South Texas orbital fracture protocol for emergency department evaluation of orbital fractures.

PURPOSE: Facial trauma and orbital fractures are common reasons for ophthalmology consultation in the emergency department (ED). The purpose of this study is to assess intervention rates and evaluate the acuity of ophthalmology consultation for orbital fractures in the ED. BASIC PROCEDURES: A retrospective chart review of orbital fractures was conducted over a 23-month period. 379 cases of orbital fractures were identified in a single-center study. All patients that received an ophthalmology consultation in the ED were included. Demographics, mechanism and location of orbital fracture, ophthalmic complications, and surgical and non-surgical ophthalmic interventions were recorded. The primary study outcome was the rate of ophthalmic consultation and intervention with and without retrospective application of our proposed South Texas Orbital Fracture Protocol (STOP). RESULTS: Immediate ophthalmic intervention was performed in 18.7% of patients. Statistically significant subjective, radiographic, and physical exam features correlating with ophthalmic intervention were identified and included globe rupture, concern for entrapment, orbital roof fractures, and retrobulbar hematoma. Retrospective application of our proposed South Texas Orbital Fracture Protocol (STOP) would have resulted in 186 of 379 patients requiring ophthalmology consultation, thus reducing consultation rate by 51% with an improved rate of intervention from 18.7% to 37.6%. CONCLUSIONS: Orbital fractures can be associated with severe ocular complications. Most cases, however, do not require emergent evaluation by an ophthalmologist. We propose the South Texas Orbital Fracture Protocol (STOP) for proper assessment and triaging of orbital fractures in the ED. While this clinical decision-making tool requires validation, it may offer improved healthcare efficiency, reduced costs, fewer unnecessary inter-facility transfers, and less burnout for ophthalmology residents.

Acute Reconstruction of Periorbital Trauma Resulting in Eyelid Anterior Lamella Loss With Simultaneous Full-thickness Skin Grafting and Amniotic Membrane Grafting: A Case Report.

Complex facial lacerations are frequently encountered in the combat environment. Trauma with soft-tissue loss of the periorbital region offers particular challenges in terms of operative reconstruction. Cicatricial changes in the sub-acute phase can lead to eyelid malposition and lagophthalmos. The authors present a novel technique for acute reconstruction of periorbital trauma with eyelid soft-tissue loss with simultaneous full-thickness skin grafting and amniotic membrane grafting. The technique involves standard preparation of the surgical area of injury and infiltration with local anesthetic. Initially, the area of injury is copiously irrigated, and debridement of any necrotic tissue is accomplished. Amniotic membrane grafting is then performed over the defect. Approximately 2 mm × 2 mm full-thickness skin grafts are procured and distributed over the initial amniotic membrane graft. A second amniotic membrane graft is then secured over the skin graft-amniotic membrane graft complex with cyanoacrylate tissue adhesive. A bolstered suture tarsorrhaphy is performed to minimize tissue trauma during the healing process. The operative and postsurgical outcomes were assessed. The graft site healed well without cicatricial changes or lagophthalmos. Peripheral small papillomatous lesions did develop requiring excision for cosmesis, but ultimately the graft site demonstrated appropriate coverage and healthy re-epithelialization over the previous defect. This case demonstrates the viability of simultaneous full-thickness skin grafting with concomitant amniotic membrane grafting for the acute reconstruction of periorbital trauma with eyelid anterior lamella tissue loss. An excellent cosmetic and functional outcome was attained. By providing acute reconstruction, the risk of damage secondary to cicatricial periorbital changes may be avoided.

In vitro stability and in vivo pharmacokinetics of the novel ketogenic ester, bis hexanoyl (R)-1,3-butanediol.

