Inflammation and inflammatory processes play a major role in the pathophysiology of numerous diseases and conditions. Conditions of the brain in which increased levels of inflammation mediators were found include severe traumatic brain injury, relapsing-remitting multiple sclerosis, cerebral artery occlusion, ischemia, and stroke. Conditions of the heart in which mediators such as the selectins are suggested to play a role include acute myocardial infarct, arterial injury, such as produced by angioplasty, and ischemia. Similarly, selectins are involved in conditions of the kidneys, such as renal injury from ischemia and reperfusion, and renal failure. Furthermore, selectins appear to play a role in organ transplant rejection, cold ischemia, hemorrhagic shock, septic shock, tumour metastasis, chronic inflammation, rheumatoid arthritis, inflammatory bowel disease, atherosclerosis, restenosis, angiogenesis, disseminated intravascular coagulation, adult respiratory stress syndrome, and circulatory shock.
Cell surface adhesion molecules have become recognised as key mediators in numerous cellular processes including cell growth, differentiation, immune cell transmigration and response, and cancer metastasis. Four major categories of adhesion molecules have been identified: the immunoglobulin superfamily cell adhesion molecules (CAMs), cadherins, integrins, and selecting. The selectins represent a family of presently three transmembraneous, carbohydrate-binding glycoproteins: “endothelial” E-selectin, “leukocyte” L-selectin, and “platelet” P-selectin. All three selectins are divalent cation (e.g. calcium) dependent and possess an extracellular domain with a carbohydrate recognition motif, an epidermal growth factor-like motif, and some smaller domains related to complement-regulatory proteins.
Human P-selectin (also referred to as GMP-140, LECAM-3, PADGEM, CD62, CD62P) is expressed by platelets and endothelial cells. When expressed on these cell surfaces, its most notable effect is the slowing of leukocytes as these leave the capillaries and enter the postcapillary venules, the latter representing the major site of leukocyte-endothelium adhesion. The slowing process is observed as leukocyte rolling, signifying an initial adhesion with relatively low affinity. The firm adhesion of rolling leukocytes is primarily mediated by integrins.
In endothelial cells, P-selectin is stored on Weibel-Palade bodies; in platelets, it is found in the α-granules. Following activation, P-selectin is mobilised to the cell surfaces within a few minutes in response to a variety of inflammatory or thrombogenic agents. The endothelial P-selectin's primary function is to recruit leukocytes into postcapillary venules, while platelet P-selectin also results in the formation of thrombi. One of the presently known natural ligands of P-selectin is PSGL-1 (P-selectin glycoprotein ligand-1), a 160 kDa sialoprotein expressed on the surface of leukocytes where it is concentrated at the uropod. More detailed descriptions of the structure and functions of p-selectin are found in numerous publications, such as J. Panes, Pathophysiology 5: 271 (1999); F. Chamoun et al., Frontiers in Bioscience 5: e103 (Nov. 1, 2000); S.-I. Hayashi, Circulation 102: 1710 (2000).
P-selectin also appears to be involved more directly in platelet aggregation, as was shown recently by studies of the Ca-independent interactions of P-selectin with 3-sulfated galactosyl ceramide (also referred to as sulfatides). This interaction probably takes place at a different binding site of P-selectin, as the binding can be inhibited by the antibody WASP12.2, but not by AK4, whereas the binding of the natural P-selectin ligand PSGL-1, which is involved in leukocyte adhesion, is blocked by both WASP12.2 and AK4. However, it appears that the binding sites are overlapping. It is assumed that sulfatide interactions stabilise platelet aggregates.
On the one hand, it would seem feasible to improve these and other conditions involving the activation of endothelial cells and leukocytes, and specifically the mobilisation and expression of P-selectin by specifically interrupting the P-selectin cascades. This can be done, for instance, by the administration of ligands which selectively bind to human P-selectin, but which do not possess its bioactivity. By this method, mobilised P-selectin could be inactivated and leukocyte-induced tissue damage prevented. Potentially, the same effect could be achieved by gene therapy, provided the P-selectin ligand or antagonist is a peptide or modified peptide. According to this method, somatic cells of a person in need of the therapy would be transfected with an expression vector carrying a DNA sequence encoding a P-selectin antagonist.
