Reversible cysteine protease inhibitors show promise for a Chagas disease cure.

The cysteine protease cruzipain is essential for the viability, infectivity, and virulence of Trypanosoma cruzi, the causative agent of Chagas disease. Thus, inhibitors of cruzipain are considered promising anti-T. cruzi chemotherapeutic agents. Reversible cruzipain inhibitors containing a nitrile "warhead" were prepared and demonstrated 50% inhibitory concentrations (IC50s) as potent as 1 nM in baculovirus-generated cruzipain enzyme assays. In epimastigote and intracellular amastigote in vitro assays, the most potent compounds demonstrated antiparasitic behavior in the 5 to 10 µM IC50 range; however, trypomastigote production from the amastigote form was ∼90 to 95% inhibited at 2 µM. Two key compounds, Cz007 and Cz008, with IC50s of 1.1 and 1.8 nM, respectively, against the recombinant enzyme were tested in a murine model of acute T. cruzi infection, with oral dosing in chow for 28 days at doses from 3 to 50 mg/kg of body weight. At 3 mg/kg of Cz007 and 3 mg/kg of Cz008, the blood parasitemia areas under the concentration-time curves were 16% and 25% of the untreated group, respectively. At sacrifice, 24 days after immunosuppression with cyclophosphamide, parasite presence in blood, heart, and esophagus was evaluated. Based on negative quantitative PCR results in all three tissues, cure rates in surviving animals were 90% for Cz007 at 3 mg/kg, 78% for Cz008 at 3 mg/kg, and 71% for benznidazole, the control compound, at 50 mg/kg.

Using human recombinant UDP-glucuronosyltransferase isoforms and a relative activity factor approach to model total body clearance of laropiprant (MK-0524) in humans.

A major pathway of elimination of the prostaglandin D2 receptor 1 antagonist laropiprant in humans is by uridine diphosphate-glucuronosyltransferase (UGT)-mediated biotransformation. In this study, liver and kidney relative activity factors were developed for UGT1A1, 1A9 and 2B7 to allow for in vitro-in vivo extrapolation of intrinsic clearance data to whole organ clearance using recombinant human UGT isoforms applying this to laropiprant as a model substrate. The total body metabolic clearance of laropiprant determined using this approach (5.0 L/hr) agreed well with the value determined in vivo following intravenous administration to healthy human volunteers (5.1 L/hr). The results suggest that approximately 36%, 36% and 28% of the hepatic metabolic clearance of laropiprant was mediated by UGT1A1, 1A9 and 2B7, respectively. Likewise, 80% and 20% of the renal metabolic clearance was mediated by UGT1A9 and 2B7, respectively. Furthermore, the data suggested that the contribution of the kidney to the overall total metabolic clearance was minor relative to the liver (≈ 12%).

The discovery of MK-0674, an orally bioavailable cathepsin K inhibitor.

MK-0674 is a potent and selective cathepsin K inhibitor from the same structural class as odanacatib with a comparable inhibitory potency profile against Cat K. It is orally bioavailable and exhibits long half-life in pre-clinical species. In vivo studies using deuterated MK-0674 show stereoselective epimerization of the alcohol stereocenter via an oxidation/reduction cycle. From in vitro incubations, two metabolites could be identified: the hydroxyleucine and the glucuronide conjugate which were confirmed using authentic synthetic standards.

Alkyl-bridged substituted 8-arylquinolines as highly potent PDE IV inhibitors.

Substituted 8-arylquinoline analogs bearing alkyl-linked side chain were identified as potent inhibitors of type 4 phophodiesterase. These compounds address the potential liabilities of the clinical candidate L-454560. The pharmacokinetic profile of the best analogs and the in vivo efficacy in an ovalbumin-induced bronchoconstriction assay in conscious guinea pigs are reported.

Design, synthesis, and biological evaluation of 8-biarylquinolines: a novel class of PDE4 inhibitors.

The structure-activity relationship of a novel series of 8-biarylquinolines acting as type 4 phosphodiesterase (PDE4) inhibitors is described herein. Prototypical compounds from this series are potent and non-selective inhibitors of the four distinct PDE4 (IC(50)<10 nM) isozymes (A-D). In a human whole blood in vitro assay, they inhibit (IC(50)<0.5 microM) the LPS-induced release of the cytokine TNF-alpha. Optimized inhibitors were evaluated in vivo for efficacy in an ovalbumin-induced bronchoconstriction model in conscious guinea pigs. Their propensity to produce an emetic response was evaluated by performing pharmacokinetic studies in squirrel monkeys. This work has led to the identification of several compounds with excellent in vitro and in vivo profiles, including a good therapeutic window of efficacy over emesis.

