Investigation of pharmacokinetic drug interaction between clesacostat and DGAT2 inhibitor ervogastat in healthy adult participants.

Co-administration of clesacostat (acetyl-CoA carboxylase inhibitor, PF-05221304) and ervogastat (diacylglycerol O-acyltransferase inhibitor, PF-06865571) in laboratory models improved non-alcoholic fatty liver disease (NAFLD)/non-alcoholic steatohepatitis (NASH) end points and mitigated clesacostat-induced elevations in circulating triglycerides. Clesacostat is cleared via organic anion-transporting polypeptide-mediated hepatic uptake and cytochrome P450 family 3A (CYP3A); in vitro clesacostat is identified as a potential CYP3A time-dependent inactivator. In vitro ervogastat is identified as a substrate and potential inducer of CYP3A. Prior to longer-term efficacy trials in participants with NAFLD, safety and pharmacokinetics (PK) were evaluated in a phase I, non-randomized, open-label, fixed-sequence trial in healthy participants. In Cohort 1, participants (n = 7) received clesacostat 15 mg twice daily (b.i.d.) alone (Days 1-7) and co-administered with ervogastat 300 mg b.i.d. (Days 8-14). Mean systemic clesacostat exposures, when co-administered with ervogastat, decreased by 12% and 19%, based on maximum plasma drug concentration and area under the plasma drug concentration-time curve during the dosing interval, respectively. In Cohort 2, participants (n = 9) received ervogastat 300 mg b.i.d. alone (Days 1-7) and co-administered with clesacostat 15 mg b.i.d. (Days 8-14). There were no meaningful differences in systemic ervogastat exposures when administered alone or with clesacostat. Clesacostat 15 mg b.i.d. and ervogastat 300 mg b.i.d. co-administration was overall safe and well tolerated in healthy participants. Cumulative safety and no clinically meaningful PK drug interactions observed in this study supported co-administration of these two novel agents in additional studies exploring efficacy and safety in the management of NAFLD.

Assessing trends in cytokine-CYP drug interaction and relevance to drug dosing.

The regulation of drug-metabolizing enzymes and transporters by cytokines has been extensively studied, in vitro and in clinic. Cytokine-mediated suppression of CYPs or drug transporters may increase or decrease the systemic clearance of drug substrates that are primarily cleared via these pathways; neutralization of cytokines by therapeutic proteins may thereby alter systemic exposures of such drug substrates. The FDA recommends evaluating such clinical drug interactions during clinical development and has provided labeling recommendations for therapeutic proteins. To determine the clinical relevance of these drug interactions to dose adjustments, trends in steady-state exposures (AUCss) of CYP-sensitive substrates co-administered with cytokine modulators as reported in the UW DIDB were extracted and examined for each of the CYPs. Co-administration of CYP3A (midazolam/simvastatin), CYP2C19 (omeprazole), or CYP1A2 (caffeine/tizanidine) substrates with anti-IL-6 and with anti-IL-23 therapeutics led to changes in systemic exposures of CYP substrates ranging from ~ -58% to ~35%; no significant trends were observed for CYP2D6 (dextromethorphan) and CYP2C9 (warfarin) substrates. Although none of these changes in systemic exposures have been reported as clinically meaningful, dose adjustment of midazolam for optimal sedation in acute care settings has been reported. Simulated concentration-time profiles of midazolam under conditions of elevated cytokine levels when co-administered with tocilizumab, suggest a ~6-7 fold increase in midazolam clearance suggesting potential implications of cytokine- CYP drug interactions on dose adjustments of sensitive CYP3A substrates in acute care settings. Additionally, this article also provides a brief overview of non-clinical and clinical assessments of cytokine-CYP drug interactions, in drug discovery and development. Significance Statement Significance statement: There has been significant progress in understanding cytokine-mediated drug interactions for CYP-sensitive substrates. This article provides an overview of the progress in this field, including a trend analysis of systemic exposures of CYP-sensitive substrates co-administered with anti-IL-x therapeutics. In addition, the review also provides a perspective of current methods used to assess these drug interactions during drug development, and a focus on individualized medicine, particularly in acute care settings.

