Radiolabel Uncovers Nonintuitive Metabolites of BIIB104: Novel Release of [14C]Cyanide from 2-Cyanothiophene and Subsequent Formation of [14C]Thiocyanate.

BIIB104 (formerly PF-04958242), N-((3S,4S)-4-(4-(5-cyanothiophen-2-yl)phenoxy)tetrahydrofuran-3-yl)propane-2-sulfonamide, is an α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor potentiator investigated for the treatment of cognitive impairment associated with schizophrenia. Preliminary in vitro metabolism studies with non-radiolabeled BIIB104 in rat, dog, and human liver microsomes (RLM, DLM, and HLM) showed O-dealkylation in all three species, tetrahydrofuran hydroxylation dominating in DLM and HLM, and thiophene hydroxylation prevalent in RLM. However, a subsequent rat mass balance study with [nitrile-14C]BIIB104 showed incomplete recovery of administered radioactivity (∼80%) from urine and feces over 7 days following an oral dose, and an exceptionally long plasma total radioactivity half-life. Radiochromatographic metabolite profiling and identification, including chemical derivation, revealed that [14C]cyanide was a major metabolite of [nitrile-14C]BIIB104 in RLM, but a minor and trace metabolite in DLM and HLM, respectively. Correspondingly in bile duct-cannulated rats, [14C]thiocyanate accounted for ∼53% of total radioactivity excreted over 48 hours postdose and it, as an endogenous substance, explained the exceptionally long plasma radioactivity half-life. The release of [14C]cyanide from the 2-cyanothiophene moiety is postulated to follow an epoxidation-initiated thiophene-opening based on the detection of non-radiolabeled counterpart metabolites in RLM. This unusual biotransformation serves as a lesson regarding placement of the radioactive label on an aryl nitrile when material will be used for evaluating the metabolism of a new drug candidate. Additionally, the potential cyanide metabolite of nitrile-containing drug molecules may be detected in liver microsomes with liquid chromatography-mass spectrometry following a chemical derivatization. SIGNIFICANCE STATEMENT: Using [nitrile-14C]BIIB104, non-intuitive metabolites of BIIB104 were discovered involving a novel cyanide release from the 2-cyanothiophene motif via a postulated epoxidation-initiated thiophene-opening. This unusual biotransformation serves as a lesson regarding placement of the radioactive label on an aryl nitrile when material will be used for evaluating the metabolism of a new drug candidate.

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.

Student Preparation for PGY1 Residency Training by US Colleges of Pharmacy: Survey of the Residency Program Director Perspective.

Purpose: To evaluate current residents' level of preparation by US colleges of pharmacy for postgraduate year 1 (PGY1) residency training from the perspective of residency program directors (RPDs). Methods: RPDs were asked in an electronic survey questionnaire to rate PGY1 pharmacy residents' abilities in 4 domains: communication, clinical knowledge, interpersonal/time-management skills, and professionalism/leadership. Results: One hundred ninety-seven RPDs of the American Society of Health-System Pharmacists (ASHP)-accredited PGY1 programs completed the survey. The majority of RPDs strongly agreed or agreed that residents were prepared as students to effectively communicate both verbally and nonverbally, were able to appropriately respond to drug inquiries using drug resources and literature searches, and consistently displayed professionalism. Respondents were more likely to disagree or give a neutral response when asked about residents' understanding of biostatistics and their ability to provide enteral and parenteral nutritional support for patients. Conclusion: Overall, RPDs agreed that residents were prepared to perform the majority of the tasks of each of the 4 domains assessed in this survey relating to PGY1 training. RPDs may use the results of this survey to provide additional support for their residents in the areas in which residents lack adequate preparation, while colleges of pharmacy may focus on incorporating more time in their curriculum for certain areas to better prepare their students for residency training.

Drug-induced Skin Lesions in Cynomolgus Macaques Treated with Metabotropic Glutamate Receptor 5 (mGluR5) Negative Allosteric Modulators.