The discovery of odanacatib (MK-0822), a selective inhibitor of cathepsin K.

Odanacatib is a potent, selective, and neutral cathepsin K inhibitor which was developed to address the metabolic liabilities of the Cat K inhibitor L-873724. Substituting P1 and modifying the P2 side chain led to a metabolically robust inhibitor with a long half-life in preclinical species. Odanacatib was more selective in whole cell assays than the published Cat K inhibitors balicatib and relacatib. Evaluation in dermal fibroblast culture showed minimal intracellular collagen accumulation relative to less selective Cat K inhibitors.

Discovery of a potent and selective prostaglandin D2 receptor antagonist, [(3R)-4-(4-chloro-benzyl)-7-fluoro-5-(methylsulfonyl)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl]-acetic acid (MK-0524).

The discovery of the potent and selective prostaglandin D2 (PGD2) receptor (DP) antagonist [(3R)-4-(4-chlorobenzyl)-7-fluoro-5-(methylsulfonyl)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl]-acetic acid (13) is presented. Initial lead antagonists 6 and 7 were found to be potent and selective DP antagonists (DP Ki = 2.0 nM for each); however, they both suffered from poor pharmacokinetic profiles, short half-lives and high clearance rates in rats. Rat bile duct cannulation studies revealed that high concentrations of parent drug were present in the biliary fluid (Cmax = 1100 microM for 6 and 3900 microM for 7). This pharmacokinetic liability was circumvented by replacing the 7-methylsulfone substituent present in 6 and 7 with a fluorine atom resulting in antagonists with diminished propensity for biliary excretion and with superior pharmacokinetic profiles. Further optimization led to the discovery of the potent and selective DP antagonist 13.

Use of a fluorescent substrate for the selective quantification of rat CYP3A in the liver and the intestine.

INTRODUCTION: Quantification of cytochrome P450 is a major issue in the development of new drugs. Different assays have been reported, but few are very selective for the 3A isoform or cytochrome P450. The benzyloxy-substituted lactone cyclooxygenase-2 inhibitor 3-[(3, 4-difluorobenzyl)oxy]-5,5-dimethyl-4-[4-methylsulfonyl) phenyl] furan-2(5H)-one has recently been used successfully to probe isoform 3A of cytochrome P450 in the liver. However, its selectivity for the rat isoform remains to be established as well as its applicability in other tissue, such as the intestine. The purpose of this study was to ascertain the specificity of this substrate for the rat 3A isoform of cytochrome P450 using Supersomes and its application in non-hepatic tissue (e.g., intestine). METHODS: Specificity of the 3-[(3,4-difluorobenzyl)oxy]-5,5-dimethyl-4-[4-methylsulfonyl)phenyl] furan-2(5H)-one for the isoform 3A of rat cytochrome P450 was established by using either isoform-specific inhibitory antibody or microsomes expressing only one cytochrome P450 isoform. Activity was assayed in rat liver and intestinal microsomal protein preparations. RESULTS: Experiments with inhibitory antibodies revealed that in liver and intestinal microsomes, more than 90% of the substrate metabolism was inhibited by antibodies against isoform 3A. Selectivity of the substrate for rat 3A isoform was further determined by testing the metabolic activity of various Supersomes preparations. DISCUSSION: In conclusion, our results validate the usefulness of 3-[(3,4-difluorobenzyl)oxy]-5,5-dimethyl-4-[4-methylsulfonyl)phenyl] furan-2(5H)-one as a simple and specific substrate to study the activity of the isoform 3A of cytochrome P450 in the rat liver and intestine.

Design, synthesis, structure-function relationship, bioconversion, and pharmacokinetic evaluation of ertapenem prodrugs.

