Renato J. Scialis

Discovery of novel hepatoselective HMG-CoA reductase inhibitors for treating hypercholesterolemia: a bench-to-bedside case study on tissue selective drug distribution.

The design of drugs with selective tissue distribution can be an effective strategy for enhancing efficacy and safety, but understanding the translation of preclinical tissue distribution data to the clinic remains an important challenge. As part of a discovery program to identify next generation liver selective HMG-CoA reductase inhibitors we report the identification of (3R,5R)-7-(4-((3-fluorobenzyl)carbamoyl)-5-cyclopropyl-2-(4-fluorophenyl)-1H-imidazol-1-yl)-3,5-dihydroxyheptanoic acid (26) as a candidate for treating hypercholesterlemia. Clinical evaluation of 26 (PF-03491165), as well as the previously reported 2 (PF-03052334), provided an opportunity for a case study comparison of the preclinical and clinical pharmacokinetics as well as pharmacodynamics of tissue targeted HMG-CoA reductase inhibitors.

Metabolism and Disposition of a Selective α7 Nicotinic Acetylcholine Receptor Agonist in Humans

The metabolism and disposition of N-(3R)-1-azabicyclo[2.2.2]oct-3-ylfuro[2,3-c]pyridine-5-carboxamide (1), an α7 nicotinic acetylcholinergic receptor agonist, were elucidated in humans (4 female, 4 male; all white) after an oral dose of [3H]1. Overall, 1 was well tolerated, with >94% of administered radioactivity excreted renally by 48 h postdose; lyophilization of all urine and plasma samples confirmed 3H stability within [3H]1. Across genders, 1 underwent low-to-moderate oral clearance comprising both renal (67%) and metabolic (33%) components, with the biotransformation of 1 occurring predominantly via oxidation of its furanopyridine moiety to carboxylic acid 2, and minimally by modification of its quinuclidine nitrogen to N-oxide 4 or N-glucuronide M5. Experiments using human in vitro systems were undertaken to better understand the enzyme(s) involved in the phase 1 biotransformation pathways. The formation of 2 was found to be mediated by CYP2D6, a polymorphically expressed enzyme absent in 5 to 10% of white people, whereas the generation of 4 was catalyzed by CYP2D6, FAD-containing monooxygenase 1 (FMO1), and FMO3. It is of interest that, although no overall gender-related differences in excretory routes, mass recoveries, pharmacokinetics, or metabolite profiles of 1 were evident, the observation of one of eight subjects (13%) showing disparate (relative to all other volunteers) systemic exposures to 1, and urinary and plasma quantitative profiles nearly devoid of 2 with the highest levels of 1, seem consistent with both the identification of CYP2D6 as the only major recombinant cytochrome P450 transforming 1 to 2 and the demographics of white CYP2D6 poor metabolizers. Data also reported herein suggest that 4 is generated predominantly by renal FMO1 in humans.

Hepatic Disposition of Gemfibrozil and Its Major Metabolite Gemfibrozil 1-O-β-Glucuronide.

Gemfibrozil (GEM), which decreases serum triglycerides and low density lipoprotein, perpetrates drug-drug interactions (DDIs) with several drugs. These DDIs are primarily attributed to the inhibition of drug transporters and metabolic enzymes, particularly cytochrome P450 (CYP) 2C8 by the major circulating metabolite gemfibrozil 1-O-β-glucuronide (GG). Here, we characterized the transporter-mediated hepatic disposition of GEM and GG using sandwich-cultured human hepatocytes (SCHH) and transporter-transfect systems. Significant active uptake was noted in SCHH for the metabolite. GG, but not GEM, showed substrate affinity to organic anion transporting polypeptide (OATP) 1B1, 1B3, and 2B1. In SCHH, glucuronidation was characterized affinity constants (Km) of 7.9 and 61.4 μM, and biliary excretion of GG was observed. Furthermore, GG showed active basolateral efflux from preloaded SCHH and ATP-dependent uptake into membrane vesicles overexpressing multidrug resistance-associated protein (MRP) 2, MRP3, and MRP4. A mathematical model was developed to estimate hepatic uptake and efflux kinetics of GEM and GG based on SCHH studies. Collectively, the hepatic transporters play a key role in the disposition and thus determine the local concentrations of GEM and more so for GG, which is the predominant inhibitory species against CYP2C8 and OATP1B1.