Three orally administered metabotropic glutamate receptor 5 (mGluR5) negative allosteric modulators caused skin lesions consistent with delayed type-IV hypersensitivity in cynomolgus macaques in 2- and 12-week toxicity studies. Several monkeys developed macroscopic skin lesions in multiple locations after 8 to 9 days of dosing; the most prominent effects involved the genital region of males and generalized erythema occurred in both sexes. Microscopic lesions occurred in both clinically affected and unaffected areas and were characterized by lymphocytic interface inflammation, subepidermal bullae, and individual keratinocyte vacuolation/necrosis. In the 12-week study, clinical effects in 2 animals resolved with continued dosing, whereas in others the inflammatory process progressed with 1 female exhibiting systemic lymphocytic inflammation in multiple tissues. The inflammatory infiltrate consisted of CD3 and CD4 positive T lymphocytes with minimal CD68 positive macrophages and only rare CD8 positive T lymphocytes. A subset of animals given a dosing holiday was subsequently rechallenged with similar lesions developing but with a more rapid clinical onset. These skin lesions were consistent with type-IV delayed hypersensitivity with some features comparable to bullous drug eruptions in humans. A relationship between these findings and the intended mode of action for these compounds could not be ruled out, given the occurrence across different chemotypes.

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.

GB1 hairpin kinetics: capturing the folding pathway with molecular dynamics, replica exchange and optimal dimensionality reduction.

We have performed molecular dynamics (MD) and replica-exchange (REMD) simulations of folding of the GB1 hairpin peptide in aqueous solution. REMD results were consistent with a cooperative zipper folding model. 120 µs MD trajectories at 320 K yielded relaxation times of 1.8 µs and 100 ns, with the slower assigned to global folding. The MD folding/unfolding transitions also followed the cooperative zipper model, specifying nucleation at the central turn followed by consecutive hydrogen bond formation. Formation of hydrogen bonds and hydrophobic contacts were highly correlated. Coarse-grained kinetic models constructed with the Optimal Dimensionality Reduction (ODR) approach found a folding time of 3.3 µs and unfolding time of 4.0 µs. Additionally, relaxation times in the 130-170 ns range could be assigned to formation of the transition state and off-path intermediates. The unfolded state was the most highly populated and, significantly, most heterogenous, assembling the largest number of microstates, primarily composed of extended and turn structures. The folded state was also heterogenous, but a to a lesser degree, involving the fully folded and partially folded in-register hairpins at early stages of the zipper pathway. The transition state corresponded to the nucleated hairpin, with central turn and first beta-sheet hydrogen bond, while the off-path intermediates were off-register partial hairpins. Our simulation results were in excellent agreement with experimental data on folded fraction, relaxation time and folding mechanism. The new findings from this work suggest a highly cooperative zipper folding mechanism, nascent hairpin transition state and ∼100 ns relaxation related to intermediate formation.Communicated by Ramaswamy H. Sarma.

Chemotherapy in pediatric brain tumor and the challenge of the blood-brain barrier.

BACKGROUND: Pediatric brain tumors (PBT) stand as the leading cause of cancer-related deaths in children. Chemoradiation protocols have improved survival rates, even for non-resectable tumors. Nonetheless, radiation therapy carries the risk of numerous adverse effects that can have long-lasting, detrimental effects on the quality of life for survivors. The pursuit of chemotherapeutics that could obviate the need for radiotherapy remains ongoing. Several anti-tumor agents, including sunitinib, valproic acid, carboplatin, and panobinostat, have shown effectiveness in various malignancies but have not proven effective in treating PBT. The presence of the blood-brain barrier (BBB) plays a pivotal role in maintaining suboptimal concentrations of anti-cancer drugs in the central nervous system (CNS). Ongoing research aims to modulate the integrity of the BBB to attain clinically effective drug concentrations in the CNS. However, current findings on the interaction of exogenous chemical agents with the BBB remain limited and do not provide a comprehensive explanation for the ineffectiveness of established anti-cancer drugs in PBT. METHODS: We conducted our search for chemotherapeutic agents associated with the blood-brain barrier (BBB) using the following keywords: Chemotherapy in Cancer, Chemotherapy in Brain Cancer, Chemotherapy in PBT, BBB Inhibition of Drugs into CNS, Suboptimal Concentration of CNS Drugs, PBT Drugs and BBB, and Potential PBT Drugs. We reviewed each relevant article before compiling the information in our manuscript. For the generation of figures, we utilized BioRender software. FOCUS: We focused our article search on chemical agents for PBT and subsequently investigated the role of the BBB in this context. Our search criteria included clinical trials, both randomized and non-randomized studies, preclinical research, review articles, and research papers. FINDING: Our research suggests that, despite the availability of potent chemotherapeutic agents for several types of cancer, the effectiveness of these chemical agents in treating PBT has not been comprehensively explored. Additionally, there is a scarcity of studies examining the role of the BBB in the suboptimal outcomes of PBT treatment, despite the effectiveness of these drugs for other types of tumors.

