Dipeptidyl Peptidase 1 Inhibitor AZD7986 Induces a Sustained, Exposure-Dependent Reduction in Neutrophil Elastase Activity in Healthy Subjects.

Neutrophil serine proteases (NSPs), such as neutrophil elastase (NE), are activated by dipeptidyl peptidase 1 (DPP1) during neutrophil maturation. High NSP levels can be detrimental, particularly in lung tissue, and inhibition of NSPs is therefore an interesting therapeutic opportunity in multiple lung diseases, including chronic obstructive pulmonary disease (COPD) and bronchiectasis. We conducted a randomized, placebo-controlled, first-in-human study to assess the safety, tolerability, pharmacokinetics, and pharmacodynamics of single and multiple oral doses of the DPP1 inhibitor AZD7986 in healthy subjects. Pharmacokinetic and pharmacodynamic data were analyzed using nonlinear mixed effects modeling and showed that AZD7986 inhibits whole blood NE activity in an exposure-dependent, indirect manner-consistent with in vitro and preclinical predictions. Several dose-dependent, possibly DPP1-related, nonserious skin findings were observed, but these were not considered to prevent further clinical development. Overall, the study results provided confidence to progress AZD7986 to phase II and supported selection of a clinically relevant dose.

Serine protease activity and residual LEKTI expression determine phenotype in Netherton syndrome.

Mutations in the SPINK5 gene encoding the serine protease (SP) inhibitor, lymphoepithelial-Kazal-type 5 inhibitor (LEKTI), cause Netherton syndrome (NS), a life-threatening disease, owing to proteolysis of the stratum corneum (SC). We assessed here the basis for phenotypic variations in nine patients with "mild", "moderate", and "severe" NS. The magnitude of SP activation correlated with both the barrier defect and clinical severity, and inversely with residual LEKTI expression. LEKTI co-localizes within the SC with kallikreins 5 and 7 and inhibits both SP. The permeability barrier abnormality in NS was further linked to SC thinning and proteolysis of two lipid hydrolases (beta-glucocerebrosidase and acidic sphingomyelinase), with resultant disorganization of extracellular lamellar membranes. SC attenuation correlated with phenotype-dependent, SP activation, and loss of corneodesmosomes, owing to desmoglein (DSG)1 and desmocollin (DSC)1 degradation. Although excess SP activity extended into the nucleated layers in NS, degrading desmosomal mid-line structures with loss of DSG1/DSC1, the integrity of the nucleated epidermis appears to be maintained by compensatory upregulation of DSG3/DSC3. Maintenance of sufficient permeability barrier function for survival correlated with a compensatory acceleration of lamellar body secretion, providing a partial permeability barrier in NS. These studies provide a mechanistic basis for phenotypic variations in NS, and describe compensatory mechanisms that permit survival of NS patients in the face of unrelenting SP attack.

A proteolytic cascade of kallikreins in the stratum corneum.

Serine proteases belonging to the kallikrein group may play a central role in desquamation. We have identified human kallikreins 5, 7, and 14 (hK5, hK7, hK14) in catalytically active form in stratum corneum. All three enzymes are produced as inactive precursors. In this work, we prepared recombinant enzymes and enzyme precursors and characterized the catalytic properties of hK5 and hK14. With peptide substrates hK5 and hK14 both showed trypsin-like specificity and alkaline pH-optima. For the substrates tested, hK14 was superior to hK5 as regards maximum catalytic rate as well as catalytic efficiency. hK5, but not hK14, could activate pro-hK7 in a reaction which was optimal at pH 5-7. hK5 could activate its own precursor as well as pro-hK14. This was in contrast to hK14, which could activate pro-hK5 but not its own precursor. The activation of pro-hK5 either by auto-activation or by hK14 occurred at maximum rate at neutral or weakly alkaline pH, whereas activation of pro-hK14 by hK5 was optimal at pH 6-7. We conclude that the enzymes studied may be part of a protease cascade in the stratum corneum, and that the observed pH effects may have physiological relevance.

Degradation of corneodesmosome proteins by two serine proteases of the kallikrein family, SCTE/KLK5/hK5 and SCCE/KLK7/hK7.