Described here are synthesis and biological evaluations of diversified groups of over 57 ertapenem prodrugs which include alkyl, methylenedioxy, carbonate, cyclic carbonate, carbamate esters, and esters containing active transport groups (e.g., carboxyl, amino acid, fatty acids, cholesterol) and macrocyclic lactones linking the two carboxyl groups. Many of the prodrugs were rapidly hydrolyzed in rat plasma but not in human plasma and were stable in simulated gastrointestinal fluid. The diethyl ester prodrug showed the best total absorption (>30%) by intredeudenal dosing in dogs, which could potentially be improved by formulation development. However, its slow rate of the hydrolysis to ertapenem also led to the presence of large amounts of circulating monoester metabolites, which pose significant development challenges. This study also suggests that the size of susbtituents at C-2 of carbapenem (e.g., benzoic acid of ertapenem) has significant impact on the absorption and the hydrolysis of the prodrugs.

Design, synthesis, and evaluation of prodrugs of ertapenem.

Carbapenems are intravenous lifesaving hospital antibiotics. Once patients leave the hospital, they are sent home with antibiotics other than carbapenems since they cannot be administered orally due to lack of oral absorption primarily because of very highly polarity. A prodrug approach is a bona fide strategy to improve oral absorption of compounds. Design and synthesis, in vitro and in vivo evaluation of diversified prodrugs of ertapenem, one of the only once daily dosed carbapenems is described. Many of the prodrugs prepared for evaluation are rapidly hydrolyzed in rat plasma. Only bis-(5-methyl-2-oxo-1,3-dioxol-4-yl)methyl (medoxomil) ester prodrug was rapidly hydrolyzed in most of the plasmas including rat, human, dog, and monkey. Although the rate of conversion of ertapenem diethyl ester prodrug (6) was slow in in vitro plasma hydrolysis, it showed the best in vivo pharmacokinetic profile in dog by an intraduodenal dosing giving >31% total oral absorption.

Use of cysteine-reactive small molecules in drug discovery for trypanosomal disease.

INTRODUCTION: The roles of cysteine protease (CP) enzymes in the biochemistry and infectivity of the three trypanosomal parasitic infections, Chagas' disease, leishmaniasis and human African trypanosomiasis, which have been elucidated over the last three decades are summarized. Inhibitors of these enzymes, which act through trapping the active site cysteine with an electrophilic warhead, hold huge potential as therapeutic agents but the promise of these has yet to be realized in clinical studies. The article addresses aspects that ought to be considered in order to develop orally active CP inhibitors that are safe and effective therapies for trypanosomiasis. AREAS COVERED: This article reviews learnings from CP research in the trypanosomal field and recent advances in developing cysteine protease inhibitors (CPIs) of human cathepsin K, a related enzyme. Considerations such as intra- and extracellular localization of the CPs, off-target activities against human cathepsin enzymes, basic versus neutral and potential pro-drug inhibitors are reviewed. A description of odanacatib, a cathepsin K inhibitor currently in late stage development, is made to illustrate the attributes of a clinically viable CPI. EXPERT OPINION: The emerging role of CPs in a wide array of parasitic diseases is highlighted with the vision that CP inhibitors could become the 'ß-lactams' of anti-parasitic treatments in the coming decades. New CPI research will see the optimization of intra- and extracellular enzyme targeting, reduction of off-target activities and better understanding of pharmacokinetic-pharmacodynamic interactions which will all lead to compounds with much improved efficacy and viability as clinical therapies.

Managing the challenge of chemically reactive metabolites in drug development.

The normal metabolism of drugs can generate metabolites that have intrinsic chemical reactivity towards cellular molecules, and therefore have the potential to alter biological function and initiate serious adverse drug reactions. Here, we present an assessment of the current approaches used for the evaluation of chemically reactive metabolites. We also describe how these approaches are being used within the pharmaceutical industry to assess and minimize the potential of drug candidates to cause toxicity. At early stages of drug discovery, iteration between medicinal chemistry and drug metabolism can eliminate perceived reactive metabolite-mediated chemical liabilities without compromising pharmacological activity or the need for extensive safety evaluation beyond standard practices. In the future, reactive metabolite evaluation may also be useful during clinical development for improving clinical risk assessment and risk management. Currently, there remains a huge gap in our understanding of the basic mechanisms that underlie chemical stress-mediated adverse reactions in humans. This review summarizes our views on this complex topic, and includes insights into practices considered by the pharmaceutical industry.

High-throughput cytochrome P450 inhibition assays using laser diode thermal desorption-atmospheric pressure chemical ionization-tandem mass spectrometry.