Using Simcyp to project human oral pharmacokinetic variability in early drug research to mitigate mechanism‐based adverse events

Positive allosteric modulators (‘potentiators’) of the α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid receptor (AMPAR) have been shown to display a mechanism‐based exposure–response continuum in preclinical species with procognitive electrophysiological and behavioral effects (‘efficacy’) at low exposures and motor coordination disruptions at progressively higher exposures. Due to the dose‐capping nature of such motor coordination deficits, an exposure threshold‐mediated adverse event (CAE), the adequacy of separation between the maximal total plasma compound concentration (Cmax) at a predicted clinically efficacious oral dose and this adverse event (AE) was explored in early drug research with three AMPAR potentiators considered potential candidates for clinical trials. In vitro metabolism studies in human liver microsomes and human hepatocytes demonstrated the metabolic clearance for each compound was predominately due to cytochromes P450 (CYP). Thus, for each compounds anticipated clinically efficacious dose, human Cmax variability following oral administration was assessed using Simcyp software, which combines its virtual human populations database using extensive demographic, physiological and genomic information with routinely collected compound‐specific in vitro biochemical data to simulate and predict drug disposition. Using a combination of experimentally determined recombinant human CYP intrinsic clearances for CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP3A4, human binding factors, expected fraction absorbed and estimated steady‐state volume of distribution, Simcyp simulations demonstrated that two of the three potentiators had acceptable projected Cmax variability (i.e. the 95th percentile Cmax did not breach CAE). This evaluation aided in the selection of compounds for preclinical progression, and represents a novel application of pharmacologically based pharmacokinetic (PBPK) software approaches to predict interpatient variability. Copyright

Positive Allosteric Modulation of AMPA Receptors from Efficacy to Toxicity: The Interspecies Exposure-Response Continuum of the Novel Potentiator PF-4778574

α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) positive allosteric modulation (i.e., “potentiation”) has been proposed to overcome cognitive impairments in schizophrenia, but AMPAR overstimulation can be excitotoxic. Thus, it is critical to define carefully a potentiator’s mechanism-based therapeutic index (TI) and to determine confidently its translatability from rodents to higher-order species. Accordingly, the novel AMPAR potentiator N-{(3R,4S)-3-[4-(5-cyano-2-thienyl)phenyl]tetrahydro-2H-pyran-4-yl}propane-2-sulfonamide (PF-4778574) was characterized in a series of in vitro assays and single-dose animal studies evaluating AMPAR-mediated activities related to cognition and safety to afford an unbound brain compound concentration (Cb,u)–normalized interspecies exposure-response relationship. Because it is unknown which AMPAR subtype(s) may be selectively potentiated for an optimal TI, PF-4778574 binding affinity and functional potency were determined in rodent tissues expected to express a native mixture of AMPAR subunits and their associated proteins to afford composite pharmacological values. Functional activity was also quantified in recombinant cell lines stably expressing human GluA2 flip or flop homotetramers. Procognitive effects of PF-4778574 were evaluated in both rat electrophysiological and nonhuman primate (nhp) behavioral models of pharmacologically induced N-methyl-d-aspartate receptor hypofunction. Safety studies assessed cerebellum-based AMPAR activation (mouse) and motor coordination disruptions (mouse, dog, and nhp), as well as convulsion (mouse, rat, and dog). The resulting empirically derived exposure-response continuum for PF-4778574 defines a single-dose-based TI of 8- to 16-fold for self-limiting tremor, a readily monitorable clinical adverse event. Importantly, the Cb,u mediating each physiological effect were highly consistent across species, with efficacy and convulsion occurring at just fractions of the in vitro–derived pharmacological values.