Investigating CNS distribution of PF-05212377, a P-glycoprotein substrate, by translation of 5-HT6 receptor occupancy from non-human primates to humans.

PF-05212377 (SAM760) is a potent and selective 5-HT6 antagonist, previously under development for the treatment of Alzheimer's disease. In vitro, PF-05212377 was determined to be a P-gp/non-BCRP human transporter substrate. Species differences were observed in the in vivo brain penetration of PF-05212377 with a ratio of the unbound concentration in brain/unbound concentration in plasma (Cbu /Cpu ) of 0.05 in rat and 0.64 in non-human primates (NHP). Based on pre-clinical evidence, brain penetration and target engagement of PF-05212377 was confirmed in NHP using positron emission tomography (PET) measured 5-HT6 receptor occupancy (%RO). The NHP Cpu EC50 of PF-05212377 was 0.31 nM (consistent with the in vitro human 5HT6 Ki : 0.32 nM). P-gp has been reported to be expressed in higher abundance at the rat BBB and in similar abundance at the BBB of non-human primates and human; brain penetration of PF-05212377 in humans was postulated to be similar to that in non-human primates. In humans, PF-05212377 demonstrated dose and concentration dependent increases in 5-HT6 RO; maximal 5-HT6 RO of ∼80% was measured in humans at doses of ≥15 mg with an estimated unbound plasma EC50 of 0.37 nM (which was similar to the in vitro human 5HT6 binding Ki 0.32 nM). In conclusion, cumulative evidence from NHP and human PET RO assessments confirmed that NHP is more appropriate than the rat for the prediction of human brain penetration of PF-05212377, a P-gp/non-BCRP substrate. Clinical trial number: NCT01258751.

Investigation of CYP3A induction by PF-05251749 in early clinical development: comparison of linear slope physiologically based pharmacokinetic prediction and biomarker response.

PF-05251749 is a dual inhibitor of casein kinase 1 δ/ε under clinical development to treat disruption of circadian rhythm in Alzheimer's and Parkinson's diseases. In vitro, PF-05251749 (0.3-100 µM) induced CYP3A in cryopreserved human hepatocytes, demonstrating non-saturable, dose-dependent CYP3A mRNA increases, with induction slopes in the range 0.036-0.39 µM-1 . In a multiple-dose study (B8001002) in healthy participants, CYP3A activity was explored by measuring changes in 4ß-hydroxycholesterol/cholesterol ratio. Following repeated oral administration of PF-05251749, up to 400 mg q.d., no significant changes were observed in 4ß-hydroxycholesterol/cholesterol ratio; this ratio increased significantly (~1.5-fold) following administration of PF-05251749 at 750 mg q.d., suggesting potential CYP3A induction at this dose. Physiologically based pharmacokinetic (PBPK) models were developed to characterize the observed clinical pharmacokinetics (PK) of PF-05251749 at 400 and 750 mg q.d.; the PBPK induction model was calibrated using the in vitro linear fit induction slope, with rifampin as reference compound (Indmax  = 8, EC50  = 0.32 µM). Clinical trial simulation following co-administration of PF-05251749, 400 mg q.d. with oral midazolam 2 mg, predicted no significant drug interaction risk. PBPK model predicted weak drug interaction following co-administration of PF-05251749, 750 mg q.d. with midazolam 2 mg. In conclusion, good agreement was obtained between CYP3A drug interaction risk predicted using linear-slope PBPK model and exploratory biomarker trends. This agreement between two orthogonal approaches enabled assessment of drug interaction risks of PF-05251749 in early clinical development, in the absence of a clinical drug-drug interaction study.

Emerging Models of Drug Metabolism, Transporters, and Toxicity.

