
CA 02379661 2002-03-28
(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.
32
CA 02379661 2002-03-28
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
33
CA 02379661 2002-03-28
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 (--
CH2-S--
); 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
34
CA 02379661 2002-03-28
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
CA 02379661 2002-03-28
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
36
CA 02379661 2002-03-28
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
37
CA 02379661 2002-03-28
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
38
CA 02379661 2002-03-28
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.
39
CA 02379661 2002-03-28
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
CA 02379661 2002-03-28
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
41
CA 02379661 2002-03-28
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,
42
CA 02379661 2002-03-28
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.
43
CA 02379661 2002-03-28
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);
44
CA 02379661 2002-03-28
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.
CA 02379661 2002-03-28
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.
46
CA 02379661 2002-03-28
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
47
CA 02379661 2002-03-28
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
48
CA 02379661 2002-03-28
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.
49
CA 02379661 2002-03-28
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
CA 02379661 2002-03-28
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
51
.r~. . .__...«..~~.,.._.~._.._ _ _.. .. .. _. _.
CA 02379661 2002-03-28
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.
52
CA 02379661 2002-03-28
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:
53
CA 02379661 2002-03-28
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.
L5
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-
4584
(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
54
CA 02379661 2002-03-28
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)
CA 02379661 2002-03-28
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
56
CA 02379661 2002-03-28
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
57
CA 02379661 2002-03-28
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
58
CA 02379661 2002-03-28
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).
59
CA 02379661 2002-03-28
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.
39
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
CA 02379661 2002-03-28
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.
61
CA 02379661 2002-03-28
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
62
CA 02379661 2002-03-28
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
63
CA 02379661 2002-03-28
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).
64
CA 02379661 2002-03-28
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
CA 02379661 2002-03-28
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.
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CA 02379661 2002-03-28
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
67
CA 02379661 2002-03-28
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
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CA 02379661 2002-03-28
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.
69
