Karen Owens

Chemical genetics define the roles of p38alpha and p38beta in acute and chronic inflammation.

The p38 MAP kinase signal transduction pathway is an important regulator of proinflammatory cytokine production and inflammation. Defining the roles of the various p38 family members, specifically p38α and p38β, in these processes has been difficult. Here we use a chemical genetics approach using knock-in mice in which either p38α or p38β kinase has been rendered resistant to the effects of specific inhibitors along with p38β knock-out mice to dissect the biological function of these specific kinase isoforms. Mice harboring a T106M mutation in p38α are resistant to pharmacological inhibition of LPS-induced TNF production and collagen antibody-induced arthritis, indicating that p38β activity is not required for acute or chronic inflammatory responses. LPS-induced TNF production, however, is still completely sensitive to p38 inhibitors in mice with a T106M point mutation in p38β. Similarly, p38β knock-out mice respond normally to inflammatory stimuli. These results demonstrate conclusively that specific inhibition of the p38α isoform is necessary and sufficient for anti-inflammatory efficacy in vivo.

The discovery of sulfonylated dipeptides as Potent VLA-4 antagonists

Directed screening of a carboxylic acid-containing combinatorial library led to the discovery of potent inhibitors of the integrin VLA-4. Subsequent optimization by solid-phase synthesis afforded a series of sulfonylated dipeptide inhibitors with structural components that when combined in a single hybrid molecule gave a sub-nanomolar inhibitor as a lead for medicinal chemistry. Preliminary metabolic studies led to the discovery of substituted biphenyl derivatives with low picomolar activities. SAR and pharmacokinetic characterization of this series are presented.

Characterization of Multidrug Resistance 1a/P-Glycoprotein Knockout Rats Generated by Zinc Finger Nucleases

The development of zinc finger nuclease (ZFN) technology has enabled the genetic engineering of the rat genome. The ability to manipulate the rat genome has great promise to augment the utility of rats for biological and pharmacological studies. A Wistar Hannover rat model lacking the multidrug resistance protein Mdr1a P-glycoprotein (P-gp) was generated using a rat Mdr1a-specific ZFN. Mdr1a was completely absent in tissues, including brain and small intestine, of the knockout rat. Pharmacokinetic studies with the Mdr1a P-gp substrates loperamide, indinavir, and talinolol indicated that Mdr1a was functionally inactive in the blood-brain barrier and intestine in Mdr1a(−/−) rats. To identify possible compensatory mechanisms in Mdr1a(−/−) rats, the expression levels of drug-metabolizing enzyme and transporter-related genes were compared in brain, liver, kidney, and intestine of male and female Mdr1a(−/−) and control rats. In general, alterations in gene expression of these genes in Mdr1a(−/−) rats seemed to be modest, with more changes in female than in male rats. Taken together, our studies demonstrate that the ZFN-generated Mdr1a(−/−) rat will be a valuable tool for central nervous system drug target validation and determining the role of P-gp in drug absorption and disposition.

Evaluation of Cynomolgus Monkeys for the Identification of Endogenous Biomarkers for Hepatic Transporter Inhibition and as a Translatable Model to Predict Pharmacokinetic Interactions with Statins in Humans

Inhibition of hepatic transporters such as organic anion transporting polypeptides (OATPs) 1B can cause drug-drug interactions (DDIs). Determining the impact of perpetrator drugs on the plasma exposure of endogenous substrates for OATP1B could be valuable to assess the risk for DDIs early in drug development. As OATP1B orthologs are well conserved between human and monkey, we assessed in cynomolgus monkeys the endogenous OATP1B substrates that are potentially suitable to assess DDI risk in humans. The effect of rifampin (RIF), a potent inhibitor for OATP1B, on plasma exposure of endogenous substrates of hepatic transporters was measured. From the 18 biomarkers tested, RIF (18 mg/kg, oral) caused significant elevation of plasma unconjugated and conjugated bilirubin, which may be attributed to inhibition of cOATP1B1 and cOATP1B3 based on in vitro to in vivo extrapolation analysis. To further evaluate whether cynomolgus monkeys are a suitable translational model to study OATP1B-mediated DDIs, we determined the inhibitory effect of RIF on in vitro transport and pharmacokinetics of rosuvastatin (RSV) and atorvastatin (ATV). RIF strongly inhibited the uptake of RSV and ATV by cOATP1B1 and cOATP1B3 in vitro. In agreement with clinical observations, RIF (18 mg/kg, oral) significantly decreased plasma clearance and increased the area under the plasma concentration curve (AUC) of intravenously administered RSV by 2.8- and 2.7-fold, and increased the AUC and maximum plasma concentration of orally administered RSV by 6- and 10.3-fold, respectively. In contrast to clinical findings, RIF did not significantly increase plasma exposure of either intravenous or orally administered ATV, indicating species differences in the rate-limiting elimination pathways.