An evaluation of using rat-derived single-dose neuropharmacokinetic parameters to project accurately large animal unbound brain drug concentrations.

Previous publications suggest that interstitial fluid compound concentrations (C(ISF)) best determine quantitative neurotherapeutic pharmacology relationships, although confirming large animal C(ISF) remains elusive. Therefore, this work primarily evaluated using respective acute dose, rat-derived unbound brain compound concentration-to-unbound plasma compound concentration ratios (C(b,u)/C(p,u)) to project accurately dog and nonhuman primate (nhp) C(b,u), a C(ISF) surrogate, from measured C(p,u) for the highly permeable non-P-glycoprotein substrates N-{(3R,4S)-3-[4-(5-cyano-2-thienyl)phenyl]tetrahydro-2H-pyran-4-yl}propane-2-sulfonamide (PF-4778574) and [4-chloro-5-fluoro-2-(3-methoxy-2-methyl-phenoxy)-benzyl]-methylamine (CE-157119) and the P-glycoprotein substrates risperidone and 9-hydroxyrisperidone. First, in rats, it was determined for eight of nine commercial compounds that their single-dose-derived C(b,u)/C(p,u) were ≤2.5-fold different from their steady-state values; for all nine drugs, their C(b,u)/C(p,u) were ≤2.5-fold different from their steady-state C(ISF)/C(p,u) (Drug Metab Dispos 37:787-793, 2009). Subsequently, PF-4778574, CE-157119 and risperidone underwent rat, dog, and nhp neuropharmacokinetics studies. In large animals at each measured C(p,u), the methodology adequately predicted [estimated mean (95% confidence interval) of 1.02 (0.80, 1.29)] the observed C(b,u) for PF-4778574 and CE-157119 but underpredicted [0.17 (0.12, 0.22)] C(b,u) for risperidone and 9-hydroxyrisperidone. The data imply that forecasting higher species C(b,u) from a measured C(p,u) and rat acute dose-determined C(b,u):C(p,u) is of high confidence for nonefflux transporter substrates that show net passive diffusion (PF-4778574) or net active influx (CE-157119) at the blood-brain barrier in rats. However, this methodology appears ineffective for correctly predicting large animal C(b,u) for P-glycoprotein substrates (risperidone and 9-hydroxyrisperidone) because of their apparently much greater C(p,u)-favoring C(b,u):C(p,u) asymmetry in rats versus dogs or nhp. Instead, for such P-glycoprotein substrates, large animal-specific cerebrospinal fluid compound concentrations (C(CSF)) seemingly best represent C(b,u).

Phosphodiesterase type 5 (PDE5) inhibition improves object recognition memory: indications for central and peripheral mechanisms.