Transporter‐Mediated Hepatic Uptake Plays an Important Role in the Pharmacokinetics and Drug–Drug Interactions of Montelukast

Montelukast, a leukotriene receptor antagonist commonly prescribed for treatment of asthma, is primarily metabolized by cytochrome P450 (CYP)2C8, and has been suggested as a probe substrate for investigating CYP2C8 activity in vivo. We evaluated the quantitative role of hepatic uptake transport in its pharmacokinetics and drug–drug interactions (DDIs). Montelukast was characterized with significant active uptake in human hepatocytes, and showed affinity towards organic anion transporting polypeptides (OATPs) in transfected cell systems. Single‐dose rifampicin, an OATP inhibitor, decreased montelukast clearance in rats and monkeys. Clinical DDIs of montelukast were evaluated using physiologically based pharmacokinetic modeling; and simulation of the interactions with gemfibrozil–CYP2C8 and OATP1B1/1B3 inhibitor, clarithromycin–CYP3A and OATP1B1/1B3 inhibitor, and itraconazole–CYP3A inhibitor, implicated OATPs‐CYP2C8‐CYP2C8 interplay as the primary determinant of montelukast pharmacokinetics. In conclusion, hepatic uptake plays a key role in the pharmacokinetics of montelukast, which should be taken into account when interpreting clinical interactions.

Detection of Statin Cytotoxicity Is Increased in Cells Expressing the OATP1B1 Transporter

Cytotoxicity of a compound is determined by the intracellular concentration mediated both by passive permeability and active uptake through drug transporters. However, the major liver uptake transporters were either absent or expressed at significantly lower levels in human liver cell lines than in human liver. When comparing cytotoxicity of five statins, the organic anion transporting polypeptide 1B1 expressing HEK cells showed a significantly higher sensitivity than the wild-type HEK cells. The IC50 shifts ranged from 9- to >100-fold, and the potency shifts collapsed in the presence of rifampicin, the inhibitor for OATPs. The extent of the IC50 shift correlated with the permeability of the statins with high permeable compounds having smaller shifts and low permeable compounds having larger shifts. The changes in statin potency in transporter-transfected cells reflect the active uptake of statins into the cells, and the increased intracellular drug concentration lead to increased toxicity. The data suggested that uptake transporters have a significant impact on the outcomes of a cell-based assay and should be considered during the early stages of compound toxicity screening in drug discovery. For compounds with low permeability that are likely to undergo transporter-mediated uptake, it is important to test them in transporter-competent cell models.

The Antimicrobial Agent Fusidic Acid Inhibits Organic Anion Transporting Polypeptide–Mediated Hepatic Clearance and May Potentiate Statin-Induced Myopathy

Chronic treatment of methicillin-resistant Staphylococcus aureus strains with the bacteriostatic agent fusidic acid (FA) is frequently associated with myopathy including rhabdomyolysis upon coadministration with statins. Because adverse effects with statins are usually the result of drug–drug interactions, we evaluated the inhibitory effects of FA against human CYP3A4 and clinically relevant drug transporters such as organic anion transporting polypeptides OATP1B1 and OATP1B3, multidrug resistant protein 1, and breast cancer resistance protein, which are involved in the oral absorption and/or systemic clearance of statins including atorvastatin, rosuvastatin, and simvastatin. FA was a weak reversible (IC50 = 295 ± 1.0 μM) and time-dependent (KI = 216 ± 41 μM and kinact = 0.0179 ± 0.001 min−1) inhibitor of CYP3A4-catalyzed midazolam-1′-hydroxylase activity in human liver microsomes. FA demonstrated inhibition of multidrug resistant protein 1–mediated digoxin transport with an IC50 value of 157 ± 1.0 μM and was devoid of breast cancer resistance protein inhibition (IC50 > 500 μM). In contrast, FA showed potent inhibition of OATP1B1- and OATP1B3-specific rosuvastatin transport with IC50 values of 1.59 μM and 2.47 μM, respectively. Furthermore, coadministration of oral rosuvastatin and FA to rats led to an approximately 19.3-fold and 24.6-fold increase in the rosuvastatin maximum plasma concentration and area under the plasma concentration–time curve, respectively, which could be potentially mediated through inhibitory effects of FA on rat Oatp1a4 (IC50 = 2.26 μM) and Oatp1b2 (IC50 = 4.38 μM) transporters, which are responsible for rosuvastatin uptake in rat liver. The potent inhibition of human OATP1B1/OATP1B3 by FA could attenuate hepatic uptake of statins, resulting in increased blood and tissue concentrations, potentially manifesting in musculoskeletal toxicity.