A novel ketone ester, bis hexanoyl (R)-1,3-butanediol (BH-BD), has been developed as a means to elevate blood ketones, for use as an energy substrate and a signaling metabolite. The metabolism of BH-BD and its effects on blood beta-hydroxybutyrate (BHB) levels was evaluated in various in vitro matrices and through analysis of plasma collected from Sprague Dawley rats and C57/BL6 mice in two oral gavage studies. A well-characterized ketone ester, (R)-3-hydroxybutyl (R)-3-hydroxybutyrate (HB-BHB), was used as an active control throughout. In vitro assay results demonstrated that BH-BD likely remains intact in the stomach and is hydrolyzed in the small intestine into hexanoate and (R)-1,3-butanediol. If absorbed intact, BH-BD is subject to hydrolysis by non-CYP enzymes in liver and esterases in plasma. If BH-BD reaches the lower intestine it is metabolized by gut flora. Plasma BHB delivery increased in a dose-dependent manner in rats and mice following oral administration of BH-BD. All doses of BH-BD were well tolerated. At doses over 3 g/kg, BHB delivery was similar between BH-BD and HB-BHB. The results of these studies support the hydrolysis of BH-BD into hexanoate and (R)-1,3-butanediol which are metabolized into BHB, delivering a well-tolerated, sustained and dose-dependent increase in plasma BHB in rodents.

Modulating inhibitory response control through potentiation of GluN2D subunit-containing NMDA receptors.

NMDA receptors containing GluN2D subunits are expressed in the subthalamic nucleus and external globus pallidus, key nuclei of the indirect and hyperdirect pathways of the basal ganglia. This circuitry integrates cortical input with dopaminergic signaling to select advantageous behaviors among available choices. In the experiments described here, we characterized the effects of PTC-174, a novel positive allosteric modulator (PAM) of GluN2D subunit-containing NMDA receptors, on response control regulated by this circuitry. The indirect pathway suppresses less advantageous behavioral choices, a manifestation of which is suppression of locomotor activity in rats. Systemic administration of PTC-174 produced a dose-dependent reduction in activity in rats placed in a novel open field or administered the stimulants MK-801 or amphetamine. The hyperdirect pathway controls release of decisions from the basal ganglia to the cortex to optimize choice processing. Such response control was modeled in rats as premature responding in the 5-choice serial reaction time (5-CSRT) task. PTC-174 produced a dose-dependent reduction in premature responding in this task. These data suggest that potentiation of GluN2D receptor activity by PTC-174 facilitates the complex basal ganglia information processing that underlies response control. The behavioral effects occurred at estimated free PTC-174 brain concentrations predicted to induce 10-50% increases in GluN2D activity. The present findings suggest the potential of GluN2D PAMs to modulate basal ganglia function and to treat neurological disorders related to dysfunctional response control.

Posterior Segment Findings in Patients on Extracorporeal Membrane Oxygenation.

Purpose: Extracorporeal membrane oxygenation (ECMO) is an established treatment modality for critically ill patients with cardiopulmonary failure, yet little is known of the ocular pathology in this population. The aim of this study is to characterize the posterior segment findings of ECMO patients. Methods: This study is a retrospective analysis of 20 ECMO patients evaluated by ophthalmology from September 2012 to May 2019 at a level 1 trauma center. Comprehensive examinations assessed for intraocular pathology. Demographic data, exam findings, and mortality were analyzed. Results: The sample size consisted of 20 patients; a majority were male (75%), and mean age was 37.4 years (interquartile range, 26.75-50 years). All patients received ECMO for care of acute respiratory distress syndrome (ARDS). Average duration of ECMO therapy was 9.6 ± 6.5 days. Eleven (55%) patients had acute retinal pathology, including Purtscher-like retinopathy (20%), intraocular hemorrhage (50%), and septic chorioretinitis (bacterial or fungal, 10%). Location of hemorrhage included the retina (40%), vitreous (30%), and optic disc (15%). Sixty percent (n = 12) of patients were unable to provide a subjective history on initial assessment. Ultimately, 5 out of 20 patients (25%) died of systemic illness during their hospital stay. Conclusion: This study demonstrates high rates of retinal pathology, most commonly vitreous and/or retinal hemorrhage alongside a Purtscher-like retinopathy. This is likely secondary to complications of anticoagulation, microthrombi, septicemia, and hemodynamic instability. We found a mortality rate slightly lower than that of prior ECMO studies. Prospective studies with pre-ECMO and post-ECMO fundus photography is warranted for better understanding of these medically complex patients.

Circumpolar permafrost maps and geohazard indices for near-future infrastructure risk assessments.