Pharmacologic Inhibition of the Renal Outer Medullary Potassium Channel Causes Diuresis and Natriuresis in the Absence of Kaliuresis

The renal outer medullary potassium (ROMK) channel, which is located at the apical membrane of epithelial cells lining the thick ascending loop of Henle and cortical collecting duct, plays an important role in kidney physiology by regulating salt reabsorption. Loss-of-function mutations in the human ROMK channel are associated with antenatal type II Bartter’s syndrome, an autosomal recessive life-threatening salt-wasting disorder with mild hypokalemia. Similar observations have been reported from studies with ROMK knockout mice and rats. It is noteworthy that heterozygous carriers of Kir1.1 mutations associated with antenatal Bartter’s syndrome have reduced blood pressure and a decreased risk of developing hypertension by age 60. Although selective ROMK inhibitors would be expected to represent a new class of diuretics, this hypothesis has not been pharmacologically tested. Compound A [5-(2-(4-(2-(4-(1H-tetrazol-1-yl)phenyl)acetyl)piperazin-1-yl)ethyl)isobenzofuran-1(3H)-one)], a potent ROMK inhibitor with appropriate selectivity and characteristics for in vivo testing, has been identified. Compound A accesses the channel through the cytoplasmic side and binds to residues lining the pore within the transmembrane region below the selectivity filter. In normotensive rats and dogs, short-term oral administration of compound A caused concentration-dependent diuresis and natriuresis that were comparable to hydrochlorothiazide. Unlike hydrochlorothiazide, however, compound A did not cause any significant urinary potassium losses or changes in plasma electrolyte levels. These data indicate that pharmacologic inhibition of ROMK has the potential for affording diuretic/natriuretic efficacy similar to that of clinically used diuretics but without the dose-limiting hypokalemia associated with the use of loop and thiazide-like diuretics.

Discovery of Selective Small Molecule ROMK Inhibitors as Potential New Mechanism Diuretics.

The renal outer medullary potassium channel (ROMK or Kir1.1) is a putative drug target for a novel class of diuretics that could be used for the treatment of hypertension and edematous states such as heart failure. An internal high-throughput screening campaign identified 1,4-bis(4-nitrophenethyl)piperazine (5) as a potent ROMK inhibitor. It is worth noting that this compound was identified as a minor impurity in a screening hit that was responsible for all of the initially observed ROMK activity. Structure-activity studies resulted in analogues with improved rat pharmacokinetic properties and selectivity over the hERG channel, providing tool compounds that can be used for in vivo pharmacological assessment. The featured ROMK inhibitors were also selective against other members of the inward rectifier family of potassium channels.

Discovery of a novel sub-class of ROMK channel inhibitors typified by 5-(2-(4-(2-(4-(1H-Tetrazol-1-yl)phenyl)acetyl)piperazin-1-yl)ethyl)isobenzofuran-1(3H)-one.

A sub-class of distinct small molecule ROMK inhibitors were developed from the original lead 1. Medicinal chemistry endeavors led to novel ROMK inhibitors with good ROMK functional potency and improved hERG selectivity. Two of the described ROMK inhibitors were characterized for the first in vivo proof-of-concept biology studies, and results from an acute rat diuresis model confirmed the hypothesis that ROMK inhibitors represent new mechanism diuretic and natriuretic agents.

Substituted N-(3,5-dichlorobenzenesulfonyl)-L-prolyl-phenylalanine analogues as potent VLA-4 antagonists.

A series of substituted N-(3,5-dichlorobenzenesulfonyl)-L-prolyl- and alpha-methyl-L-prolyl-phenylalanine derivatives was prepared as VLA-4/VCAM antagonists. The compounds showed excellent potency with a wide variety of neutral, polar, electron withdrawing or donating groups on the phenylalanine ring (IC50 approximately 1 nM). Heteroaryl ring substitution for phenylalanine was also well tolerated. Pharmacokinetic studies in rat were performed on a representative set of compounds in both series.

Design and synthesis of a new class of malonyl-CoA decarboxylase inhibitors with anti-obesity and anti-diabetic activities

A new series of thiazole-substituted 1,1,1,3,3,3-hexafluoro-2-propanols were prepared and evaluated as malonyl-CoA decarboxylase (MCD) inhibitors. Key analogs caused dose-dependent decreases in food intake and body weight in obese mice. Acute treatment with these compounds also led to a drop in elevated blood glucose in a murine model of type II diabetes.

Discovery of a Potent and Selective ROMK Inhibitor with Pharmacokinetic Properties Suitable for Preclinical Evaluation

A new subseries of ROMK inhibitors exemplified by 28 has been developed from the initial screening hit 1. The excellent selectivity for ROMK inhibition over related ion channels and pharmacokinetic properties across preclinical species support further preclinical evaluation of 28 as a new mechanism diuretic. Robust pharmacodynamic effects in both SD rats and dogs have been demonstrated.