This paper describes the development of a high-throughput method for the analysis of cytochrome P450 (CYP) inhibition assay incubation samples using laser diode thermal desorption interfaced with atmospheric pressure chemical ionization mass spectrometry (LDTD-APCI-MS). Data for the CYP isoforms 3A4, 2D6, 2C9, and 1A2 from competitive inhibition assays are shown. The potential for inhibition of the CYP isoforms was measured by monitoring the level of the metabolites 6beta-hydroxytestosterone (3A4), dextrorphan (2D6), 4'-hydroxydiclofenac (2C9), and acetaminophen (1A2) formed in the presence of drug candidates using an eight-point titration. The analytical method involves plating of the inhibition samples on specially designed 96-well plates with stainless steel bottoms, followed by direct analysis using the LDTD source. Validation of the LDTD-MS method was performed by testing for interferences, reproducibility, dynamic range, ion suppression, and the ability of the source to produce comparable results to previously validated LC-MS methods. IC50 values for each CYP isoform using 33 different test compounds showed excellent agreement between LDTD-APCI-MS and LC-MS methods and literature values where available. Assay analysis time using the LDTD-APCI source is reduced to less than 30 min for a single 96-well plate compared to greater than 10 h using the LC-MS method. The LDTD-APCI-MS and LC-MS methods and results are compared and limitations and future potential for LDTD-APCI-MS are discussed.

MSE with mass defect filtering for in vitro and in vivo metabolite identification.

Metabolite identification studies involve the detection and structural characterization of the biotransformation products of drug candidates. These experiments are necessary throughout the drug discovery and development process. The use of high-resolution chromatography and high-resolution mass spectrometry together with data processing using mass defect filtering is described for in vitro and in vivo metabolite identification studies. Data collection was done using UPLC coupled with an orthogonal hybrid quadrupole time-of-flight mass spectrometer. This experimental approach enabled the use of MS(E) data collection (where E represents collision energy) which has previously been shown to be a powerful approach for metabolite identification studies. Post-acquisition processing with a prototype mass defect filtering program was used to eliminate endogenous interferences in the study samples, greatly enhancing the discovery of metabolites. The ease of this approach is illustrated by results showing the detection and structural characterization of metabolites in plasma from a preclinical rat pharmacokinetic study.

In vitro biotransformations of the prostaglandin D2 (DP) antagonist MK-0524 and synthesis of metabolites.

Metabolites of the potent DP antagonist, MK-0524, were generated using in vitro systems including hepatic microsomes and hepatocytes. Four metabolites (two hydroxylated diastereomers, a ketone and an acyl glucuronide) were characterized by LC-MS/MS and 1H NMR. Larger quantities of these metabolites were prepared by either organic synthesis or biosynthetically to be used as standards in other studies. The propensity for covalent binding was assessed and was found to be acceptable (<50 pmol-equiv/mg protein).

Metabolic activation of indole-containing prostaglandin D2 receptor 1 antagonists: impacts of glutathione trapping and glucuronide conjugation on covalent binding.

Some DP1 receptor antagonists from an indole-containing series were shown to cause in vitro covalent binding to protein in rat and human liver microsomes. Glutathione trapping experiments along with in vitro labeling assays confirmed that the presence of a strong electron withdrawing group was necessary to abrogate in vitro covalent binding, leading to the discovery of MK-0524. Hepatocyte incubations and in vivo studies showed that acyl-glucuronide formation did not translate into covalent binding.

A strategy for identification of drug metabolites from dried blood spots using triple-quadrupole/linear ion trap hybrid mass spectrometry.

Discovery stage studies that address issues of absorption, distribution, metabolism and excretion (ADME) are vital for lead optimization resulting in new drug candidates. Often pharmacokinetics (PK) is assessed in these experiments without regard for the metabolism of the compound or the potential for metabolites to circulate in vivo. This work presents a strategy for drug level determination and detection of metabolites using dried blood spots for sample collection. Initially, metabolites are detected from microsomal incubations and characterized using tandem mass spectrometry. Data dependent enhanced MS and enhanced product ion (EMS-EPI) scanning with dynamic background subtraction was used on a hybrid quadruple linear ion trap mass spectrometer. On-the-fly background subtraction greatly improved the detection of metabolites. These data were used to build a multiple reaction monitoring (MRM) method for the parent and metabolites. MRM-EPI scanning was used to analyze the extracted dried blood spots from the PK study. Circulating metabolites were detected using MRM and their identities confirmed on the basis of fragment ion spectra collected simultaneously. The use of dried blood spots provides a means for re-analysis of PK samples for metabolite identification without the need for complex sample storage and preparation. Both parent compound and metabolite information can be collected in these studies, resulting in a savings of time and resources.