Corneodesmosin (CDSN), desmoglein 1 (DSG1), and desmocollin 1 (DSC1) are adhesive proteins of the extracellular part of the corneodesmosomes, the junctional structures that mediate corneocyte cohesion. The degradation of these proteins at the epidermis surface is necessary for desquamation. Two serine proteases of the kallikrein family synthesized as inactive precursors have been implicated in this process: the stratum corneum chymotryptic enzyme (SCCE/KLK7/hK7) and the stratum corneum tryptic enzyme (SCTE/KLK5/hK5). Here, we analyzed the capacity of these enzymes to cleave DSG1, DSC1, and epidermal or recombinant forms of CDSN, at an acidic pH close to that of the stratum corneum. SCCE directly cleaved CDSN and DSC1 but was unable to degrade DSG1. But incubation with SCTE induced degradation of the three corneodesmosomal components. Using the recombinant form of CDSN, either with its N-glycan chain or enzymatically deglycosylated, we also demonstrated that oligosaccharide residues do not protect CDSN against proteolysis by SCCE. Moreover, our results suggest that SCTE is able to activate the proform of SCCE. These results strongly suggest that the two kalikreins are involved in desquamation. A model is proposed for desquamation that could be regulated by a precisely controlled protease-protease inhibitor balance.

Epidermal overexpression of stratum corneum chymotryptic enzyme in mice: a model for chronic itchy dermatitis.

Identification of tissue-specific mechanisms involved in the pathophysiology of inflammatory skin diseases could offer new possibilities to develop effective therapies with fewer systemic effects. The serine protease stratum corneum chymotryptic enzyme is preferentially expressed in cornifying epithelia. We have previously reported on increased expression of the stratum corneum chymotryptic enzyme in psoriasis. Here is reported an increased epidermal expression of stratum corneum chymotryptic enzyme also found in chronic lesions of atopic dermatitis. Transgenic mice expressing human stratum corneum chymotryptic enzyme in suprabasal epidermal keratinocytes were found to develop pathologic skin changes with increased epidermal thickness, hyperkeratosis, dermal inflammation, and severe pruritus. The results suggest that stratum corneum chymotryptic enzyme may be involved in the pathogenesis of inflammatory skin diseases, and that stratum corneum chymotryptic enzyme and related enzymes should be evaluated as potential targets for new therapies.

SPINK9: a selective, skin-specific Kazal-type serine protease inhibitor.

A previously unreported Kazal-type serine protease inhibitor, serine protease inhibitor Kazal type 9 (SPINK9), was identified in human skin. SPINK9 expression was strong in palmar epidermis, but not detectable or very low in non palmoplantar skin. Analysis of a human cDNA panel showed intermediate expression in thymus, pancreas, liver, and brain, and low or undetectable expression in other tissues. Using kallikrein-related peptidases (KLKs) 5, 7, 8, and 14, thrombin, trypsin, and chymotrypsin, inhibition with recombinant SPINK9 was seen only for KLK5 using low molecular weight substrates, with an apparent K(i) of 65 nM. Also KLK5 degradation of fibrinogen was totally inhibited by SPINK9. Slight inhibition of KLK8 using fibrinogen substrate could be observed using high concentrations of SPINK9. Analyses by surface plasmon resonance showed heterogeneous binding to SPINK9 of KLK5 and KLK8, but no binding of KLK7 or KLK14. KLK5 has been suggested to play a central role in skin desquamation as an initiating activating enzyme in proteolytic cascades formed by KLKs. An apparently KLK5-specific inhibitor, such as SPINK9, may play a significant regulatory role in such cascades. We suggest a possible role for SPINK9 in the site-specific epidermal differentiation of palms and soles.

Activation of proteinase-activated receptor-2 by human kallikrein-related peptidases.

Proteinase-activated receptor-2 (PAR2) is a seven transmembrane spanning, G-protein-coupled receptor, present on the membrane of many cell types including keratinocytes. In skin, PAR2 is suggested to play a regulatory role during inflammation, epidermal barrier function, and pruritus. PAR2 is activated by trypsin-like proteases by a unique mechanism where cleavage of the receptor leads to the release of a small peptide, which activates the receptor as a tethered ligand. The endogenous activators of PAR2 on keratinocytes have not been identified as of yet. Potential candidates are kallikrein-related peptidases (KLKs) expressed by epidermal cells. Therefore, the ability of four human skin-derived KLKs was examined with regard to their capacity to activate PAR2 in vitro. PAR2 cleavage was followed by immunofluorescence analysis and functional activation by measurements of changes in intracellular calcium levels. We found that KLK5 and KLK14, but neither KLK7 nor KLK8, induced PAR2 signalling. We conclude that certain, but not all, epidermal KLKs are capable of activating PAR2. We could also show the coexpression of KLK14 and PAR2 receptor in inflammatory skin disorders. These in vitro results suggest that KLKs may take part in PAR2 activation in the epidermis and thereby in PAR2-mediated inflammatory responses, including epidermal barrier repair and pruritus. The role of KLKs in PAR2 activation in vivo remains to be elucidated.

