ORALLY ACTIVE INHIBITORS OF HUMAN LEUKOCYTE ELASTASE. II. DISPOSITION OF L-694,458 IN RATS AND RHESUS MONKEYS

ABSTRACT: The disposition of L-694,458, a potent monocyclic ␤-lactam inhibitor of human leukocyte elastase, was studied in male SpragueDawley rats and rhesus monkeys. After iv dosing, L-694,458 exhibited similar pharmacokinetic parameters in rats and rhesus monkeys. The mean values for its plasma clearance, terminal halflife, and volume of distribution at steady state were 27 ml/min/kg, 1.8 hr, and 4.0 liters/kg in rats and 34 ml/min/kg, 2.3 hr, and 5 liters/kg in rhesus monkeys. The bioavailability of a 10 mg/kg oral dose was higher in rats (65%) than in rhesus monkeys (39%). In both species, concentrations of L-694,458 in plasma increased more than proportionally when the oral dose was increased from 10 mg/kg to 40 mg/kg. In monkeys a protracted plasma concentration-time profile was observed at 40 mg/kg, characterized by a delayed T max (8-24 hr) and a long terminal half-life (6 hr). [ 3 H]L-694,458 was well absorbed after oral dosing to rats at 10 mg/kg, as indicated by the high recovery of radioactivity in bile (83%) and urine (6%) of bile duct-cannulated rats. Only ϳ5% or less of the radioactivity in bile, urine, and feces was a result of intact L-694,458, indicating that the compound was being eliminated by metabolism, followed by excretion of the metabolites in feces, via bile. Demethylenation of the methylenedioxyphenyl group resulting in the catechol was the primary metabolic pathway in human and rhesus monkey liver microsomes. In rat liver microsomes, the major metabolite was the N-oxide of the methyl-substituted piperazine nitrogen. In rats dosed iv and orally with [ 3 H]L-694,458, concentrations of radioactivity were highest in the lung (the primary target tissue), adrenals, and liver. L-694,458 was unstable in rat blood and plasma, degrading via a pathway believed to be catalyzed by B-esterases and to involve cleavage of the ␤-lactam ring and loss of the methylpiperazine phenoxy group. In vitro studies indicated that in human liver, L-694,458 was metabolized by CYP3A and 2C isozymes, and in both monkey and human liver microsomes the compound acted as an inhibitor of testosterone 6␤-hydroxylation. Leukocyte elastase is a serine protease capable of proteolytic degradation of a variety of substrates, including elastin and collagen, which are components of connective tissue. Specific inhibitors of leukocyte elastase are being explored as potential therapeutic agents for the treatment of inflammatory diseases, such as cystic fibrosis and rheumatoid arthritis where high amounts of extracellular elastase, either free or bound to its natural inhibitors, ␣ 1 -proteinase inhibitor and secretory leukocyte proteinase inhibitor, have been detected extracellularly (1-3). Several classes of inhibitors of elastase have been synthesized and evaluated to date (4 -19). L-694,458 Materials and Methods Chemicals. L-694,458 ( Microsomes. Fresh rat liver and frozen human and rhesus monkey liver tissue were used for the preparation of microsomes. Microsomes containing 1 Abbreviations used are: L-694,458, N-[1(R)-(1,3-benzodioxol-5-yl

Identification and characterization of sebaceous gland atrophy-sparing DGAT1 inhibitors.

Inhibition of Diacylglycerol O-acyltransferase 1 (DGAT1) has been a mechanism of interest for metabolic disorders. DGAT1 inhibition has been shown to be a key regulator in an array of metabolic pathways; however, based on the DGAT1 KO mouse phenotype the anticipation is that pharmacological inhibition of DGAT1 could potentially lead to skin related adverse effects. One of the aims in developing small molecule DGAT1 inhibitors that target key metabolic tissues is to avoid activity on skin-localized DGAT1 enzyme. In this report we describe a modeling-based approach to identify molecules with physical properties leading to differential exposure distribution. In addition, we demonstrate histological and RNA based biomarker approaches that can detect sebaceous gland atrophy pre-clinically that could be used as potential biomarkers in a clinical setting

RNA biomarkers for sebaceous gland atrophy in skin. Listed are the 41 unique genes from the 42 probesets identified in the Training Set as shown in Figure 4 (Cxcl16 had 2 probesets).

<p>Fold change and ANOVA p values for compound treatments compared to their respective vehicle treatments, for both the Training and Test Sets, are included. The 26 probesets that are also significantly regulated in the Test Set are shown in bold. **  = ANOVA p<0.01; *  = ANOVA p<0.05; $ = ANOVA p<0.1.</p

DGAT1 inhibitors with high lipophilicity induce sebaceous gland atrophy.

<p>Shown are hematoxylin and eosin stains of dorsal skin biopsies from DIO mice treated with either vehicle (A), Cpd1 (B), Cpd2 (C), or Cpd3 (D) for 14 days at doses indicated. Scoring refers to the histological adverse effect score as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088908#pone-0088908-t001" target="_blank">Table 1</a>. Bar  = 50 µm. The corresponding sebaceous gland sizes (area) are plotted in (E) (and shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088908#pone.0088908.s002" target="_blank">Table S1</a>).</p

RNA biomarkers for sebaceous gland atrophy in skin.

<p>Shown are the 42 probesets, identified in the Training Set (Studies 1 and 2), that were regulated by skin-positive compound treatments (those that produced sebaceous gland atrophy) but not by the skin-negative compound treatments (the one that did <i>not</i> produce sebaceous gland atrophy). After excluding the absent probes (low intensity), these 42 probesets met the following cutoffs: 1.2 fold change and ANOVA p<0.01 between all 3 skin-positive compound treatments (red arrows) and their respective vehicle treatments, and ANOVA p>0.1 between the skin negative compound treatment (black arrow) and its respective vehicle treatment. The probesets for RIKEN genes were excluded. Plotted are the LogRatio values (+/− 4 fold fold scale) with magenta representing up-regulated probesets and cyan representing down-regulated probesets. Treatments from the independent Test Set (Study 4) are included for comparison but were not used to identify the 42 probesets.</p

Histopathology versus lipophilicity.

<p>Calculated logD (clogD, A) or measured logD (mlogD, B) versus observed skin adverse effects. Data is jittered on the y-axis to improve visualization.</p

Representative compound structures from three chemical compound series as shown in Table 1.

<p>Representative compound structures from three chemical compound series as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088908#pone-0088908-t001" target="_blank">Table 1</a>.</p

Immune-regulated genes are up-regulated, while lipid metabolism genes are down-regulated, with sebaceous gland atrophy.

<p>Box plots of probesets regulated by the skin-positive DGAT1 inhibitors (those that produce sebaceous gland atrophy) but not by the skin-negative compounds (those that do <i>not</i> produce sebaceous gland atrophy). Plotted are the LogIntensity values across the replicates in each group, and across the three studies. Ccl1 (A; chemokine (C-C motif) ligand 1) is involved in the recruitment of T cells in skin inflammation; and Scd3 (B; stearoyl-coenzyme A desaturase 3) is a sebaceous gland specific gene.</p