A promising target for memory improvement is phosphodiesterase type 5 (PDE5), which selectively hydrolyzes cyclic guanosine monophosphate (cGMP). In rodents, PDE5 inhibitors (PDE5-Is) have been shown to improve memory performance in many behavioral paradigms. However, it is questioned whether the positive effects in animal studies result from PDE5 inhibition in the central nervous system or the periphery. Therefore, we studied the effects of PDE5 inhibition on memory and determined whether compound penetration of the blood-brain barrier (BBB) is required for this activity. Two selective PDE5-Is, vardenafil and UK-343,664, were tested in the object recognition task (ORT) in both a MK-801- and scopolamine-induced memory deficit model, and a time-delay model without pharmacological intervention. Compounds were dosed 30 min before the learning trial of the task. To determine if the PDE5-Is crossed the BBB, their concentrations were determined in plasma and brain tissue collected 30 min after oral administration. Vardenafil improved object recognition memory in all three variants of the ORT. UK-343,664 was ineffective at either preventing MK-801-induced memory disruption or time-dependent memory decay. However, UK-343,664 attenuated the memory impairment of scopolamine. Vardenafil crossed the BBB whereas UK-343,664 did not. Further, co-administration of UK-343,664 and scopolamine did not alter the brain partitioning of either molecule. This suggests that the positive effect of UK-343,664 on scopolamine-induced memory decay might arise from peripheral PDE5 inhibition. The results herein suggest that there may be multiple mechanisms that mediate the efficacy of PDE5 inhibition to improve memory performance in tasks such as the ORT and that these involve PDE5 located both within and outside of the brain. To further elucidate the underlying mechanisms, the cellular and subcellular localization of PDE5 needs to be determined.

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 (C(AE) ), the adequacy of separation between the maximal total plasma compound concentration (C(max) ) 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 compound's anticipated clinically efficacious dose, human C(max) 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 C(max) variability (i.e. the 95th percentile C(max) did not breach C(AE) ). 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.

The Use of Renal Biomarkers in Pediatric Cardiac Patients With Acute Kidney Injury.

Acute kidney injury (AKI) is a common and serious condition that occurs in approximately 30% to 50% of pediatric patients that undergo cardiac surgery. Currently used parameters to measure kidney function (serum creatinine and urine output) are often unreliable and delay the prediction of AKI, despite their adoption into clinical guidelines. Emerging evidence suggests that biomarkers such as neutrophil gelatinase-associated lipocalin, cystatin C, interleukin-18, kidney injury molecule 1, and liver-type fatty acid- binding protein may be useful in the identification and location of pediatric renal injury. Ontogeny-related changes in tubular function and nephrogenesis result in reference values that differ based on age and sex. In addition, changes in endogenous concentrations may result from factors such as cardiopulmonary bypass. The use of urine samples to measure renal biomarkers offers a significant advantage compared with routine blood sampling, especially in the neonatal patient population. Future research is warranted to determine age-dependent changes in AKI biomarkers and the relationship with pharmacokinetic clearance of commonly used medications in the postoperative cardiac patient.

Antimicrobial prescribing for treatment of serious infections caused by Staphylococcus aureus and methicillin-resistant Staphylococcus aureus in pediatrics: an expert review.

Introduction: Staphylococcus aureus, including methicillin-resistant S. aureus (MRSA), remains a significant pathogen in children. Despite evidence of decreasing prevalence, MRSA bacteremia has been closely associated with complications, including certain infections (i.e. musculoskeletal and endovascular) linked to increased treatment failures.Areas covered: This expert review summarized recent published literature on the role of treatment, dosing and administration of antibiotics used to combat serious S. aureus infections in children. The pertinent antibiotics presented were vancomycin, oxazolidinones, semi-synthetic glycopeptides, daptomycin, tigecycline, novel cephalosporins, fosfomycin and lefamulin. Vancomycin has been the most commonly used antibiotic in empiric therapy for serious MRSA infection, with new key recommendations emphasizing a different approach to dosing and therapeutic monitoring. For other antibiotics, data remain limited or clinical trials are underway.Expert opinion: MRSA remains a significant pathogen in the pediatric population. As numerous therapeutic agents are available, many agents have limited data on usage in pediatric patients. Future studies require pharmacokinetic, safety and efficacy studies in pediatric patients to ensure appropriate therapeutic treatment and outcomes. Phage therapy has been used to treat deep-seated MRSA infections and is an emerging investigational treatment option.

Species differences in the biotransformation of an alpha 4 beta 2 nicotinic acetylcholine receptor partial agonist: the effects of distinct glucuronide metabolites on overall compound disposition.