Kallikrein-related peptidase 14 may be a major contributor to trypsin-like proteolytic activity in human stratum corneum.

We have previously presented evidence that two human kallikrein-related peptidases, KLK5 (hK5, stratum corneum tryptic enzyme, SCTE) and KLK7 (hK7, stratum corneum chymotryptic enzyme, SCCE), which are abundant in the stratum corneum, may be involved in desquamation. Since we had noted that not all trypsin-like activity in the plantar stratum corneum could be ascribed to KLK5, we set out to identify other skin proteases with similar primary substrate specificity. Here we describe purification of a protease identified as KLK14 from plantar stratum corneum, and show that this enzyme may be responsible for as much as 50% of the total trypsin-like activity in this tissue, measured as activity towards a chromogenic substrate cleaved by a wide variety of enzymes with trypsin-like specificity. This was in spite of very low levels of KLK14 protein compared to KLK5 and KLK7. KLK14 could be detected by immunoblotting in normal superficial stratum corneum of all individuals examined. The majority of KLK14 in the plantar stratum corneum is present in its catalytically active form. KLK14 could be immunohistochemically detected in sweat ducts, preferentially in the intraepidermal parts (the acrosyringium), and in sweat glands. The role played by this very efficient protease under normal and disease conditions in the skin remains to be elucidated.

Transgenic mice over-expressing a serine protease in the skin: evidence of interferon gamma-independent MHC II expression by epidermal keratinocytes.

Stratum corneum chymotryptic enzyme (SCCE; also known as kallikrein 7) is a serine protease that is preferentially expressed in cornifying epithelia and possibly involved in the desquamation process. We have recently described transgenic mice over-expressing human SCCE in the epidermis showing increased epidermal thickness, hyperkeratosis, and an apparent dermal inflammation with pruritus. This suggests that SCCE may be involved in the pathophysiology of inflammatory skin diseases. We therefore carried out a further characterization of the skin changes observed in scce-transgenic mice. An increase in number of dermal cells was verified by stereological measurements showing a more than twofold increase of the volume fraction of dermis occupied by cell nuclei. In some, but not all, animals the number of dermal mast cells was increased. The dermal cell infiltrate was shown to consist mainly of macrophages and granulocytes. The number of epidermal and dermal T-lymphocytes was not increased. Dermal changes were found in transgenic animals before the age they became pruritic. No increase in interferon-gamma expression could be detected in the skin of transgenic animals. In spite of this, keratinocytes of adult transgenic mice were found to express MHC II antigen. We suggest that increased expression and/or activity of epidermal SCCE may lead to skin changes that contribute to development and maintenance of inflammatory skin diseases.

Epidermal hyperproliferation and decreased skin barrier function in mice overexpressing stratum corneum chymotryptic enzyme.

Stratum corneum chymotryptic enzyme (SCCE; also known as kallikrein 7) is a serine protease that may have an important role in the skin desquamation process. We have recently described transgenic mice overexpressing human SCCE in suprabasal epidermal keratinocytes, leading to increased epidermal thickness, hyperkeratosis, dermal inflammation and signs of severe pruritus in older animals. In order to further evaluate the scce-transgenic mice as a potential disease model, we compared transgenic animals and wild-type littermates for patterns of epidermal keratin expression, in situ hybridization of scce-mRNA, scratching behaviour and measurements of transepidermal water loss (TEWL). In 3-day-old mice, despite readily detectable amounts of human scce-mRNA in the epidermis of transgenic animals, there were no histological differences in skin appearance, and no differences could be found in epidermal expression of the keratins 5, 6 and 10. In mice 7-8 weeks of age and older, there was strong suprabasal expression of keratins 5 and 6 in the epidermis of transgenic animals, suggesting that the thickened epidermis in these animals is the result of keratinocyte hyperproliferation. In transgenic animals 11 weeks of age and older there was an increased frequency of scratching, suggestive of pruritus, and also signs of a deteriorating skin barrier function, as reflected by an increased TEWL. There was no correlation between increased TEWL and increased frequency of scratching in individual animals, suggesting that the defect barrier function was not an effect of skin damage caused by scratching.