Ongoing climate change is causing fundamental changes in the Arctic, some of which can be hazardous to nature and human activity. In the context of Earth surface systems, warming climate may lead to rising ground temperatures and thaw of permafrost. This Data Descriptor presents circumpolar permafrost maps and geohazard indices depicting zones of varying potential for development of hazards related to near-surface permafrost degradation, such as ground subsidence. Statistical models were used to predict ground temperature and the thickness of the seasonally thawed (active) layer using geospatial data on environmental conditions at 30 arc-second resolution. These predictions, together with data on factors (ground ice content, soil grain size and slope gradient) affecting permafrost stability, were used to formulate geohazard indices. Using climate-forcing scenarios (Representative Concentration Pathways 2.6, 4.5 and 8.5), permafrost extent and hazard potential were projected for the 2041-2060 and 2061-2080 time periods. The resulting data (seven permafrost and 24 geohazard maps) are relevant to near-future infrastructure risk assessments and for targeting localized geohazard analyses.

Degrading permafrost puts Arctic infrastructure at risk by mid-century.

Degradation of near-surface permafrost can pose a serious threat to the utilization of natural resources, and to the sustainable development of Arctic communities. Here we identify at unprecedentedly high spatial resolution infrastructure hazard areas in the Northern Hemisphere's permafrost regions under projected climatic changes and quantify fundamental engineering structures at risk by 2050. We show that nearly four million people and 70% of current infrastructure in the permafrost domain are in areas with high potential for thaw of near-surface permafrost. Our results demonstrate that one-third of pan-Arctic infrastructure and 45% of the hydrocarbon extraction fields in the Russian Arctic are in regions where thaw-related ground instability can cause severe damage to the built environment. Alarmingly, these figures are not reduced substantially even if the climate change targets of the Paris Agreement are reached.

Antarctic emerald rockcod have the capacity to compensate for warming when uncoupled from CO2 -acidification.

Increases in atmospheric CO2 levels and associated ocean changes are expected to have dramatic impacts on marine ecosystems. Although the Southern Ocean is experiencing some of the fastest rates of change, few studies have explored how Antarctic fishes may be affected by co-occurring ocean changes, and even fewer have examined early life stages. To date, no studies have characterized potential trade-offs in physiology and behavior in response to projected multiple climate change stressors (ocean acidification and warming) on Antarctic fishes. We exposed juvenile emerald rockcod Trematomus bernacchii to three PCO2 treatments (~450, ~850, and ~1,200 µatm PCO2 ) at two temperatures (-1 or 2°C). After 2, 7, 14, and 28 days, metrics of physiological performance including cardiorespiratory function (heart rate [fH ] and ventilation rate [fV ]), metabolic rate (M˙O2), and cellular enzyme activity were measured. Behavioral responses, including scototaxis, activity, exploration, and escape response were assessed after 7 and 14 days. Elevated PCO2 independently had little impact on either physiology or behavior in juvenile rockcod, whereas warming resulted in significant changes across acclimation time. After 14 days, fH , fV and M˙O2 significantly increased with warming, but not with elevated PCO2 . Increased physiological costs were accompanied by behavioral alterations including increased dark zone preference up to 14%, reduced activity by 12%, as well as reduced escape time suggesting potential trade-offs in energetics. After 28 days, juvenile rockcod demonstrated a degree of temperature compensation as fV , M˙O2, and cellular metabolism significantly decreased following the peak at 14 days; however, temperature compensation was only evident in the absence of elevated PCO2 . Sustained increases in fV and M˙O2 after 28 days exposure to elevated PCO2 indicate additive (fV ) and synergistic (M˙O2) interactions occurred in combination with warming. Stressor-induced energetic trade-offs in physiology and behavior may be an important mechanism leading to vulnerability of Antarctic fishes to future ocean change.

Application of structure-based drug design and parallel chemistry to identify selective, brain penetrant, in vivo active phosphodiesterase 9A inhibitors.

