Mark Niosi

Plasma Protein Binding of Challenging Compounds

Accurately determining fraction unbound (fu ) with currently available methods has been challenging for certain compounds. Inaccurate fu values can lead to the misinterpretation of important attributes of a drug candidate. Our analysis of over 2000 Pfizer drug discovery compounds showed no systematic bias in low or high fu precision using the equilibrium dialysis method. However, the accuracy of the plasma protein binding (PPB) estimate for highly bound compounds may be poor, should equilibrium not be fully achieved in the equilibrium dialysis assay. Here, a dilution method and a presaturation method were applied to accelerate equilibration for a set of challenging compounds. These improved methods demonstrate the ability to provide an accurate measurement of PPB for highly bound compounds with fu values much less than 1%. Therefore, we recommend that the actual experimental fu value be used for the prediction of drug-drug interaction potential and for the characterization of important drug candidate properties. Our recommendation calls into question the need for current regulatory guidelines that restrict the reporting of fu values below 1%.

Discovery and characterization of novel inhibitors of the sodium-coupled citrate transporter (NaCT or SLC13A5)

Citrate is a key regulatory metabolic intermediate as it facilitates the integration of the glycolysis and lipid synthesis pathways. Inhibition of hepatic extracellular citrate uptake, by blocking the sodium-coupled citrate transporter (NaCT or SLC13A5), has been suggested as a potential therapeutic approach to treat metabolic disorders. NaCT transports citrate from the blood into the cell coupled to the transport of sodium ions. The studies herein report the identification and characterization of a novel small dicarboxylate molecule (compound 2) capable of selectively and potently inhibiting citrate transport through NaCT, both in vitro and in vivo. Binding and transport experiments indicate that 2 specifically binds NaCT in a competitive and stereosensitive manner, and is recognized as a substrate for transport by NaCT. The favorable pharmacokinetic properties of 2 permitted in vivo experiments to evaluate the effect of inhibiting hepatic citrate uptake on metabolic endpoints.

Discovery and Optimization of Imidazopyridine-Based Inhibitors of Diacylglycerol Acyltransferase 2 (DGAT2)

The medicinal chemistry and preclinical biology of imidazopyridine-based inhibitors of diacylglycerol acyltransferase 2 (DGAT2) is described. A screening hit 1 with low lipophilic efficiency (LipE) was optimized through two key structural modifications: (1) identification of the pyrrolidine amide group for a significant LipE improvement, and (2) insertion of a sp(3)-hybridized carbon center in the core of the molecule for simultaneous improvement of N-glucuronidation metabolic liability and off-target pharmacology. The preclinical candidate 9 (PF-06424439) demonstrated excellent ADMET properties and decreased circulating and hepatic lipids when orally administered to dyslipidemic rodent models.

Leveraging of Rifampicin-Dosed Cynomolgus Monkeys to Identify Bile Acid 3-O-Sulfate Conjugates as Potential Novel Biomarkers for Organic Anion-Transporting Polypeptides

In the search for novel bile acid (BA) biomarkers of liver organic anion-transporting polypeptides (OATPs), cynomolgus monkeys received oral rifampicin (RIF) at four dose levels (1, 3, 10, and 30 mg/kg) that generated plasma-free Cmax values (0.06, 0.66, 2.57, and 7.79 µM, respectively) spanning the reported in vitro IC50 values for OATP1B1 and OATP1B3 (≤1.7 μM). As expected, the area under the plasma concentration-time curve (AUC) of an OATP probe drug (i.v. 2H4-pitavastatin, 0.2 mg/kg) was increased 1.2-, 2.4-, 3.8-, and 4.5-fold, respectively. Plasma of RIF-dosed cynomolgus monkeys was subjected to a liquid chromatography-tandem mass spectrometry method that supported the analysis of 30 different BAs. Monkey urine was profiled, and we also determined that the impact of RIF on BA renal clearance was minimal. Although sulfated BAs comprised only 1% of the plasma BA pool, a robust RIF dose response (maximal ≥50-fold increase in plasma AUC) was observed for the sulfates of five BAs [glycodeoxycholate (GDCA-S), glycochenodeoxycholate (GCDCA-S), taurochenodeoxycholate, deoxycholate (DCA-S), and taurodeoxycholate (TDCA-S)]. In vitro, RIF (≤100 μM) did not inhibit cynomolgus monkey liver cytosol-catalyzed BA sulfation and cynomolgus monkey hepatocyte-mediated uptake of representative sulfated BAs (GDCA-S, GCDCA-S, DCA-S, and TDCA-S) was sodium-independent and inhibited (≥70%) by RIF (5 μM); uptake of taurocholic acid was sensitive to sodium removal (74% decrease) and relatively refractory to RIF (≤21% inhibition). We concluded that sulfated BAs may serve as sensitive biomarkers of cynomolgus monkey OATPs and that exploration of their utility as circulating human OATP biomarkers is warranted.