This commentary summarizes expert mini-reviews and original research articles that have been assembled in a special issue on novel models of drug metabolism and disposition. The special issue consists of research articles or reviews on novel static or micro-flow based models of the intestine, liver, eye, and kidney. This issue reviews static intestinal systems like mucosal scrapings and cryopreserved intestinal enterocytes, as well as novel bioengineered or chemically engineered intestinal models derived from primary human tissue, iPSCs, enteroids, and crypts. Experts have reviewed hepatic systems like cryopermeabilized Metmax hepatocytes and longer term, hepatocyte coculture system from HµREL, yielding in vivo-like primary and secondary drug metabolite profiles. Additional liver models, including micropattern hepatocyte coculture, 3D liver spheroids, and microflow systems, applicable to the study of drug disposition and toxicology have also been reviewed. In this commentary, we have outlined expert opinions and current efforts on hepatic- and nephrotoxicity models. Ocular disposition models including corneal permeability models have been included within this special issue. This commentary provides a summary of in vivo mini-reviews of the issue, which have discussed the applications and drawbacks of pig and humanized mice models of P450, UGT, and rat organic anionic transporting polypeptide 1a4. While not extensively reviewed, novel positron emissions tomography imaging-based approaches to study the distribution of xenobiotics have been highlighted. This commentary also outlines in vitro and in vivo models of drug metabolism derived from breakthrough genetic, chromosomal, and tissue engineering techniques. The commentary concludes by providing a futuristic view of the novel models discussed in this issue.

Physiologically Relevant, Humanized Intestinal Systems to Study Metabolism and Transport of Small Molecule Therapeutics.

Intestinal disposition of small molecules involves interplay of drug metabolizing enzymes (DMEs), transporters, and host-microbiome interactions, which has spurred the development of in vitro intestinal models derived from primary tissue sources. Such models have been bioengineered from intestinal crypts, mucosal extracts, induced pluripotent stem cell (iPSC)-derived organoids, and human intestinal tissue. This minireview discusses the utility and limitations of these human-derived models in support of small molecule drug metabolism and disposition. Enteroids from human intestinal crypts, organoids derived from iPSCs using growth factors or small molecule compounds, and enterocytes extracted from mucosal scrapings show key absorptive cell morphology while are limited in quantitative applications due to the lack of accessibility to the apical compartment, the lack of monolayers, or low expression of key DMEs, transporters, and nuclear hormone receptors. Despite morphogenesis to epithelial cells, similar challenges have been reported by more advanced technologies that have explored the impact of flow and mechanical stretch on proliferation and differentiation of Caco-2 cells. Most recently, bioengineered human intestinal epithelial or ileal cells have overcome many of the challenges, as the DME and transporter expression pattern resembles that of native intestinal tissue. Engineering advances may improve such models to support longer-term applications and meet end-user needs. Biochemical characterization and transcriptomic, proteomic, and functional endpoints of emerging novel intestinal models, when referenced to native human tissue, can provide greater confidence and increased utility in drug discovery and development.

Quantitative Translational Analysis of Brain Kynurenic Acid Modulation via Irreversible Kynurenine Aminotransferase II Inhibition.

Kynurenic acid (KYNA) plays a significant role in maintaining normal brain function, and abnormalities in KYNA levels have been associated with various central nervous system disorders. Confirmation of its causality in human diseases requires safe and effective modulation of central KYNA levels in the clinic. The kynurenine aminotransferases (KAT) II enzyme represents an attractive target for pharmacologic modulation of central KYNA levels; however, KAT II and KYNA turnover kinetics, which could contribute to the duration of pharmacologic effect, have not been reported. In this study, the kinetics of central KYNA-lowering effect in rats and nonhuman primates (NHPs, Cynomolgus macaques) was investigated using multiple KAT II irreversible inhibitors as pharmacologic probes. Mechanistic pharmacokinetic-pharmacodynamic analysis of in vivo responses to irreversible inhibition quantitatively revealed that 1) KAT II turnover is relatively slow [16-76 hours' half-life (t1/2)], whereas KYNA is cleared more rapidly from the brain (<1 hour t1/2) in both rats and NHPs, 2) KAT II turnover is slower in NHPs than in rats (76 hours vs. 16 hours t1/2, respectively), and 3) the percent contribution of KAT II to KYNA formation is constant (∼80%) across rats and NHPs. Additionally, modeling results enabled establishment of in vitro-in vivo correlation for both enzyme turnover rates and drug potencies. In summary, quantitative translational analysis confirmed the feasibility of central KYNA modulation in humans. Model-based analysis, where system-specific properties and drug-specific properties are mechanistically separated from in vivo responses, enabled quantitative understanding of the KAT II-KYNA pathway, as well as assisted development of promising candidates to test KYNA hypothesis in humans.