The metabolism and disposition of (1R,5S)-2,3,4,5-tetrahydro-7-(trifluoromethyl)-1,5-methano-1H-3-benzazepine (1), an alpha(4)beta(2) nicotinic acetylcholine receptor partial agonist, was investigated in Sprague-Dawley rats and cynomolgus monkeys receiving (1R,5S)-2,3,4,5-tetrahydro-7-(trifluoromethyl)-1,5-methano-1H-4[(14)C]-3- benzazepine hydrochloride ([(14)C]1) orally. Although both species chiefly (>or=62%) cleared 1 metabolically, species-specific dispositional profiles were observed for both 1 and total radioactivity. Radioactivity was excreted equally in the urine and feces of intact rats but largely (72%) in bile in bile duct-cannulated animals. In monkeys, radioactivity recoveries were 50-fold greater in urine than feces and minimal (<5%) in bile. Both species metabolized 1 similarly: four-electron oxidation to one of four amino acids or two lactams (minor) and glucuronide formation (major). In rats, the latter pathway predominantly formed an N-carbamoyl glucuronide (M6), exclusively present in bile (69% of dose), whereas in monkeys it afforded an N-O-glucuronide (M5), a minor biliary component (4%) but the major plasma (62%) and urinary (42%) entity. In rats, first-pass hepatic conversion of 1 to M6, which was confirmed in rat hepatocytes, and its biliary secretion resulted in the indirect enterohepatic cycling of 1 via M6 and manifested in double-humped plasma concentration-time curves and long t(1/2) for both 1 and total radioactivity. In monkeys, in which only M5 was formed, double-humped plasma concentration-time curves were absent, and moderate t(1/2) for both 1 and total radioactivity were observed. A seemingly subtle, yet critical, difference in the chemical structures of these two glucuronide metabolites considerably affected the overall disposition of 1 in rats versus monkeys.

Biotransformation of an alpha4beta2 nicotinic acetylcholine receptor partial agonist in sprague-dawley rats and the dispositional characterization of its N-carbamoyl glucuronide metabolite.

The metabolism and disposition of (1S,5R)-2,3,4,5-tetrahydro-7-(trifluoromethyl)-1,5-methano-1H-3-benzazepine (1), an alpha(4)beta(2) nicotinic acetylcholine receptor partial agonist, was determined in Sprague-Dawley rats after oral administration of [(14)C]1. In intact animals, mass balance was achieved within 48 h, with 5 times more radioactivity excreted in urine than in feces. Compound 1 underwent renal and metabolic clearance equally and exhibited a very long half-life attributable to a secondary peak occurring 8 h postdose in its serum concentration-time curve. In bile duct-cannulated (BDC) rats, mass balance was also achieved within 48 h with 73.7, 23.4, and 5.5% of the dose detected in bile, urine, and feces, respectively. Rats metabolized 1 by two primary routes: four-electron oxidation to either four amino acids or a lactam and formation of an N-carbamoyl glucuronide (M6), which was only detected in bile. The presence of M6 solely in bile and the double-humped serum concentration-time curve of 1 suggested the indirect enterohepatic cycling of 1 via M6 after oral administration. To explore this mechanistic hypothesis further, intravenous studies were conducted with 1 in both intact and BDC rats to determine the extent of 1 undergoing indirect enterohepatic cycling via M6. Compared with the pharmacokinetics in intact rats, total serum clearance was higher (1.7-fold) and volume of distribution was lower (1.6-fold) in BDC rats, resulting in a correspondingly shorter (2.5-fold) half-life, with 56% of administered 1 undergoing recirculation, an amount consistent with that (68% of dose) of M6 observed in bile from rats dosed orally with [(14)C]1.

Pharmacological evaluation of clinically relevant concentrations of (2R,6R)-hydroxynorketamine.

