Neural signatures of sleep in zebrafish.

Slow-wave sleep and rapid eye movement (or paradoxical) sleep have been found in mammals, birds and lizards, but it is unclear whether these neuronal signatures are found in non-amniotic vertebrates. Here we develop non-invasive fluorescence-based polysomnography for zebrafish, and show-using unbiased, brain-wide activity recording coupled with assessment of eye movement, muscle dynamics and heart rate-that there are at least two major sleep signatures in zebrafish. These signatures, which we term slow bursting sleep and propagating wave sleep, share commonalities with those of slow-wave sleep and paradoxical or rapid eye movement sleep, respectively. Further, we find that melanin-concentrating hormone signalling (which is involved in mammalian sleep) also regulates propagating wave sleep signatures and the overall amount of sleep in zebrafish, probably via activation of ependymal cells. These observations suggest that common neural signatures of sleep may have emerged in the vertebrate brain over 450 million years ago.

Moving beyond Binary Predictions of Human Drug-Induced Liver Injury (DILI) toward Contrasting Relative Risk Potential.

The hepatic risk matrix (HRM) was developed and used to differentiate lead clinical and back-up drug candidates against competitor/marketed drugs within the same pharmaceutical class for their potential to cause human drug-induced liver injury (DILI). The hybrid HRM scoring system blends physicochemical properties (Rule of Two Model: dose and lipophilicity or Partition Model: dose, ionization state, lipophilicity, and fractional carbon bond saturation) with common toxicity mechanisms (cytotoxicity, mitochondrial dysfunction, and bile salt export pump (BSEP) inhibition) that promote DILI. HRM scores are based on bracketed safety margins (<1, 1-10, 10-100, and >100× clinical Cmax,total). On the basis of well-established clinical safety experience of marketed/withdrawn drug candidates, the background analysis consists of 200 drugs from the Liver Toxicity Knowledge Base annotated as Most-DILI- (79), Less-DILI- (56), No-DILI- (47), and Ambiguous-DILI-concern (18) drugs. Scores were generated for over 21 internal and 7 external drug candidates discontinued for unacceptable incidence/magnitude of liver transaminase elevations during clinical trials or withdrawn for liver injury severity. Both hybrid scoring systems identified 70-80% Most-DILI-concern drugs, but more importantly, stratified successful/unsuccessful drug candidates for liver safety (incidence/severity of transaminase elevations and approved drug labels). Incorporating other mechanisms (reactive metabolite and cytotoxic metabolite generation and hepatic efflux transport inhibition, other than BSEP) to the HRM had minimal beneficial impact in DILI prediction/stratification. As is, the hybrid scoring system was positioned for portfolio assessments to contrast DILI risk potential of small molecule drug candidates in early clinical development. This stratified approach for DILI prediction aided decisions regarding drug candidate progression, follow-up mechanistic work, back-up selection, clinical dose selection, and due diligence assessments in favor of compounds with less implied clinical hepatotoxicity risk.

Clearance Prediction of Targeted Covalent Inhibitors by In Vitro-In Vivo Extrapolation of Hepatic and Extrahepatic Clearance Mechanisms.

The concept of target-specific covalent enzyme inhibitors appears attractive from both an efficacy and a selectivity viewpoint considering the potential for enhanced biochemical efficiency associated with an irreversible mechanism. Aside from potential safety concerns, clearance prediction of covalent inhibitors represents a unique challenge due to the inclusion of nontraditional metabolic pathways of direct conjugation with glutathione (GSH) or via GSH S-transferase-mediated processes. In this article, a novel pharmacokinetic algorithm was developed using a series of Pfizer kinase selective acrylamide covalent inhibitors based on their in vitro-in vivo extrapolation of systemic clearance in rats. The algorithm encompasses the use of hepatocytes as an in vitro model for hepatic clearance due to oxidative metabolism and GSH conjugation, and the use of whole blood as an in vitro surrogate for GSH conjugation in extrahepatic tissues. Initial evaluations with clinical covalent inhibitors suggested that the scaling algorithm developed from rats may also be useful for human clearance prediction when species-specific parameters, such as hepatocyte and blood stability and blood binding, were considered. With careful consideration of clearance mechanisms, the described in vitro-in vivo extrapolation approach may be useful to facilitate candidate optimization, selection, and prediction of human pharmacokinetic clearance during the discovery and development of targeted covalent inhibitors.

Evaluation of a New Molecular Entity as a Victim of Metabolic Drug-Drug Interactions-an Industry Perspective.