Metabolism of a 5HT6 Antagonist, 2-Methyl-1-(Phenylsulfonyl)-4-(Piperazin-1-yl)-1H-Benzo[d]imidazole (SAM-760): Impact of Sulfonamide Metabolism on Diminution of a Ketoconazole-Mediated Clinical Drug-Drug Interaction.

SAM-760 [(2-methyl-1-(phenylsulfonyl)-4-(piperazin-1-yl)-1H-benzo[d]imidazole)], a 5HT6 antagonist, was investigated in humans for the treatment of Alzheimer's disease. In liver microsomes and recombinant cytochrome P450 (P450) isozymes, SAM-760 was predominantly metabolized by CYP3A (∼85%). Based on these observations and an expectation of a 5-fold magnitude of interaction with moderate to strong CYP3A inhibitors, a clinical DDI study was performed. In the presence of ketoconazole, the mean Cmax and area under the plasma concentration-time curve from time zero extrapolated to infinite time values of SAM-760 showed only a modest increase by 30% and 38%, respectively. In vitro investigation of this unexpectedly low interaction was undertaken using [14C]SAM-760. Radiometric profiling in human hepatocytes confirmed all oxidative metabolites previously observed with unlabeled SAM-760; however, the predominant radiometric peak was an unexpected polar metabolite that was insensitive to the pan-P450 inhibitor 1-aminobenzotriazole. In human hepatocytes, radiometric integration attributed 43% of the total metabolism of SAM-760 to this non-P450 pathway. Using an authentic standard, this predominant metabolite was confirmed as benzenesulfinic acid. Additional investigation revealed that the benzenesulfinic acid metabolite may be a novel, nonenzymatic, thiol-mediated reductive cleavage of an aryl sulfonamide group of SAM-760. We also determined the relative contribution of P450 to the metabolism of SAM-760 in human hepatocytes by following the rate of formation of oxidative metabolites in the presence and absence of P450 isoform-specific inhibitors. The P450-mediated oxidative metabolism of SAM-760 was still primarily attributed to CYP3A (33%), with minor contributions from P450 isoforms CYP2C19 and CYP2D6. Thus, the disposition of [14C]SAM-760 in human hepatocytes via novel sulfonamide metabolism and CYP3A verified the lower than expected clinical DDI when SAM-760 was coadministered with ketoconazole.

Estimation of Circulating Drug Metabolite Exposure in Human Using In Vitro Data and Physiologically Based Pharmacokinetic Modeling: Example of a High Metabolite/Parent Drug Ratio.

(R)-4-((4-(((4-((tetrahydrofuran-3-yl)oxy)benzo[d]isoxazol-3-yl)oxy)methyl)piperidin-1-yl)methyl)tetrahydro-2H-pyran-4-ol (TBPT), a serotonin-4 receptor partial agonist, is metabolized to two metabolites: an N-dealkylation product [(R)-3-(piperidin-4-ylmethoxy)-4-((tetrahydrofuran-3-yl)oxy)benzo[d]isoxazole (M1)] and a cyclized oxazolidine structure [7-(((4-(((R)-tetrahydrofuran-3-yl)oxy)benzo[d]isoxazol-3-yl)oxy)methyl)octahydro-3H (M2)]. After administration of TBPT to humans the exposure to M1 was low and the exposure to M2 was high, relative to the parent drug, despite this being the opposite in vitro. In this study, projection of the plasma metabolite/parent (M/P) ratios for M1 and M2 was attempted using in vitro metabolism, binding, and permeability data in static and dynamic physiologically based pharmacokinetic (PBPK) models. In the static model, the fraction of parent clearance yielding the metabolite (which also required taking into account secondary metabolites of M1 and M2), the clearance of the metabolites and parent, and an estimate of the availability of the metabolites from the liver were combined to yield estimated parent/metabolite ratios of 0.32 and 23 for M1 and M2, respectively. PBPK modeling that used in vitro and physicochemical data input yielded estimates of 0.26 and 20, respectively. The actual values were 0.12 for M1/TBPT and 58 for M2/TBPT. Thus, the ratio for M1 was overpredicted, albeit at values less than unity. The ratio for M2/TBPT was underpredicted, and the high ratio of 58 may exceed a limiting ceiling of the approach. Nevertheless, when considered in the context of determining whether a potential circulating metabolite may be quantitatively important prior to administration of a drug for the first time to humans, the approaches succeeded in highlighting the importance of M2 (M/P ratio >> 1) relative to M1, despite M1 being much greater than M2 in vitro.