Ketamine is a rapid-onset antidepressant whose efficacy long outlasts its pharmacokinetics. Multiple studies suggest ketamine's antidepressant effects require increased α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR)-dependent currents, which have recently been exclusively attributed to its N-methyl-d-aspartate receptor-inactive metabolite (2R,6R)-hydroxynorketamine ((2R,6R)-HNK). To investigate this AMPAR-activation claim further, we estimated and evaluated preclinically and clinically relevant unbound brain HNK concentrations (Cb,u). (2S,6S)-HNK and (2R,6R)-HNK were novelly synthesized, and their neuropharmacokinetic profiles were determined to project relevant Cb,u. Using concentrations (0.01-10 µM) bracketing the pertinent cross-species Cb,u, both compounds' AMPAR modulation was assessed in vitro by electrophysiological recordings and GluA1 surface expression. Neither (2S,6S)-HNK nor (2R,6R)-HNK bound orthosterically to or directly functionally activated AMPARs. (2R,6R)-HNK failed to evoke AMPAR-centric changes in any electrophysiological endpoint from adult rodent hippocampal slices. Conversely, time- and concentration-dependent increases in GluA1 expression occurred only with (2R,6R)-HNK (≥0.1 µM at ≥90 min). The (2R,6R)-HNK concentrations that increased GluA1 expression are consistent with its maximal Cb,u (0.92-4.84 µM) at reportedly efficacious doses of ketamine or (2R,6R)-HNK in mouse depression models, but ≥3-fold above its projected maximal human Cb,u (≤37.8 ±â€¯14.3 nM) following ketamine's clinically antidepressant infusion. These findings provide insight into the observed AMPAR-affecting (2R,6R)-HNK concentrations versus its exposures attained clinically at an antidepressant ketamine dose. To optimize any clinical study with (2R,6R)-HNK to fully assess its translational pharmacology, future preclinical work should test (2R,6R)-HNK concentrations and/or Cb,u of 0.01-0.1 µM to parallel its projected human Cb,u at a clinically antidepressant ketamine dose.

Metabolism and disposition of a gamma-aminobutyric acid type A receptor partial agonist in humans.

The metabolism and disposition of N-[3-fluoro-4-[2-(propylamino)ethoxy]phenyl]-4,5,6,7-tetrahydro-4-oxo-1H-indole-3-carboxamide (1), a potent subtype-selective partial agonist at the gamma-aminobutyric acid type A receptor complex, were elucidated in humans following a p.o. dose of N-[3-fluoro-4-[2-(propylamino)ethoxy]phenyl]-4,5,6,7-tetrahydro-4-oxo-1H-[3-(14)C]indole-3-carboxamide monomethane-sulfonate ([(14)C]1). Overall, 1 was well tolerated, with approximately twice as much radioactivity excreted in feces (64.8 +/- 13.3%) as in urine (28.4 +/- 8.8%). Across subjects, the oral clearance of 1 was composed of both renal (10%) and metabolic (< or =90%) components, with the biotransformation of 1 happening predominately via oxidative deamination to either 2-fluoro-4-[(4-oxo-4,5,6,7-tetrahydro-1H-indole-3-carbonyl)-amino]-phenoxy acetic acid (2) or 4-oxo-4,5,6,7-tetrahydro-1H-indole-3-carboxylic acid [3-fluoro-4-(2-hydroxy-ethoxy)-phenyl]-amide (3) and minimally by aliphatic hydroxylation and carbamate formation. Active renal secretion of 1 was observed as its unbound renal clearance was 6-fold greater than the glomerular filtration rate. Experiments using human hepatic in vitro systems were undertaken to better understand the enzyme(s) involved in the clinically observed oxidative biotransformation pathways. N-Dealkylation of 1, the principal metabolic route observed in vivo, was found to be predominately monoamine oxidase-B-mediated with the resulting putative aldehyde intermediate undergoing subsequent oxidation to 2 or reduction to 3.

Reverse and Forward Translational Neuropharmacology in Psychiatric Drug Discovery.

The probability of achieving marketing approval of a novel therapeutic for psychiatric indications is extremely low due largely to the inability to demonstrate durable and reproducible efficacy in phase II trials and beyond. These failures are often attributed to the lack of translation of the underlying neuropharmacology from animal model(s) to the disease population. However, how assured is such a conclusion considering the clinical efficacy path rarely meticulously parallels the preclinical experiment(s) that underwrote it?

Prediction of Human Brain Penetration of P-glycoprotein and Breast Cancer Resistance Protein Substrates Using In Vitro Transporter Studies and Animal Models.