Under the guidance of the International Consortium for Innovation and Quality in Pharmaceutical Development (IQ), scientists from 20 pharmaceutical companies formed a Victim Drug-Drug Interactions Working Group. This working group has conducted a review of the literature and the practices of each company on the approaches to clearance pathway identification (fCL), estimation of fractional contribution of metabolizing enzyme toward metabolism (fm), along with modeling and simulation-aided strategy in predicting the victim drug-drug interaction (DDI) liability due to modulation of drug metabolizing enzymes. Presented in this perspective are the recommendations from this working group on: 1) strategic and experimental approaches to identify fCL and fm, 2) whether those assessments may be quantitative for certain enzymes (e.g., cytochrome P450, P450, and limited uridine diphosphoglucuronosyltransferase, UGT enzymes) or qualitative (for most of other drug metabolism enzymes), and the impact due to the lack of quantitative information on the latter. Multiple decision trees are presented with stepwise approaches to identify specific enzymes that are involved in the metabolism of a given drug and to aid the prediction and risk assessment of drug as a victim in DDI. Modeling and simulation approaches are also discussed to better predict DDI risk in humans. Variability and parameter sensitivity analysis were emphasized when applying modeling and simulation to capture the differences within the population used and to characterize the parameters that have the most influence on the prediction outcome.

Apical migration of nuclei during G2 is a prerequisite for all nuclear motion in zebrafish neuroepithelia.

Nuclei in the proliferative pseudostratified epithelia of vastly different organisms exhibit a characteristic dynamics - the so-called interkinetic nuclear migration (IKNM). Although these movements are thought to be intimately tied to the cell cycle, little is known about the relationship between IKNM and distinct phases of the cell cycle and the role that this association plays in ensuring balanced proliferation and subsequent differentiation. Here, we perform a quantitative analysis of modes of nuclear migration during the cell cycle using a marker that enables the first unequivocal differentiation of all four phases in proliferating neuroepithelial cells in vivo. In zebrafish neuroepithelia, nuclei spend the majority of the cell cycle in S phase, less time in G1, with G2 and M being noticeably shorter still in comparison. Correlating cell cycle phases with nuclear movements shows that IKNM comprises rapid apical nuclear migration during G2 phase and stochastic nuclear motion during G1 and S phases. The rapid apical migration coincides with the onset of G2, during which we find basal actomyosin accumulation. Inhibiting the transition from G2 to M phase induces a complete stalling of nuclei, indicating that IKNM and cell cycle continuation cannot be uncoupled and that progression from G2 to M is a prerequisite for rapid apical migration. Taken together, these results suggest that IKNM involves an actomyosin-driven contraction of cytoplasm basal to the nucleus during G2, and that the stochastic nuclear movements observed in other phases arise passively due to apical migration in neighboring cells.

Disposition and metabolic profiling of [(14)C]cerlapirdine using accelerator mass spectrometry.

Cerlapirdine (SAM-531, PF-05212365) is a selective, potent, full antagonist of the 5-hydroxytryptamine 6 (5-HT6) receptor. Cerlapirdine and other 5-HT6 receptor antagonists have been in clinical development for the symptomatic treatment of Alzheimer's disease. A human absorption, distribution, metabolism, and excretion study was conducted to gain further understanding of the metabolism and disposition of cerlapirdine. Because of the low amount of radioactivity administered, total (14)C content and metabolic profiles in plasma, urine, and feces were determined using accelerator mass spectrometry (AMS). After a single, oral 5-mg dose of [(14)C]cerlapirdine (177 nCi), recovery of total (14)C was almost complete, with feces being the major route of elimination of the administered dose, whereas urinary excretion played a lesser role. The extent of absorption was estimated to be at least 70%. Metabolite profiling in pooled plasma samples showed that unchanged cerlapirdine was the major drug-related component in circulation, representing 51% of total (14)C exposure in plasma. One metabolite (M1, desmethylcerlapirdine) was detected in plasma, and represented 9% of the total (14)C exposure. In vitro cytochrome P450 reaction phenotyping studies showed that M1 was formed primarily by CYP2C8 and CYP3A4. In pooled urine samples, three major drug-related peaks were detected, corresponding to cerlapirdine-N-oxide (M3), cerlapirdine, and desmethylcerlapirdine. In feces, cerlapirdine was the major (14)C component excreted, followed by desmethylcerlapirdine. The results of this study demonstrate that the use of the AMS technique enables comprehensive quantitative elucidation of the disposition and metabolic profiles of compounds administered at a low radioactive dose.

Slit1b-Robo3 signaling and N-cadherin regulate apical process retraction in developing retinal ganglion cells.