Phosphodiesterase 9A inhibitors have shown activity in preclinical models of cognition with potential application as novel therapies for treating Alzheimer's disease. Our clinical candidate, PF-04447943 (2), demonstrated acceptable CNS permeability in rats with modest asymmetry between central and peripheral compartments (free brain/free plasma = 0.32; CSF/free plasma = 0.19) yet had physicochemical properties outside the range associated with traditional CNS drugs. To address the potential risk of restricted CNS penetration with 2 in human clinical trials, we sought to identify a preclinical candidate with no asymmetry in rat brain penetration and that could advance into development. Merging the medicinal chemistry strategies of structure-based design with parallel chemistry, a novel series of PDE9A inhibitors was identified that showed improved selectivity over PDE1C. Optimization afforded preclinical candidate 19 that demonstrated free brain/free plasma ≥ 1 in rat and reduced microsomal clearance along with the ability to increase cyclic guanosine monophosphosphate levels in rat CSF.

Design and discovery of 6-[(3S,4S)-4-methyl-1-(pyrimidin-2-ylmethyl)pyrrolidin-3-yl]-1-(tetrahydro-2H-pyran-4-yl)-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one (PF-04447943), a selective brain penetrant PDE9A inhibitor for the treatment of cognitive disorders.

6-[(3S,4S)-4-Methyl-1-(pyrimidin-2-ylmethyl)pyrrolidin-3-yl]-1-(tetrahydro-2H-pyran-4-yl)-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one (PF-04447943) is a novel PDE9A inhibitor identified using parallel synthetic chemistry and structure-based drug design (SBDD) and has advanced into clinical trials. Selectivity for PDE9A over other PDE family members was achieved by targeting key residue differences between the PDE9A and PDE1C catalytic site. The physicochemical properties of the series were optimized to provide excellent in vitro and in vivo pharmacokinetics properties in multiple species including humans. It has been reported to elevate central cGMP levels in the brain and CSF of rodents. In addition, it exhibits procognitive activity in several rodent models and synaptic stabilization in an amyloid precursor protein (APP) transgenic mouse model. Recent disclosures from clinical trials confirm that it is well tolerated in humans and elevates cGMP in cerebral spinal fluid of healthy volunteers, confirming that it is a quality pharmacological tool for testing clinical hypotheses in disease states associated with impairment of cGMP signaling or cognition.

Discovery of two clinical histamine H(3) receptor antagonists: trans-N-ethyl-3-fluoro-3-[3-fluoro-4-(pyrrolidinylmethyl)phenyl]cyclobutanecarboxamide (PF-03654746) and trans-3-fluoro-3-[3-fluoro-4-(pyrrolidin-1-ylmethyl)phenyl]-N-(2-methylpropyl)cyclobutanecarboxamide (PF-03654764).

The discovery of two histamine H(3) antagonist clinical candidates is disclosed. The pathway to identification of the two clinical candidates, 6 (PF-03654746) and 7 (PF-03654764) required five hypothesis driven design cycles. The key to success in identifying these clinical candidates was the development of a compound design strategy that leveraged medicinal chemistry knowledge and traditional assays in conjunction with computational and in vitro safety tools. Overall, clinical compounds 6 and 7 exceeded conservative safety margins and possessed optimal pharmacological and pharmacokinetic profiles, thus achieving our initial goal of identifying compounds with fully aligned oral drug attributes, "best-in-class" molecules.

Use of structure-based design to discover a potent, selective, in vivo active phosphodiesterase 10A inhibitor lead series for the treatment of schizophrenia.

Utilizing structure-based virtual library design and scoring, a novel chimeric series of phosphodiesterase 10A (PDE10A) inhibitors was discovered by synergizing binding site interactions and ADME properties of two chemotypes. Virtual libraries were docked and scored for potential binding ability, followed by visual inspection to prioritize analogs for parallel and directed synthesis. The process yielded highly potent and selective compounds such as 16. New X-ray cocrystal structures enabled rational design of substituents that resulted in the successful optimization of physical properties to produce in vivo activity and to modulate microsomal clearance and permeability.

Identification of a brain penetrant PDE9A inhibitor utilizing prospective design and chemical enablement as a rapid lead optimization strategy.

By use of chemical enablement and prospective design, a novel series of selective, brain penetrant PDE9A inhibitors have been identified that are capable of producing in vivo elevations of brain cGMP.