Novel monobactams utilizing a siderophore uptake mechanism for the treatment of gram-negative infections

Novel siderophore-linked monobactams with in vitro and in vivo anti-microbial activity against MDR Gram-negative pathogens are described.

Defining the key pharmacophore elements of PF-04620110: Discovery of a potent, orally-active, neutral DGAT-1 inhibitor

DGAT-1 is an enzyme that catalyzes the final step in triglyceride synthesis. mRNA knockout experiments in rodent models suggest that inhibitors of this enzyme could be of value in the treatment of obesity and type II diabetes. The carboxylic acid-based DGAT-1 inhibitor 1 was advanced to clinical trials for the treatment of type 2 diabetes, despite of the low passive permeability of 1. Because of questions relating to the potential attenuation of distribution and efficacy of a poorly permeable agent, efforts were initiated to identify compounds with improved permeability. Replacement of the acid moiety in 1 with an oxadiazole led to the discovery of 52, which possesses substantially improved passive permeability. The resulting pharmacodynamic profile of this neutral DGAT-1 inhibitor was found to be similar to 1 at comparable plasma exposures.

Quantitative Characterization of Major Hepatic UDP-Glucuronosyltransferase Enzymes in Human Liver Microsomes: Comparison of Two Proteomic Methods and Correlation with Catalytic Activity

Quantitative characterization of UDP-glucuronosyltransferase (UGT) enzymes is valuable in glucuronidation reaction phenotyping, predicting metabolic clearance and drug-drug interactions using extrapolation exercises based on pharmacokinetic modeling. Different quantitative proteomic workflows have been employed to quantify UGT enzymes in various systems, with reports indicating large variability in expression, which cannot be explained by interindividual variability alone. To evaluate the effect of methodological differences on end-point UGT abundance quantification, eight UGT enzymes were quantified in 24 matched liver microsomal samples by two laboratories using stable isotope-labeled (SIL) peptides or quantitative concatemer (QconCAT) standard, and measurements were assessed against catalytic activity in seven enzymes (n = 59). There was little agreement between individual abundance levels reported by the two methods; only UGT1A1 showed strong correlation [Spearman rank order correlation (Rs) = 0.73, P < 0.0001; R2 = 0.30; n = 24]. SIL-based abundance measurements correlated well with enzyme activities, with correlations ranging from moderate for UGTs 1A6, 1A9, and 2B15 (Rs = 0.52–0.59, P < 0.0001; R2 = 0.34–0.58; n = 59) to strong correlations for UGTs 1A1, 1A3, 1A4, and 2B7 (Rs = 0.79–0.90, P < 0.0001; R2 = 0.69–0.79). QconCAT-based data revealed generally poor correlation with activity, whereas moderate correlations were shown for UGTs 1A1, 1A3, and 2B7. Spurious abundance-activity correlations were identified in the cases of UGT1A4/2B4 and UGT2B7/2B15, which could be explained by correlations of protein expression between these enzymes. Consistent correlation of UGT abundance with catalytic activity, demonstrated by the SIL-based dataset, suggests that quantitative proteomic data should be validated against catalytic activity whenever possible. In addition, metabolic reaction phenotyping exercises should consider spurious abundance-activity correlations to avoid misleading conclusions.