When neurons exit the cell cycle after their terminal mitosis, they detach from the apical surface of the neuroepithelium. Despite the fact that this detachment is crucial for further neurogenesis and neuronal migration, the underlying mechanisms are still not understood. Here, taking advantage of the genetics and imaging possibilities of the zebrafish retina as a model system, we show by knockdown experiments that the guidance molecule Slit1b and its receptor Robo3 are required for apical retraction of retinal ganglion cells (RGCs). In contrast, N-cadherin seems to be responsible for maintenance of apical attachment, as expression of dominant-negative N-cadherin causes RGCs to lose apical attachments prematurely and rescues retraction in slit1b morphants. These results suggest that Slit-Robo signaling downregulates N-cadherin activity to allow apical retraction in newly generated RGCs.

Drug metabolites as cytochrome p450 inhibitors: a retrospective analysis and proposed algorithm for evaluation of the pharmacokinetic interaction potential of metabolites in drug discovery and development.

Understanding drug-drug interactions (DDIs) is a key component of clinical practice ensuring patient safety and efficacy of medicines. The role of drug metabolites in DDIs is a developing area of science, and has been recently highlighted in a draft regulatory guidance. The guidance states that metabolites representing ≥25% of the parent drug's area under the plasma concentration/time curve and/or >10% of exposure of total drug-related material should trigger in vitro characterization of metabolites for cytochrome P450 inhibition and propensity for DDIs. The relationship between in vitro cytochrome P450 inhibitory potency, systemic exposure, and DDI potential of drug metabolites was examined using the Pfizer development database to identify compounds with pre-existing in vivo biotransformation data, where circulating metabolites were identified in humans. The database yielded 33 structurally diverse compounds with collectively 115 distinct circulating metabolites. Of these, 52% (60/115) achieved exposures >25% of parent drug levels as judged from mass balance/metabolite identification studies. It was noted that 14 metabolite standards for 12 parent drugs had been synthesized, monitored in clinical studies, and examined for cytochrome P450 inhibition. For the 14 metabolite/parent drug pairs, no clinically relevant DDIs were expected to occur against the major human cytochrome P450 isoforms. A review of the literature for parent/metabolite DDI information was also conducted to examine trends using a larger data set. Leveraging the analysis of both internal and literature-based data sets, an algorithm was devised for use in drug discovery/early development to assess cytochrome P450 inhibitory potential of drug metabolites and the propensity to cause a clinically relevant DDI.

Cerebrospinal fluid ß-Amyloid turnover in the mouse, dog, monkey and human evaluated by systematic quantitative analyses.

BACKGROUND: Reducing brain ß-amyloid (Aß) via inhibition of ß-secretase, or inhibition/modulation of γ-secretase, has been widely pursued as a potential disease-modifying treatment for Alzheimer's disease. Compounds that act through these mechanisms have been screened and characterized with Aß lowering in the brain and/or cerebrospinal fluid (CSF) as the primary pharmacological end point. Interpretation and translation of the pharmacokinetic (PK)/pharmacodynamic (PD) relationship for these compounds is complicated by the relatively slow Aß turnover process in these compartments. OBJECTIVE: To understand Aß turnover kinetics in preclinical species and humans. METHODS: We collected CSF Aß dynamic data after ß- or γ-secretase inhibitor treatment from in-house experiments and the public domain, and analyzed the data using PK/PD modeling to obtain CSF Aß turnover rates (kout) in the mouse, dog, monkey and human. RESULTS: The kout for CSF Aß40 follows allometry (kout = 0.395 × body weight(-0.351)). The kout for CSF Aß40 is approximately 2-fold higher than the turnover of CSF in rodents, but in higher species, the two are comparable. CONCLUSION: The turnover of CSF Aß40 was systematically examined, for the first time, in multiple species through quantitative modeling of multiple data sets. Our result suggests that the clearance mechanisms for CSF Aß in rodents may be different from those in the higher species. The understanding of Aß turnover has considerable implications for the discovery and development of Aß-lowering therapeutics, as illustrated from the perspectives of preclinical PK/PD characterization and preclinical-to-clinical translation.

Metabolic activation in drug-induced liver injury.