Four P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) substrates with human cerebrospinal fluid (CSF) concentrations and preclinical neuropharmacokinetics were used to assess in vitro-in vivo extrapolation of brain penetration in preclinical species and the ability to predict human brain penetration. Unbound brain (Cb,u), unbound plasma (Cp,u), and CSF compound concentrations (CCSF) were measured in rats and nonhuman primates (NHPs), and the unbound partition coefficients (Cb,u/Cp,u and CCSF/Cp,u) were used to assess brain penetration. The results indicated that for P-gp and BCRP dual substrates, brain penetration was severally impaired in all species. In comparison, for P-gp substrates that are weak or non-BCRP substrates, improved brain penetration was observed in NHPs and humans than in rats. Overall, NHP appears to be more predictive of human brain penetration for P-gp substrates with weak or no interaction with BCRP than rat. Although CCSF does not quantitatively correspond to Cb,u for efflux transporter substrates, it is mostly within 3-fold higher of Cb,u in rat and NHP, suggesting that CCSF can be used as a surrogate for Cb,u. Taken together, a holistic approach including both in vitro transporter and in vivo neuropharmacokinetics data enables a better estimation of human brain penetration of P-gp/BCRP substrates.

Discovery of Trifluoromethyl Glycol Carbamates as Potent and Selective Covalent Monoacylglycerol Lipase (MAGL) Inhibitors for Treatment of Neuroinflammation.

Monoacylglycerol lipase (MAGL) inhibition provides a potential treatment approach to neuroinflammation through modulation of both the endocannabinoid pathway and arachidonoyl signaling in the central nervous system (CNS). Herein we report the discovery of compound 15 (PF-06795071), a potent and selective covalent MAGL inhibitor, featuring a novel trifluoromethyl glycol leaving group that confers significant physicochemical property improvements as compared with earlier inhibitor series with more lipophilic leaving groups. The design strategy focused on identifying an optimized leaving group that delivers MAGL potency, serine hydrolase selectivity, and CNS exposure while simultaneously reducing log  D, improving solubility, and minimizing chemical lability. Compound 15 achieves excellent CNS exposure, extended 2-AG elevation effect in vivo, and decreased brain inflammatory markers in response to an inflammatory challenge.

Metabolism of a 14C/3H-labeled GABAA receptor partial agonist in rat, dog and human liver microsomes: evaluation of a dual-radiolabel strategy.

The metabolism of 2-{[2-(3-fluoropyrid-2-yl)-1H-imidazol-1-yl]methyl}-1-propyl-5-cyano-1H-benzimidazole (1), a potent subtype-selective GABA(A) receptor partial agonist, was investigated in rat, dog and human liver microsomes. Due to its significant metabolic cleavage at C(8) observed in preliminary biotransformation studies with non-radiolabeled 1, both [(14)C]1 and [(3)H]1 were synthesized with respective radioisotopes placed on either side of C(8) to determine if all microsomal metabolites formed after C(8)N-dealkylation of 1 (or its core-intact metabolites) could be detected and quantified adequately. Both radiolabeled forms of 1, used separately in mono-radiolabel studies in cross-species microsomes and concomitantly in dual-radiolabel studies in rat microsomes, permitted the detection and quantification of all metabolites of 1, and a combination of radioactive and mass spectral data allowed structural elucidation of its Phase I metabolites. As expected, the sum of (14)C-only metabolites equaled that of (3)H-only metabolites in all incubations. In-line radiometric analysis worked extremely well (and was very reproducible) for quantifying either (14)C- or (3)H-compounds within separate incubations when using mono-radiolabeled 1. However, although the in-line radiodetector provided a comprehensive qualitative metabolic profile using dual-radiolabled 1, its inability to exclude completely (14)C- from (3)H-generated counts caused a degree of ambiguity pertaining to metabolite quantification. Thus, off-line liquid scintillation counting of collected dual-radiolabeled incubation LC-fractions was employed to quantify both (14)C- and (3)H-metabolites simultaneously, while in-line radiodetection was only used for qualitative analyses accompanying MS and MS/MS experiments. These studies demonstrated the analytical feasibility of using a dual-radiolabel approach for subsequent in vivo ADME studies with 1.