Elucidation of the biochemical basis for a clinical drug–drug interaction between atorvastatin and 5-(N-(4-((4-ethylbenzyl)thio)phenyl)sulfamoyl)-2-methyl benzoic acid (CP-778 875), a subtype selective agonist of the peroxisome proliferator-activated receptor alpha

Abstract 1. 5-(N-(4-((4-ethylbenzyl)thio)phenyl)sulfamoyl)-2-methyl benzoic acid (CP-778 875), an agonist of the peroxisome proliferator-activated receptor alpha, has been evaluated in the clinic to treat dyslipidemia and type 2 diabetes mellitus. Herein, we investigate the effect of CP-778 875 on the pharmacokinetics of atorvastatin acid and its metabolites in humans. 2. The study incorporated a fixed-sequence design conducted in two groups. Group A was designed to estimate the effects of multiple doses of CP-778 875 on the single dose pharmacokinetics of atorvastatin. Subjects in group A (n = 26) received atorvastatin (40 mg) on days 1 and 9 and CP-778 875 (1.0 mg QD) on days 5–12. Group B was designed to examine the effects of multiple doses of atorvastatin on the single dose pharmacokinetics of CP-778 875. Subjects in group B (n = 29) received CP-778 875 (0.3 mg) on days 1 and 9 and atorvastatin (40 mg QD) on days 5–12. 3. Mean maximum serum concentration (Cmax) and area under the curve of atorvastatin were increased by 45% and 20%, respectively, upon co-administration with CP-778 875. Statistically significant increases in the systemic exposure of ortho- and para-hydroxyatorvastatin were also observed upon concomitant dosing with CP-778 875. CP-778 875 pharmacokinetics, however, were not impacted upon concomitant dosing with atorvastatin. 4. Inhibition of organic anion transporting polypeptide 1B1 by CP-778 875 (IC50 = 2.14 ±0.40 μM) could be the dominant cause of the pharmacokinetic interaction as CP-778 875 did not exhibit significant inhibition of cytochrome P450 3A4/3A5, multidrug resistant protein 1 or breast cancer resistant protein, which are also involved in the hepatobiliary disposition of atorvastatin.

The Discovery and Characterization of the α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptor Potentiator N-{(3S,4S)-4-[4-(5-Cyano-2-thienyl)phenoxy]tetrahydrofuran-3-yl}propane-2-sulfonamide (PF-04958242)

A unique tetrahydrofuran ether class of highly potent α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor potentiators has been identified using rational and structure-based drug design. An acyclic lead compound, containing an ether-linked isopropylsulfonamide and biphenyl group, was pharmacologically augmented by converting it to a conformationally constrained tetrahydrofuran to improve key interactions with the human GluA2 ligand-binding domain. Subsequent replacement of the distal phenyl motif with 2-cyanothiophene to enhance its potency, selectivity, and metabolic stability afforded N-{(3S,4S)-4-[4-(5-cyano-2-thienyl)phenoxy]tetrahydrofuran-3-yl}propane-2-sulfonamide (PF-04958242, 3), whose preclinical characterization suggests an adequate therapeutic index, aided by low projected human oral pharmacokinetic variability, for clinical studies exploring its ability to attenuate cognitive deficits in patients with schizophrenia.