Discovery of a novel class of phosphodiesterase 10A inhibitors and identification of clinical candidate 2-[4-(1-methyl-4-pyridin-4-yl-1H-pyrazol-3-yl)-phenoxymethyl]-quinoline (PF-2545920) for the treatment of schizophrenia.

By utilizing structure-based drug design (SBDD) knowledge, a novel class of phosphodiesterase (PDE) 10A inhibitors was identified. The structure-based drug design efforts identified a unique "selectivity pocket" for PDE10A inhibitors, and interactions within this pocket allowed the design of highly selective and potent PDE10A inhibitors. Further optimization of brain penetration and drug-like properties led to the discovery of 2-[4-(1-methyl-4-pyridin-4-yl-1H-pyrazol-3-yl)-phenoxymethyl]-quinoline (PF-2545920). This PDE10A inhibitor is the first reported clinical entry for this mechanism in the treatment of schizophrenia.

The effect of breast cancer resistance protein and P-glycoprotein on the brain penetration of flavopiridol, imatinib mesylate (Gleevec), prazosin, and 2-methoxy-3-(4-(2-(5-methyl-2-phenyloxazol-4-yl)ethoxy)phenyl)propanoic acid (PF-407288) in mice.

The role of breast cancer resistance protein (Bcrp) and the combined activities of Bcrp and P-glycoprotein (P-gp, Mdr1a/1b) in limiting the brain penetration of drugs at the blood-brain barrier (BBB) were investigated using wild-type FVB, Mdr1a/1b(-/-), (-/-), Bcrp(-/-), and Mdr1a/1b(-/-), (-/-)Bcrp(-/-) mice. Four drugs, flavopiridol, imatinib mesylate (Gleevec), PF-407288, and prazosin, with different transport specificity for BCRP/Bcrp and MDR1/Mdr1a were selected, and the drug levels in plasma, cerebrospinal fluid, and brain of mice were determined. Flavopiridol and prazosin were identified as substrates for both mouse Bcrp and Mdr1a with greater transport associated with Bcrp. The brain/plasma (B/P) ratios at 0.5 and 2 h in Mdr1a/1b(-/-), (-/-) and Bcrp(-/-) mice were 1- to 2-fold for both compounds, whereas the ratios in Mdr1a/1b(-/-), (-/-)Bcrp(-/-) mice were more than 5-fold of those observed in FVB mice. For imatinib, a better substrate of P-gp than Bcrp, the B/P ratios in Bcrp(-/-) were comparable to those in FVB mice, whereas the B/P ratios in Mdr1a/1b(-/-), (-/-) and Mdr1a/1b(-/-), (-/-)Bcrp(-/-) mice were more than 4- and 28-fold of those in FVB mice at both time points, respectively. Finally, the Bcrp-specific substrate PF-407288 exhibited comparable B/P ratios in Mdr1a/1b(-/-), (-/-) and Bcrp(-/-) mice and slightly but significantly increased B/P ratios in Mdr1a/1b(-/-), (-/-)Bcrp(-/-) mice compared with those in FVB mice. The B/P ratios of compounds in Mdr1a/1b(-/-), (-/-)Bcrp(-/-) mice compared with those in Mdr1a/1b(-/-), (-/-) mice clearly demonstrate that Bcrp impairs the brain penetration of its substrates. Moreover, P-gp and Bcrp at BBB function synergistically to limit the brain penetration of shared substrates.

P-glycoprotein contributes to the blood-brain, but not blood-cerebrospinal fluid, barrier in a spontaneous canine p-glycoprotein knockout model.

P-glycoprotein is considered to be a major factor impeding effective drug therapy for many diseases of the central nervous system (CNS). Thus, efforts are being made to gain a better understanding of P-glycoprotein's role in drug distribution to brain parenchyma and cerebrospinal fluid (CSF). The goal of this study was to validate and introduce a novel P-glycoprotein-deficient (ABCB1-1Delta) canine model for studying P-glycoprotein-mediated effects of drug distribution to brain tissue and CSF. CSF concentrations of drug are often used to correlate efficacy of CNS drug therapy as a surrogate for determining drug concentration in brain tissue. A secondary goal of this study was to investigate the validity of using CSF concentrations of P-glycoprotein substrates to predict brain tissue concentrations. Loperamide, an opioid that is excluded from the brain by P-glycoprotein, was used to confirm a P-glycoprotein-null phenotype in the dog model. ABCB1-1Delta dogs experienced CNS depression following loperamide administration, whereas ABCB1 wild-type dogs experienced no CNS depression. In summary, we have validated a novel P-glycoprotein-deficient canine model and have used the model to investigate transport of the P-glycoprotein substrate (99m)Tc-sestamibi at the blood-brain barrier and blood-CSF barrier.