Optimization of a Dicarboxylic Series for in Vivo Inhibition of Citrate Transport by the Solute Carrier 13 (SLC13) Family

Inhibition of the sodium-coupled citrate transporter (NaCT or SLC13A5) has been proposed as a new therapeutic approach for prevention and treatment of metabolic diseases. In a previous report, we discovered dicarboxylate 1a (PF-06649298) which inhibits the transport of citrate in in vitro and in vivo settings via a specific interaction with NaCT. Herein, we report the optimization of this series leading to 4a (PF-06761281), a more potent inhibitor with suitable in vivo pharmacokinetic profile for assessment of in vivo pharmacodynamics. Compound 4a was used to demonstrate dose-dependent inhibition of radioactive [(14)C]citrate uptake in liver and kidney in vivo, resulting in modest reductions in plasma glucose concentrations.

Determination of Unbound Partition Coefficient and in Vitro–in Vivo Extrapolation for SLC13A Transporter–Mediated Uptake

Unbound partition coefficient (Kpuu) is important to an understanding of the asymmetric free drug distribution of a compound between cells and medium in vitro, as well as between tissue and plasma in vivo, especially for transporter-mediated processes. Kpuu was determined for a set of compounds from the SLC13A family that are inhibitors and substrates of transporters in hepatocytes and transporter-transfected cell lines. Enantioselectivity was observed, with (R)-enantiomers achieving much higher Kpuu (>4) than the (S)-enantiomers (<1) in human hepatocytes and SLC13A5-transfected human embryonic 293 cells. The intracellular free drug concentration correlated directly with in vitro pharmacological activity rather than the nominal concentration in the assay because of the high Kpuu mediated by SLC13A5 transporter uptake. Delivery of the diacid PF-06649298 directly or via hydrolysis of the ethyl ester prodrug PF-06757303 resulted in quite different Kpuu values in human hepatocytes (Kpuu of 3 for diacid versus 59 for prodrug), which was successfully modeled on the basis of passive diffusion, active uptake, and conversion rate from ester to diacid using a compartmental model. Kpuu values changed with drug concentrations; lower values were observed at higher concentrations possibly owing to a saturation of transporters. Michaelis-Menten constant (Km) of SLC13A5 was estimated to be 24 μM for PF-06649298 in human hepatocytes. In vitro Kpuu obtained from rat suspension hepatocytes supplemented with 4% fatty acid free bovine serum albumin showed good correlation with in vivo Kpuu of liver-to-plasma, illustrating the potential of this approach to predict in vivo Kpuu from in vitro systems.

Novel Method to Predict In Vivo Liver-to-Plasma Kpuu for OATP Substrates Using Suspension Hepatocytes

The ability to predict human liver-to-plasma unbound partition coefficient (Kpuu) is of great importance to estimate unbound liver concentration, develop PK/PD relationships, predict efficacy and toxicity in the liver, and model the drug-drug interaction potential for drugs that are asymmetrically distributed into the liver. A novel in vitro method has been developed to predict in vivo Kpuu with good accuracy using cryopreserved suspension hepatocytes in InVitroGRO HI media with 4% BSA. Validation was performed using six OATP substrates with rat in vivo Kpuu data from i.v. infusion studies where a steady state was achieved. Good in vitro-in vivo correlation (IVIVE) was observed as the in vitro Kpuu values were mostly within 2-fold of in vivo Kpuu. Good Kpuu IVIVE in human was also observed with in vivo Kpuu data of dehydropravastatin from positron emission tomography and in vivo Kpuu data from PK/PD modeling for pravastatin and rosuvastatin. Under the specific Kpuu assay conditions, the drug-metabolizing enzymes and influx/efflux transporters appear to function at physiologic levels. No scaling factors are necessary to predict in vivo Kpuu from in vitro data. The novel in vitro Kpuu method provides a useful tool in drug discovery to project in vivo Kpuu.