Modifying the permeability of physiological barriers

Details Modifying the permeability of physiological barriers Modifying the permeability of physiological barriers

1999-03-29

2002-06-18

Jones, Dwayne C. (Department: 1614)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Peptide containing doai

C514S414000

Reexamination Certificate

active

06407058

ABSTRACT:

The present invention relates to substances which are useful in modifying cell—cell adhesion and in modifying the permeability of physiological barriers.
Cell—cell adhesion is of crucial importance for the development and maintenance of tissue structure. Furthermore, regulation of such adhesion plays a normal role in physiological situations such as tissue turnover. Aberrant control may contribute to the aetiology of pathologies such as cancer and inflammation.
Therefore, there has been considerable interest in the basic processes whereby cells adhere to each other and ways in which such processes may be regulated. In general, cell—cell adhesion involves proteins on neighbouring cells that bind to one another. The cytoplasmic domains of proteins actually involved in adhesion can also physically associate with other cytoplasmic proteins that may either play a mechanical role, such as providing links with other proteins, or provide a regulatory influence.
p120 and p100 are members of the armadillo protein family. The armadillo repeats found in the members of this family appear to provide means whereby proteins can interact with others. p120 and p100, intracellular, cytoplasmic proteins, are now known to associate with cadherins, which are Ca
2+
-dependent adhesion molecules that contribute in an important way to cell—cell adhesion. Cadherins are important in control of cell proliferation. If cadherins fail to function properly, cells can proliferate in an unregulated fashion and metastasize. Cadherins are also thought to be important components in controlling the permeability of physiological barriers i.e. cell tight junctions. Here, disruption of cadherin based cell—cell adhesion leads to increased tight junction permeability. This has raised the possibility that tight junctions, although potentially regulatable in themselves, may be regulated by the adhesiveness of the adherens junction. In turn, cadherin adhesiveness may be regulated by the phosphorylation state of associated catenins, including p100 and p120.
The blood-brain barrier is an important example of a cell tight junction. It serves to separate the molecular, ionic and cellular environment of the blood from that of the brain. To a major degree, this separation is achieved by inter-endothelial tight junctions of high electrical resistance which greatly diminish paracellular flux. It is clear that the permeability of the tight junctions of the blood-brain barrier is not immutable. Rather, permeability appears to undergo dynamic regulation, but the way in which this is achieved is not fully understood.
In WO95/13820 it is disclosed that tyrosine protein phosphorylation is a key regulator of the permeability of tight junctions in both epithelial and endothelial cells; tyrosine protein phosphorylation may therefore be manipulated to control the permeability of the blood-brain and other physiological barriers. Decreasing the degree of tyrosine protein phosphorylation reduces permeability of the blood-brain or other barrier, whereas increasing the degree of tyrosine protein phosphorylation increases permeability. WO95/13820 also disclosed that the proteins p100 and p120 are believed to be substrates of tyrosine kinase. Further information regarding p100 and p120 is provided in WO96/16170.
Although WO95/13820 and WO96/16170 provide important information regarding the functioning of p100 and p120 this information is not complete. Unexpectedly, the present inventors have now discovered that p100 and/or p120 participate in a cycle which involves the phosphorylation/dephosphorylation of serine/threonine residues on these proteins. Furthermore they have shown that this cycle is regulatable by agents which are known tumour promoters, inflammatory mediators and tight junction permeability modulators. Thus, agents which interfere with regulation of this cycle itself or pathways involved in its regulation could have application to a wide variety of medical situations.
According to the present invention there is provided an agent capable of inducing the phosphorylation of serine and/or threonine residues of p100 and/or p120, for use in medicine.
The present invention also provides an agent capable of inducing the dephosphorylation of phosphorylated serine and/or threonine of p100 and/or p120 residues, for use in medicine.
By using an agent as described above, the permeability of physiological barriers i.e. tight junction permeability could be modified and cell—cell adhesion could also be modified.
By way of example, one way of utilising the present invention is to adjust the activity of protein kinase C. The present inventors have found that dephosphorylation of phosphorylated serine and/or threonine residues in p120 and/or p100 can be induced by increasing protein kinase C activity (i.e. by using protein kinase C activators), and that the phosphorylation of serine and/or threonine residues present in p100 and/or p120 can be induced by decreasing protein kinase C activity (i.e. by using protein kinase C inhibitors).
Examples of protein kinase C inhibitors are phorbol diesters, bryostatins 1 and 2, (−) indolactams V and (+) indolactam V, teleocidind, DHI ([6-(N-Decylamino)-4-hydroxymethylindole]) and ADMB ([3-(N-Acetylamino)-5-(N-decyl-N-methylamino)benzyl alcohol]), lipotoxin A4 and B4, mezerein, (−)-7-octylindolactam V, resiniferatoxin, thymeleatoxin. Protein kinase C may also be activated by ligands that bind to receptors to generate diacylglyccrol. Examples of these are bombesin and other neuropeptides, platelet-derived growth factor, epidermal growth factor.
Examples of protein kinase C inhibitors are A3 ([N-(2-Aminoethyl)-5-chloronaphthalene-1-sulfonamide]—this is optionally combined with HCl), bisindolylmaleimide I (also known as GF 109203X), chelerythrine chloride, Gö6976, Gö7874, H-7 ([1-(5-isoquinolinesulfonyl)-2-methylpiperazine]—this is optionally combined with HCl), hypericin, K-252a, b and c, melittin, phloretin,pseudohypericin, rottlerin, Ro 31-8220, Ro 32-0432, LY333531, (−)balanonl. Other examples are givine in “Drug Delivery Today, 1996, vol 1, pp438-447”.
The term “protein kinase C”, which is sometimes referred to as “PKC”, is used herein to refer to a class of enzymes which catalyse the transfer of phosphate from ATP to the serine or threonine residues of polypeptides. Preferably this occurs in a specific manner so that other amino acid residues are not phosphorylated.
Activation of these enzymes can generally be inferred by assaying with MARCKS (a PKC specific substrate). MARCKS is the term used for
M
yristoylated
A
lanine
R
ich
C
K
inase
S
ubstrate. This is a protein which was the first major PKC substrate identified. If increased phosphorylation of MARCKS is observed, it can usually be inferred that activation of PKC has occurred.
Inhibitors of PKC can be identified by their ability to prevent or reduce PKC activation.
The present invention is however not limited to the use of activators/inhibitors of PKC since other agents which work independently of PKC but which can affect the level of phosphorylation of threonine and/or serine residues of p100 and/or p120 can be used.
For example, lysophosphatidic acid (LPA) or histamine may be used to induce dephosphorylation of threonine and/or serine residues present in p100 and/or p120 and agents which bind LPA or histamine or blockers of these agents may be used to block the effect of LPA or histamine.
This effect might also be achieved by the use of compositions which are hyperosmolar with respect to the physiological environment proximal to p100 and/or p120 (e.g. hyperosmolar solutions of sugars such as mannitol or arabinose).
Hyperosmotic solutions are believed by some to open up the blood-brain barrier by causing the shrinking of brain endothelial cells which results in the mechanical opening of the endothelial tight junctions. However in view of the information provided herein, it is possible that hyperosmotic treatment triggers intracellular signaling processes leading to p100/p120 dephospho

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