Peripheral elevation of IGF-1 fails to alter Abeta clearance in multiple in vivo models.

Increasing beta-amyloid (Abeta) clearance may alter the course of Alzheimer's disease progression and attenuate amyloid plaque pathology. Insulin-like growth factor I (IGF-1) augmentation has been suggested to increase Abeta clearance by facilitating transport of Abeta out of the brain. The availability of safe agents that increase IGF-1 levels therefore makes IGF-1 elevation an attractive target for disease modifying therapy in AD. The present series of studies sought to replicate published paradigms in which peripheral IGF-1 administration lowered brain Abeta acutely, with reduction in plaque pathology after chronic treatment. Thus Abeta levels were measured in several animal models following treatments that elevated IGF-1. Administration of IGF-1 to young or old rats for up to 3 days had no effect on Abeta levels in brain, CSF, or plasma. In adult beagles, 4 days of dosing with the growth hormone secretagogue, CP-424391, doubled baseline plasma IGF-1 levels, yet failed to alter CSF or plasma Abeta. 5-day treatment of young Tg2576 mice with IGF-1 produced robust elevations of IGF-1 levels in plasma, but no effects on Abeta were detected in brain, CSF, or plasma. Finally, 11-month-old Tg2576 mice were implanted with subcutaneous minipumps delivering IGF-1 for 1 month. No significant changes in Abeta (by ELISA or Western blot), plaque pathology, or phospho-tau epitopes were detected. These results do not demonstrate acute or chronic actions of peripherally administered IGF-1 on Abeta levels or the phosphorylation state of tau and therefore do not suggest any disease-modifying benefits of IGF-1 restorative therapy for AD through these mechanisms.

Central nervous system pharmacokinetics of the Mdr1 P-glycoprotein substrate CP-615,003: intersite differences and implications for human receptor occupancy projections from cerebrospinal fluid exposures.

The central nervous system (CNS) distribution and transport mechanisms of the investigational drug candidate CP-615,003 (N-[3-fluoro-4-[2-(propylamino)ethoxy]phenyl]-4,5,6,7-tetrahydro-4-oxo-1H-indole-3-carboxamide) and its active metabolite CP-900,725 have been characterized. Brain distribution of CP-615,003 and CP-900,725 was low in rats and mice (brain-to-serum ratio < 0.2). Cerebrospinal fluid (CSF)-to-serum ratios of CP-615,003 were 6- to 8-fold lower than the plasma unbound fraction in rats and dogs. In vitro, CP-615,003 displayed quinidine-like efflux in MDR1-expressing Madin-Darby canine kidney II cells. The brain-to-serum ratio of CP-615,003 in mdr1a/1b (-/-) mice was approximately 7 times that in their wild-type counterparts, confirming that impaired CNS distribution was explained by P-gp efflux transport. In contrast, P-gp efflux did not explain the impaired CNS penetration of CP-900,725. Intracerebral microdialysis was used to characterize rat brain extracellular fluid (ECF) distribution. Interestingly, the ECF-to-serum ratio of the P-gp substrate CP-615,003 was 7-fold below the CSF-to-serum ratio, whereas this disequilibrium was not observed for CP-900,725. In a clinical study, steady-state CSF exposures were measured after administration of 100 mg of CP-615,003 b.i.d. The human CSF-to-plasma ratios of CP-615,003 and CP-900,725 were both approximately 10-fold below their ex vivo plasma unbound fractions, confirming impaired human CNS penetration. Preliminary estimates of CNS receptor occupancy from human CSF concentrations were sensitive to assumptions regarding the magnitude of the CSF-ECF gradient for CP-615,003 in humans. In summary, this case provides an example of intersite differences in CNS pharmacokinetics of a P-gp substrate and potential implications for projection of human CNS receptor occupancy of transporter substrates from CSF pharmacokinetic data when direct imaging-based approaches are not feasible.

