A Potent and Selective Kallikrein-5 Inhibitor Delivers High Pharmacological Activity in Skin from Patients with Netherton Syndrome.

Regulation of proteolytic activity in the skin plays a pivotal role in epidermal homeostasis. This is best exemplified in Netherton syndrome, a severe genetic skin condition caused by loss-of-function mutations in the gene serine protease inhibitor Kazal-type 5 encoding lympho-epithelial Kazal-type-related inhibitor, a serine protease inhibitor that regulates kallikrein (KLK)-related peptidase 5, 7, and 14 activities. KLK5 plays a central role in stratum corneum shedding and inflammatory cell signaling, activates KLK7 and KLK14, and is therefore an optimal therapeutic target. We aimed to identify a potent and selective small-molecule inhibitor of KLK5 amenable to epidermal delivery. GSK951 was identified using a structure-based design strategy and showed a half maximal inhibitory concentration of 250 pM for KLK5 and greater than 100-fold selectivity over KLK7 and KLK14. Cocrystal structure analysis identified the critical catalytic site interactions to a surrogate for KLK5. Topical application of GSK951-containing cream inhibited KLK5 activity in TgKLK5 mouse skin, reduced transepidermal water loss, and decreased proinflammatory cytokine expression. GSK951 achieved high concentrations in healthy human epidermis following topical application in a cream formulation. Finally, KLK5 protease activity was increased in stratum corneum of patients with Netherton syndrome and significantly inhibited by GSK951. These findings unveil a KLK5-specific small-molecule inhibitor with a high therapeutic potential for patients with Netherton syndrome.

Navigating CYP1A Induction and Arylhydrocarbon Receptor Agonism in Drug Discovery. A Case History with S1P1 Agonists.

This article describes the finding of substantial upregulation of mRNA and enzymes of the cytochrome P450 1A family during a lead optimization campaign for small molecule S1P1 agonists. Fold changes in mRNA up to 10,000-fold for CYP1A1 in vivo in rat and cynomolgus monkey and up to 45-fold for CYP1A1 and CYP1A2 in vitro in rat and human hepatocytes were observed. Challenges observed with correlating induction in vitro and induction in vivo resulted in the implementation of a short, 4 day in vivo screening study in the rat which successfully identified noninducers. Subtle structure-activity relationships in this series of S1P1 agonists are described extending beyond planarity and lipophilicity, and the impact and considerations of AhR and CYP1A induction in the context of drug development are discussed.

Correlation of histopathology, urinary biomarkers, and gene expression responses following hexachloro-1:3-butadiene-induced acute nephrotoxicity in male Hanover Wistar rats: a 28-day time course study.

Hexachloro-1:3-butadiene (HCBD) causes segment-specific injury to the proximal renal tubule. A time course study of traditional and more recently proposed urinary biomarkers was performed in male Hanover Wistar rats receiving a single intraperitoneal (ip) injection of 45 mg/kg HCBD. Animals were killed on days 1, 2, 3, 4, 5, 6, 7, 10, 14, and 28 postdosing and the temporal response of renal biomarkers was characterized using kidney histopathology, urinary and serum biochemistry, and gene expression. Histopathologic evidence of tubular degeneration was seen from day 1 until day 3 postdosing and correlated with increased urinary levels of α-glutathione S-transferase (α-GST), albumin, glucose, and kidney injury molecule-1 (KIM-1), and increased gene expression of KIM-1, NAD(P)H dehydrogenase, quinone 1, and heme oxygenase (decycling) 1. Histopathologic evidence of tubular regeneration was seen from day 2 postdosing and correlated with raised levels of urinary KIM-1 and osteopontin and increased gene expression of KIM-1 and annexin A7. Traditional renal biomarkers generally demonstrated low sensitivity. It is concluded that in rat proximal tubular injury, measurement of a range of renal biomarkers, in conjunction with gene expression analysis, provides an understanding of the extent of degenerative changes induced in the kidney and the process of regeneration.

Urinary biomarkers in hexachloro-1:3-butadiene-induced acute kidney injury in the female Hanover Wistar rat; correlation of α-glutathione S-transferase, albumin and kidney injury molecule-1 with histopathology and gene expression.

Hexachloro-1:3-butadiene (HCBD) causes kidney injury specific to the pars recta of the proximal tubule. In the present studies, injury to the nephron was characterized at 24 h following a single dose of HCBD, using a range of quantitative urinary measurements, renal histopathology and gene expression. Multiplexed renal biomarker measurements were performed using both the Meso Scale Discovery (MSD) and Rules Based Medicine platforms. In a second study, rats were treated with a single nephrotoxic dose of HCBD and the time course release of a range of traditional and newer urinary biomarkers was followed over a 25 day period. Urinary albumin (a marker of both proximal tubular function and glomerular integrity) and α-glutathione S-transferase (α-GST, a proximal tubular cell marker of cytoplasmic leakage) showed the largest fold change at 24 h (day 1) after dosing. Most other markers measured on either the MSD or RBM platforms peaked on day 1 or 2 post-dosing, whereas levels of kidney injury molecule-1 (KIM-1), a marker of tubular regeneration, peaked on day 3/4. Therefore, in rat proximal tubular nephrotoxicity, the measurement of urinary albumin, α-GST and KIM-1 is recommended as they potentially provide useful information about the function, degree of damage and repair of the proximal tubule. Gene expression data provided useful confirmatory information regarding exposure of the kidney and liver to HCBD, and the response of these tissues to HCBD in terms of metabolism, oxidative stress, inflammation, and regeneration and repair.

Biophotonic imaging in HO-1.luc transgenic mice: real-time demonstration of gender-specific chloroform induced renal toxicity.

The metabolism of luciferin in mice transgenic for luciferase (luc) produces light that may be detected trans vivo by an intensified CCD camera (biophotonics). Thus, the generation of transgenic promoter-luciferase animals for genes regulated by specific toxic processes, coupled with real-time evaluation of site-specific gene expression may provide novel, non-invasive biomarkers which are predictive of developing toxicity in vivo. As part of a programme to evaluate the potential of biophotonics for predictive toxicology we have conducted a series of studies in HO-1.luc transgenic mice. Male and female animals were treated with chloroform (200 mg/kg, p.o., daily for 5 days) and imaged 2 and 6 h after dosing. During a 2-day washout period, female animals were treated daily with testosterone prior to repeat administration of chloroform for a further 5 days. Comparison of the in vivo response of the luciferase reporter with markers of toxicity measured ex vivo (differential gene expression of adaptive antioxidant response genes, clinical chemistry and microscopic examination) confirms the gender-specific difference in chloroform renal toxicity in HO-1.luc transgenic mice and its reversal following androgenisation of females and correlates with the expression of the endogenous haem oxygenase-1 (HO-1) gene. These studies demonstrate the capacity of biophotonics for real-time site-specific gene expression, which may be predictive of developing toxicity.