Quantitative projection of human brain penetration of the H3 antagonist PF-03654746 by integrating rat-derived brain partitioning and PET receptor occupancy.

1. Unbound brain drug concentration (Cb,u), a valid surrogate of interstitial fluid drug concentration (CISF), cannot be directly determined in humans, which limits accurately defining the human Cb,u:Cp,u of investigational molecules. 2. For the H3R antagonist (1R,3R)-N-ethyl-3-fluoro-3-[3-fluoro-4-(pyrrolidin-1-lmethyl)phenyl]cyclobutane-1-carboxamide (PF-03654746), we interrogated Cb,u:Cp,u in humans and nonhuman primate (NHP). 3. In rat, PF-03654746 achieved net blood-brain barrier (BBB) equilibrium (Cb,u:Cp,u of 2.11). 4. In NHP and humans, the PET receptor occupancy-based Cp,u IC50 of PF-03654746 was 0.99 nM and 0.31 nM, respectively, which were 2.1- and 7.4-fold lower than its in vitro human H3 Ki (2.3 nM). 5. In an attempt to understand this higher-than-expected potency in humans and NHP, rat-derived Cb,u:Cp,u of PF-03654746 was integrated with Cp,u IC50 to identify unbound (neuro) potency of PF-03654746, nIC50. 6. The nIC50 of PF-03654746 was 2.1 nM in NHP and 0.66 nM in human which better correlated (1.1- and 3.49-fold lower) with in vitro human H3 Ki (2.3 nM). 7. This correlation of the nIC50 and in vitro hH3 Ki suggested the translation of net BBB equilibrium of PF-03654746 from rat to NHP and humans, and confirmed the use of Cp,u as a reliable surrogate of Cb,u. 8. Thus, nIC50 quantitatively informed the human Cb,u:Cp,u of PF-03654746.

Determination of receptor occupancy in the presence of mass dose: [11C]GSK189254 PET imaging of histamine H3 receptor occupancy by PF-03654746.

Measurements of drug occupancies using positron emission tomography (PET) can be biased if the radioligand concentration exceeds "tracer" levels. Negative bias would also arise in successive PET scans if clearance of the radioligand is slow, resulting in a carryover effect. We developed a method to (1) estimate the in vivo dissociation constant Kd of a radioligand from PET studies displaying a non-tracer carryover (NTCO) effect and (2) correct the NTCO bias in occupancy studies taking into account the plasma concentration of the radioligand and its in vivo Kd. This method was applied in a study of healthy human subjects with the histamine H3 receptor radioligand [11C]GSK189254 to measure the PK-occupancy relationship of the H3 antagonist PF-03654746. From three test/retest studies, [11C]GSK189254 Kd was estimated to be 9.5 ± 5.9 pM. Oral administration of 0.1 to 4 mg of PF-03654746 resulted in occupancy estimates of 71%-97% and 30%-93% at 3 and 24 h post-drug, respectively. NTCO correction adjusted the occupancy estimates by 0%-15%. Analysis of the relationship between corrected occupancies and PF-03654746 plasma levels indicated that PF-03654746 can fully occupy H3 binding sites ( ROmax = 100%), and its IC50 was estimated to be 0.144 ± 0.010 ng/mL. The uncorrected IC50 was 26% higher.