An inhibitor of casein kinase I epsilon induces phase delays in circadian rhythms under free-running and entrained conditions.

Casein kinase Iepsilon (CKIepsilon) is an essential component of the biological clock, phosphorylating PER proteins, and in doing so regulating their turnover and nuclear entry in oscillator cells of the suprachiasmatic nucleus (SCN). Although hereditary decreases in PER phosphorylation have been well characterized, little is known about the consequences of acute enzyme inhibition by pharmacological means. A novel reagent, 4-[3-cyclohexyl-5-(4-fluoro-phenyl)-3H-imidazol-4-yl]-pyrimidin-2-ylamine (PF-670462), proved to be both a potent (IC(50) = 7.7 +/- 2.2 nM) and selective (>30-fold with respect to 42 additional kinases) inhibitor of CKIepsilon in isolated enzyme preparations; in transfected whole cell assays, it caused a concentration-related redistribution of nuclear versus cytosolic PER. When tested in free-running animals, 50 mg/kg s.c. PF-670462 produced robust phase delays when dosed at circadian time (CT)9 (-1.97 +/- 0.17 h). Entrained rats dosed in normal light-dark (LD) and then released to constant darkness also experienced phase delays that were dose- and time of dosing-dependent. PF-670462 yielded only phase delays across the circadian cycle with the most sensitive time at CT12 when PER levels are near their peak in the SCN. Most importantly, these drug-induced phase delays persisted in animals entrained and maintained in LD throughout the entire experiment; re-entrainment to the prevailing LD required days in contrast to the rapid elimination of the drug (t(1/2) = 0.46 +/- 0.04 h). Together, these results suggest that inhibition of CKIepsilon yields a perturbation of oscillator function that forestalls light as a zeitgeber, and they demonstrate that pharmacological tools such as PF-670462 may yield valuable insight into clock function.

Evaluation of cerebrospinal fluid concentration and plasma free concentration as a surrogate measurement for brain free concentration.

This study was designed to evaluate the use of cerebrospinal fluid (CSF) drug concentration and plasma unbound concentration (C(u,plasma)) to predict brain unbound concentration (C(u,brain)). The concentration-time profiles in CSF, plasma, and brain of seven model compounds were determined after subcutaneous administration in rats. The C(u,brain) was estimated from the product of total brain concentrations and unbound fractions, which were determined using brain tissue slice and brain homogenate methods. For theobromine, theophylline, caffeine, fluoxetine, and propranolol, which represent rapid brain penetration compounds with a simple diffusion mechanism, the ratios of the area under the curve of C(u,brain)/C(CSF) and C(u,brain)/C(u,plasma) were 0.27 to 1.5 and 0.29 to 2.1, respectively, using the brain slice method, and were 0.27 to 2.9 and 0.36 to 3.9, respectively, using the brain homogenate method. A P-glycoprotein substrate, CP-141938 (methoxy-3-[(2-phenyl-piperadinyl-3-amino)-methyl]-phenyl-N-methyl-methane-sulfonamide), had C(u,brain)/C(CSF) and C(u,brain)/C(u,plasma) ratios of 0.57 and 0.066, using the brain slice method, and 1.1 and 0.13, using the brain homogenate method, respectively. The slow brain-penetrating compound, N[3-(4'-fluorophenyl)-3-(4'-phenylphenoxy)propyl-]sarcosine, had C(u,brain)/C(CSF) and C(u,brain)/C(u,plasma) ratios of 0.94 and 0.12 using the brain slice method and 0.15 and 0.018 using the brain homogenate method, respectively. Therefore, for quick brain penetration with simple diffusion mechanism compounds, C(CSF) and C(u,plasma) represent C(u,brain) equally well; for efflux substrates or slow brain penetration compounds, C(CSF) appears to be equivalent to or more accurate than C(u,plasma) to represent C(u,brain). Thus, we hypothesize that C(CSF) is equivalent to or better than C(u,plasma) to predict C(u,brain). This hypothesis is supported by the literature data.