On the other hand, P-selectin-related diseases and conditions may also be treated or prevented by drugs which do not directly interact with P-selectin, but which suppress some of the detrimental effects of P-selectin activation in the respective cells and tissues. Among the drug substances potentially useful for therapeutic intervention are anti-inflammatory agents such as glucocorticoids.
One of the major drawbacks of any systemic therapy with highly active compounds is their distribution within the organism and the exposure of unaffected cells and tissues, potentially leading to substantial side effects. It would be most desirable to have methods and drug delivery systems available which allow the targeted delivery of active agents specifically to affected cells, without substantially exposing unaffected cells.
While there is no pharmaceutical product comprising a cell-specifically targeted drug delivery system available on the market today, a number of experimental delivery systems have been described in the scientific and patent literature. Drug targeting may be based on conjugates of active principles with target-recognising ligands, such conjugates representing molecular drug delivery systems. A general disadvantage of such conjugates is the low ration of drug substance per ligand (often only 1:1), resulting in the exposure to high levels of ligands.
As an example, Everts et al. (J. Immunol. 168: 883 (2002)) report the selective intracellular delivery of dexamethasone into activated endothelial cells using an E-selectin-directed immunoconjugate. Dexamethasone was covalently attached to an anti-E-selectin Ab, resulting in the so-called dexamethasone-anti-E-selectin conjugate. Binding of the conjugate to E-selectin was studied using surface plasmon resonance and immunohistochemistry. Furthermore, internalisation of the conjugate was studied using confocal laser scanning microscopy and immuno-transmission electron microscopy. It was demonstrated that the dexamethasone-anti-E-selectin conjugate, like the unmodified anti-E-selectin Ab, selectively bound to TNF-alpha-stimulated endothelial cells and not to resting endothelial cells. After binding, the conjugate was internalised and routed to multivesicular bodies, which is a lysosome-related cellular compartment. After intracellular degradation, pharmacologically active dexamethasone was released, as shown in endothelial cells that were transfected with a glucocorticoid-responsive reporter gene. Furthermore, intracellularly delivered dexamethasone was able to down-regulate the proinflammatory gene IL-8.
Alternatively, carrier-based drug delivery systems may be rendered target-specific by attaching appropriate target-recognising ligands to their surface. For instance, this approach has been employed using liposomes as carriers. Some of the recent developments based on this approach have been reviewed by Maruyama (Biosci. Rep. 22: 251 (2002)).
For instance, methods for E-selectin targeted drug delivery have been investigated by Spragg et al. (Proc. Nat. Acad. Sci USA 94: 8795 (1997)). According to this document, E-selectin was selected as a molecular target for endothelial-selective delivery of therapeutic drugs or genes for treating various disease states. Liposomes of various types (classical, sterically stabilised, cationic, pH-sensitive), each conjugated with mAb H18/7, a murine monoclonal antibody that recognises the extracellular domain of E-selectin, bound selectively and specifically to IL-1 beta-activated HUVEC at levels up to 275-fold higher than to unactivated HUVEC. E-selectin-targeted immunoliposomes appeared in acidic, perinuclear vesicles 2-4 hr after binding to the cell surface, consistent with internalisation via the endosome/lysosome pathway. Activated HUVEC incubated with E-selectin-targeted immunoliposomes, loaded with the cytotoxic agent doxorubicin, exhibited significantly decreased cell survival, whereas unactivated HUVEC were unaffected by such treatment.
On the other hand, there is some evidence that P-selectin may also be at least as an appropriate molecular target for activated endothelial cell involved in inflammatory processes, as was described above. Therefore, there is a need for drug delivery systems which are specifically targeted to this member of the selectin family, and thereby to cells and tissues showing (increased) P-selectin expression or presentation.
The majority of P-selectin binding compounds known today are carbohydrates, based on sialyl Lewis X (sLeX), a tetrasaccharide and natural ligand for the selecting. However, these mimics have the disadvantage of displaying low affinity (micromolar to millimolar range) and low specificity, as they tend to bind to other members of the selectin family with approximately the same affinity as they have for P-selectin.
Therefore, there also is a need for such P-selectin-directed, targeted drug delivery systems which have a high affinity and specificity for the target molecule.