Discovery of a substituted 8-arylquinoline series of PDE4 inhibitors: structure-activity relationship, optimization, and identification of a highly potent, well tolerated, PDE4 inhibitor.

The discovery and SAR of a new series of substituted 8-arylquinoline PDE4 inhibitors are herein described. This work has led to the identification of several compounds with excellent in vitro and in vivo profiles, including a good therapeutic window of emesis to efficacy in several animal models. Typical optimized compounds from this series are potent inhibitors of PDE4 (IC(50)<1nM) and also of LPS-induced TNF-alpha release in human whole blood (IC(50)<0.5microM). The same compounds are potent inhibitors of ovalbumin-induced bronchoconstriction in conscious guinea pigs (EC(50)<0.1mg/kg ip) but require a dose of about 10mg/kg po in the squirrel monkey to produce an emetic response. From this series of compounds, 23a (L-454,560) was identified as an optimized compound.

Drug-protein adducts: an industry perspective on minimizing the potential for drug bioactivation in drug discovery and development.

It is generally accepted that there is neither a well-defined nor a consistent link between the formation of drug-protein adducts and organ toxicity. Because the potential does exist, however, for these processes to be causally related, the general strategy at Merck Research Laboratories has been to minimize reactive metabolite formation to the extent possible by appropriate structural modification during the lead optimization stage. This requires a flexible approach to defining bioactivation issues in a variety of metabolism vectors and typically involves the initial use of small molecule trapping agents to define the potential for bioactivation. At some point, however, there is a requirement to synthesize a radiolabeled tracer and to undertake covalent binding studies in vitro, usually in liver microsomal (and sometimes hepatocyte) preparations from preclinical species and human, and also in vivo, typically in the rat. This paper serves to provide one pragmatic approach to addressing the issue of bioactivation from an industry viewpoint based on protocols adopted by Merck Research Laboratories. The availability of a dedicated Labeled Compound Synthesis group, coupled to a close working relationship between Drug Metabolism and Medicinal Chemistry, represents a framework within which this perspective becomes viable; the overall aim is to bring safer drugs to patients.

Use of a benzyloxy-substituted lactone cyclooxygenase-2 inhibitor as a selective fluorescent probe for CYP3A activity in primary cultured rat and human hepatocytes.

The benzyloxy-substituted lactone cyclooxygenase-2 inhibitor DFB [3-[(3,4-difluorobenzyl)oxy]-5,5-dimethyl-4-[4-(methylsulfonyl)phenyl]furan-2(5H)-one] is metabolized in human and rat liver microsomal incubations and hepatocytes to a fluorescent metabolite, DFH [3-hydroxy-5,5-dimethyl-4-[4-(methylsulfonyl)phenyl]furan-2(5H)-one]. This process is CYP3A-mediated in both species, as demonstrated by incubations with recombinant CYP3A enzymes and experiments with inhibitory antibodies. Measurement of DFH fluorescence can be used as a rapid readout of CYP3A activity following microsomal or cultured hepatocyte incubations. In rat and human hepatocytes treated with prototypical inducers, the formation of DFH was linear for the first 30 min, with no secondary metabolism of DFH, such as phase II glucuronidation, observed at early time points. Using a panel of four prototypical inducers (phenobarbital, dexamethasone, phenytoin, and rifampicin), the correlation between testosterone 6beta-hydroxylation in cultured human hepatocytes and CYP3A enzyme level in cell lysate was confirmed. DFB debenzylation was then shown to correlate well with testosterone 6beta-hydroxylation in hepatocytes treated with these four inducers. Primary cultured rat and human hepatocyte induction assays were optimized for 24- and 96-well plates, respectively. Controls were established to evaluate whether test compounds demonstrate time-dependent CYP3A inhibition to avoid false negative results. Thus, the use of DFB, a fluorogenic CYP3A-selective probe substrate, affords a fast, efficient, and robust assay for the measurement of CYP3A induction in both rat and human cultured primary hepatocytes.