It is generally believed that metabolic bioactivation of drug molecules to form reactive metabolites, followed by their covalent binding to endogenous macromolecules, is one of the mechanisms that can lead to hepatotoxicity or idiosyncratic adverse drug reactions (IADRs). Although the role of bioactivation in drug-induced liver injury has been reasonably well established and accepted, and methodologies (e.g., structural alerts, reactive metabolite trapping, and covalent binding) continue to emerge in an attempt to detect the occurrence of bioactivation, the challenge remains to accurately predict the likelihood for idiosyncratic liver toxicity. Recent advances in risk-assessment methodologies, such as by the estimate of total body burden of covalent binding or by zone classification, taking the clinical dose into consideration, are positive steps toward improving risk assessment. The ability to better predict the potential of a drug candidate to cause IADRs will further be dependent upon a better understanding of the pathophysiological mechanisms of such reactions. Until a thorough understanding of the relationship between liver toxicity and the formation of reactive metabolites is achieved, it appears, at present, that the most practical strategy in drug discovery and development to reduce the likelihood of idiosyncratic liver toxicity via metabolic activation is to minimize or eliminate the occurrence of bioactivation and, at the same time, to maximize the pharmacological potency (to minimze the clinical dose) of the drug of interest.

Cerebrospinal fluid amyloid-ß (Aß) as an effect biomarker for brain Aß lowering verified by quantitative preclinical analyses.

Reducing the generation of amyloid-ß (Aß) in the brain via inhibition of ß-secretase or inhibition/modulation of γ-secretase has been pursued as a potential disease-modifying treatment for Alzheimer's disease. For the discovery and development of ß-secretase inhibitors (BACEi), γ-secretase inhibitors (GSI), and γ-secretase modulators (GSM), Aß in cerebrospinal fluid (CSF) has been presumed to be an effect biomarker for Aß lowering in the brain. However, this presumption is challenged by the lack of quantitative understanding of the relationship between brain and CSF Aß lowering. In this study, we strived to elucidate how the intrinsic pharmacokinetic (PK)/pharmacodynamic (PD) relationship for CSF Aß lowering is related to that for brain Aß through quantitative modeling of preclinical data for numerous BACEi, GSI, and GSM across multiple species. Our results indicate that the intrinsic PK/PD relationship in CSF is predictive of that in brain, at least in the postulated pharmacologically relevant range, with excellent consistency across mechanisms and species. As such, the validity of CSF Aß as an effect biomarker for brain Aß lowering is confirmed preclinically. Meanwhile, we have been able to reproduce the dose-dependent separation between brain and CSF effect profiles using simulations. We further discuss the implications of our findings to drug discovery and development with regard to preclinical PK/PD characterization and clinical prediction of Aß lowering in the brain.

Optimization of 5-vinylaryl-3-pyridinecarbonitriles as PKCtheta inhibitors.

Analog 8, a 3-pyridinecarbonitrile with an (E)-2-[6-[(4-methylpiperazin-1-yl)methyl]pyridin-2-yl]vinyl group at C-5, had an IC(50) value of 1.1 nM for the inhibition of PKCtheta and potently blocked the production of IL-2 in both stimulated murine T cells (IC(50)=34 nM) and human whole blood (IC(50)=500 nM).

Using a homology model of cytochrome P450 2D6 to predict substrate site of metabolism.

CYP2D6 is an important enzyme that is involved in first pass metabolism and is responsible for metabolizing ~25% of currently marketed drugs. A homology model of CYP2D6 was built using X-ray structures of ligand-bound CYP2C5 complexes as templates. This homology model was used in docking studies to rationalize and predict the site of metabolism of known CYP2D6 substrates. While the homology model was generally found to be in good agreement with the recently solved apo (ligand-free) X-ray structure of CYP2D6, significant differences between the structures were observed in the B' and F-G helical region. These structural differences are similar to those observed between ligand-free and ligand-bound structures of other CYPs and suggest that these conformational changes result from induced-fit adaptations upon ligand binding. By docking to the homology model using Glide, it was possible to identify the correct site of metabolism for a set of 16 CYP2D6 substrates 85% of the time when the 5 top scoring poses were examined. On the other hand, docking to the apo CYP2D6 X-ray structure led to a loss in accuracy in predicting the sites of metabolism for many of the CYP2D6 substrates considered in this study. These results demonstrate the importance of describing substrate-induced conformational changes that occur upon binding. The best results were obtained using Glide SP with van der Waals scaling set to 0.8 for both the receptor and ligand atoms. A discussion of putative binding modes that explain the distribution of metabolic sites for substrates, as well as a relationship between the number of metabolic sites and substrate size, are also presented. In addition, analysis of these binding modes enabled us to rationalize the typical hydroxylation and O-demethylation reactions catalyzed by CYP2D6 as well as the less common N-dealkylation.

NF-protocadherin and TAF1 regulate retinal axon initiation and elongation in vivo.