Dopamine D3/D2 Receptor Antagonist PF-4363467 Attenuates Opioid Drug-Seeking Behavior without Concomitant D2 Side Effects.

Dopamine receptor antagonism is a compelling molecular target for the treatment of a range of psychiatric disorders, including substance use disorders. From our corporate compound file, we identified a structurally unique D3 receptor (D3R) antagonist scaffold, 1. Through a hybrid approach, we merged key pharmacophore elements from 1 and D3 agonist 2 to yield the novel D3R/D2R antagonist PF-4363467 (3). Compound 3 was designed to possess CNS drug-like properties as defined by its CNS MPO desirability score (≥4/6). In addition to good physicochemical properties, 3 exhibited low nanomolar affinity for the D3R (D3 Ki = 3.1 nM), good subtype selectivity over D2R (D2 Ki = 692 nM), and high selectivity for D3R versus other biogenic amine receptors. In vivo, 3 dose-dependently attenuated opioid self-administration and opioid drug-seeking behavior in a rat operant reinstatement model using animals trained to self-administer fentanyl. Further, traditional extrapyramidal symptoms (EPS), adverse side effects arising from D2R antagonism, were not observed despite high D2 receptor occupancy (RO) in rodents, suggesting that compound 3 has a unique in vivo profile. Collectively, our data support further investigation of dual D3R and D2R antagonists for the treatment of drug addiction.

Strategies toward optimization of the metabolism of a series of serotonin-4 partial agonists: investigation of azetidines as piperidine isosteres.

1.The first generation 5HT-4 partial agonist, 4-{4-[4-Tetrahydrofuran-3-yloxy)-benzo[d]isoxazol-3-yloxymethyl]-piperidin-1-ylmethyl}-tetrahydropyran-4-ol, PF-4995274 (TBPT), was metabolized to N-dealkylated (M1) and an unusual, cyclized oxazolidine (M2) metabolites. M1 and M2 demonstrated pharmacological activity at 5HT receptor subtypes warranting further investigation into their dispositional properties in humans; M2 was a minor component in vitro but was the pre-dominant metabolite identified in human plasma. 2.To shift metabolism away from the piperidine ring of TBPT, a series of heterocyclic replacements were designed, synthesized, and profiled. Groups including azetidines, pyrrolidines, as well as functionalized piperidines were evaluated with the goal of identifying an alternative group that maintained the desired potency, functional activity, and reduced turnover in human hepatocytes. 3.Activities of 4-substituted piperidines or pyrrolidine analogs at the pharmacological target were not significantly altered, but the same metabolic pathways of N-dealkylation and oxazolidine formation were still observed. Altering these to bridged ring systems lowered oxazolidine metabolite formation, but not N-dealkylation. 4.The effort concluded with identification of azetidines as second-generation 5HT4 partial agonists. These were neither metabolized via N-dealkylation nor converted to cyclized oxazolidine metabolites rather oxidized on the isoxazole ring. The use of azetidine as a replacement for aliphatic aza-heterocyclic rings in drug design to alter drug metabolism and pharmacology is discussed.

Metabolism and clinical pharmacokinetics of 2-methyl-n-(2'-(pyrrolidinyl-1-ylsulfonyl)-n-[1,1'-biphenyl]-4-yl)propran-1-amine: insights into monoamine oxidase- and CYP-mediated disposition by integration of in vitro ADME tools.