NF-protocadherin (NFPC)-mediated cell-cell adhesion plays a critical role in vertebrate neural tube formation. NFPC is also expressed during the period of axon tract formation, but little is known about its function in axonogenesis. Here we have tested the role of NFPC and its cytosolic cofactor template-activating factor 1 (TAF1) in the emergence of the Xenopus retinotectal projection. NFPC is expressed in the developing retina and optic pathway and is abundant in growing retinal axons. Inhibition of NFPC function in developing retinal ganglion cells (RGCs) severely reduces axon initiation and elongation and suppresses dendrite genesis. Furthermore, an identical phenotype occurs when TAF1 function is blocked. These data provide evidence that NFPC regulates axon initiation and elongation and indicate a conserved role for TAF1, a transcriptional regulator, as a downstream cytosolic effector of NFPC in RGCs.

C-5 substituted heteroaryl-3-pyridinecarbonitriles as PKCtheta inhibitors: part II.

We previously reported that a 3-pyridinecarbonitrile analog with a furan substituent at C-5 and a 4-methylindol-5-ylamino substituent at C-4, 1, was a potent inhibitor of PKCtheta (IC50=4.5 nM). Replacement of the C-5 furan ring of 1 with bicyclic heteroaryl rings, led to compounds with significantly improved potency against PKCtheta. Analog 6b with a 4-methylindol-5-ylamino group at C-4 and a 5-[(4-methylpiperazin-1-yl)methyl]-1-benzofuran-2-yl group at C-5 had an IC50 value of 0.28 nM for the inhibition of PKCtheta.

A single red InGaN-based light-emitting diode with a europium (III) ternary complex as mono-phosphor.

A novel europium (III) ternary complex, Eu(TPBDTFA)(3)Phen, was designed and synthesized. Photoluminescence measurements show that the energy absorbed by the organic ligands was efficiently transferred to the central Eu(3+) ions, and the complex exhibits strongly red emission due to the (5)D(0)-(7)F(j) transitions of Eu(3+) ions with appropriate CIE (Commission Internationale de l'Eclairage, International Commission on Illumination) chromaticity coordinates (x=0.66, y=0.33) under 310-420 nm light excitation. The luminescence quantum yield for the Eu(3+) complex is 0.18. Thermogravimetric analysis (TGA) confirms a high thermal stability of the complex with a decomposition temperature of 341 degrees C. All the characteristics indicate that the Eu(3+) complex is a highly efficient red phosphor suitable to be excited by near UV light. An intense red-emitting LED was fabricated by combining the mono-phosphor Eu(TPBDTFA)(3)Phen with a approximately 395 nm emitting InGaN chip.

Sleep: Short Sleepers Should Keep Count of Their Hypocretin Neurons.

Sleep durations vary greatly across animals from 2 to 20 hours with no clear explanation. A small Mexican cavefish reveals how the brain can adapt to increase its wake-stabilizing hypocretin circuit and dramatically reduce sleep, likely to allow adaptive foraging.

Identification of Cyanamide-Based Janus Kinase 3 (JAK3) Covalent Inhibitors.

Ongoing interest in the discovery of selective JAK3 inhibitors led us to design novel covalent inhibitors that engage the JAK3 residue Cys909 by cyanamide, a structurally and mechanistically differentiated electrophile from other cysteine reacting groups previously incorporated in JAK3 covalent inhibitors. Through crystallography, kinetic, and computational studies, interaction of cyanamide 12 with Cys909 was optimized leading to potent and selective JAK3 inhibitors as exemplified by 32. In relevant cell-based assays and in agreement with previous results from this group, 32 demonstrated that selective inhibition of JAK3 is sufficient to drive JAK1/JAK3-mediated cellular responses. The contribution from extrahepatic processes to the clearance of cyanamide-based covalent inhibitors was also characterized using metabolic and pharmacokinetic data for 12. This work also gave key insights into a productive approach to decrease glutathione/glutathione S-transferase-mediated clearance, a challenge typically encountered during the discovery of covalent kinase inhibitors.

Structure-based optimization of protein tyrosine phosphatase 1B inhibitors: from the active site to the second phosphotyrosine binding site.

Protein tyrosine phosphatase 1B (PTP1B) is a negative regulator of the insulin and leptin receptor pathways and thus an attractive therapeutic target for diabetes and obesity. Starting with a high micromolar lead compound, structure-based optimization of novel PTP1B inhibitors by extension of the molecule from the enzyme active site into the second phosphotyrosine binding site is described. Medicinal chemistry, guided by X-ray complex structure and molecular modeling, has yielded low nanomolar PTP1B inhibitors in an efficient manner. Compounds from this chemical series were found to be actively transported into hepatocytes. This active uptake into target tissues could be one of the possible avenues to overcome the poor membrane permeability of PTP1B inhibitors.