1. In early discovery stages, 2-methyl-N-(2'-(pyrrolidinyl-1-ylsulfonyl)-[1,1'-biphenyl]-4-yl)propan-1-amine (PBPA) demonstrated monoamine oxidase A (MAO-A) and cytochrome P450 (CYP)-mediated clearance. While human liver microsomes predicted low CL(b) PBPA demonstrated a moderate CL(p)/F in humans. The plasma pharmacokinetic (PK) of PBPA was characterized by unexpected high inter-individual variability. Hence, a retrospective analysis was undertaken to understand the disposition processes of PBPA, by applying in vitro mechanistic tools. 2. The in vitro-to-in vivo of rat CL(b) of PBPA was calculated as similar to that of human, suggesting rat to be a better predictor of a MAO-A/CYP substrate, but not dog or monkey; this is consistent with differences in expression of MAO-A in rat, dog, monkey and human. Fraction metabolized (f(m)) of human MAO A (hMAO-A) (50%), CYP3A4 (8%), CYP3A5 (16%) and CYP2D6 (29%) was determined, in vitro. 3. While the fm of CYP3A5 was <50%, Michaelis-Menten kinetics demonstrated that it was a higher capacity pathway compared with MAO-A, 2D6 and 3A4. This was consistent with strong association of dose-normalized plasma C(max) and area under the plasma concentration time curve (AUC(0-tlast)) of PBPA with CYP3A5 genotype, but not with genotype of CYP2D6. 4. This investigation demonstrates the value of integrating in vitro mechanistic tools to gain comprehensive understanding of disposition properties of drug candidates, in a discovery paradigm and prior to the investment in clinical trials.

Metabolism of a serotonin-4 receptor partial agonist 4-{4-[4-tetrahydrofuran-3-yloxy)-benzo[d]isoxazol-3-yloxymethyl]-piperidin-1-ylmethyl}-tetrahydropyran-4-ol (TBPT): identification of an unusual pharmacologically active cyclized oxazolidine metabolite in human.

4-{4-[4-Tetrahydrofuran-3-yloxy)-benzo[d]isoxazol-3-yloxymethyl]-piperidin-1-ylmethyl}-tetrahydropyran-4-ol (PF-4995274, TBPT) is a new agent that is a partial agonist of the human serotonin-4 (5-HT4) receptor and is under investigation for neurological disorders. Metabolism of TBPT was examined in vitro in human liver microsomes and human hepatocytes. Metabolites were also identified in the plasma of healthy human subjects in a phase 1 clinical study. Human-derived metabolite profiles were compared with corresponding profiles obtained in laboratory animal species. There were two major routes of metabolism in vitro: N-dealkylation of the methyltetrahydropyran moiety (M1) and hydroxylation at the seven position of the benzisoxazole moiety (M4). These were also observed in human plasma; however, in that matrix, the major metabolite was an unusual cyclized oxazolidine entity (M2). M2 was proposed to be formed via generation of an intermediate 4° iminium ion on the piperidine ring followed by spontaneous cyclization by attack of the ß-hydroxyl substituent of the tetrahydropyran ring to form a cyclized oxazolidine product. An authentic standard of the metabolite was generated using a methylene-blue-sensitized photochemical oxidation reaction as well as microbial transformation. Further investigation of this metabolite showed that it also possessed 5-HT4 agonism activity similar to the parent. The metabolite was 150-fold more highly protein bound in human plasma than TBPT, which is consistent with its presence as a major circulating metabolite while being only a minor metabolite in in vitro systems. Overall, this illustrates the importance of understanding the complex dispositional properties of a pharmacologically active metabolite.

Identification of multiple 5-HT4 partial agonist clinical candidates for the treatment of Alzheimer's disease.

The cognitive impairments observed in Alzheimer's disease (AD) are in part a consequence of reduced acetylcholine (ACh) levels resulting from a loss of cholinergic neurons. Preclinically, serotonin 4 receptor (5-HT(4)) agonists are reported to modulate cholinergic function and therefore may provide a new mechanistic approach for treating cognitive deficits associated with AD. Herein we communicate the design and synthesis of potent, selective, and brain penetrant 5-HT(4) agonists. The overall goal of the medicinal chemistry strategy was identification of structurally diverse clinical candidates with varying intrinsic activities. The exposure-response relationships between binding affinity, intrinsic activity, receptor occupancy, drug exposure, and pharmacodynamic activity in relevant preclinical models of AD were utilized as key selection criteria for advancing compounds. On the basis of their excellent balance of pharmacokinetic attributes and safety, two lead 5-HT(4) partial agonist candidates 2d and 3 were chosen for clinical development.

The 5-hydroxytryptamine4 receptor agonists prucalopride and PRX-03140 increase acetylcholine and histamine levels in the rat prefrontal cortex and the power of stimulated hippocampal θ oscillations.

5-Hydroxytryptamine (5-HT)(4) receptor agonists reportedly stimulate brain acetylcholine (ACh) release, a property that might provide a new pharmacological approach for treating cognitive deficits associated with Alzheimer's disease. The purpose of this study was to compare the binding affinities, functional activities, and effects on neuropharmacological responses associated with cognition of two highly selective 5-HT(4) receptor agonists, prucalopride and 6,7-dihydro-4-hydroxy-7-isopropyl-6-oxo-N-[3-(piperidin-1-yl)propyl]thieno[2,3-b]pyridine-5-carboxamide (PRX-03140). In vitro, prucalopride and PRX-03140 bound to native rat brain 5-HT(4) receptors with K(i) values of 30 nM and 110 nM, respectively, and increased cAMP production in human embryonic kidney-293 cells expressing recombinant rat 5-HT(4) receptors. In vivo receptor occupancy studies established that prucalopride and PRX-03140 were able to penetrate the brain and bound to 5-HT(4) receptors in rat brain, achieving 50% receptor occupancy at free brain exposures of 330 nM and 130 nM, respectively. Rat microdialysis studies revealed that prucalopride maximally increased ACh and histamine levels in the prefrontal cortex at 5 and 10 mg/kg, whereas PRX-03140 significantly increased cortical histamine levels at 50 mg/kg, failing to affect ACh release at doses lower than 150 mg/kg. In combination studies, donepezil-induced increases in cortical ACh levels were potentiated by prucalopride and PRX-03140. Electrophysiological studies in rats demonstrated that both compounds increased the power of brainstem-stimulated hippocampal θ oscillations at 5.6 mg/kg. These findings show for the first time that the 5-HT(4) receptor agonists prucalopride and PRX-03140 can increase cortical ACh and histamine levels, augment donepezil-induced ACh increases, and increase stimulated-hippocampal θ power, all neuropharmacological parameters consistent with potential positive effects on cognitive processes.

Quantitative PK-PD model-based translational pharmacology of a novel kappa opioid receptor antagonist between rats and humans.

Pharmacokinetic-pharmacodynamic (PK-PD) modeling greatly enables quantitative implementation of the "learn and confirm" paradigm across different stages of drug discovery and development. This work describes the successful prospective application of this concept in the discovery and early development of a novel κ-opioid receptor (KOR) antagonist, PF-04455242, where PK-PD understanding from preclinical biomarker responses enabled successful prediction of the clinical response in a proof of mechanism study. Preclinical data obtained in rats included time course measures of the KOR antagonist (PF-04455242), a KOR agonist (spiradoline), and a KOR-mediated biomarker response (prolactin secretion) in plasma. Clinical data included time course measures of PF-04455242 and prolactin in 24 healthy volunteers following a spiradoline challenge and single oral doses of PF-04455242 (18 and 30 mg). In both species, PF-04455242 successfully reversed spiradoline-induced prolactin response. A competitive antagonism model was developed and implemented within NONMEM to describe the effect of PF-04455242 on spiradoline-induced prolactin elevation in rats and humans. The PK-PD model-based estimate of K(i) for PF-04455242 in rats was 414 ng/mL. Accounting for species differences in unbound fraction, in vitro K(i) and brain penetration provided a predicted human K(i) of 44.4 ng/mL. This prediction was in good agreement with that estimated via the application of the proposed PK-PD model to the clinical data (i.e., 39.2 ng/mL). These results illustrate the utility of the proposed PK-PD model in supporting the quantitative translation of preclinical studies into an accurate clinical expectation. As such, the proposed PK-PD model is useful for supporting the design, selection, and early development of novel KOR antagonists.