39 results on '"Nolan CE"'
Search Results
2. Is the older perineum a safer perineum? Risk factors for obstetric anal sphincter injury.
- Author
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Nolan CE, O'Leary BD, and Ciprike V
- Subjects
- Adult, Age Factors, Female, Humans, Pregnancy, Retrospective Studies, Risk Factors, Anal Canal injuries, Delivery, Obstetric adverse effects, Obstetric Labor Complications etiology, Perineum injuries
- Abstract
Introduction: Obstetric anal sphincter injury (OASI) is the most common cause of anal incontinence. Identifying risk factors may facilitate change in labour and delivery practice, potentially reducing the risk. The objective of this study is to identify maternal, foetal and intrapartum risk factors for OASI in a regional hospital., Method: We conducted a retrospective analysis of vaginal deliveries over a 10-year period (2008-2017). Anal sphincter injury was diagnosed by an experienced clinician and classified according to RCOG recommendations. A multiple logistic regression model was created using the presence of OASI as the dependent variable. Coefficients were adjusted for relevant maternal, foetal and intrapartum risk factors., Results: During the study period, there were 23,887 vaginal deliveries. Of these births, 18,550 were spontaneous (77.66%), 3746 vacuum-assisted (15.68%), 1196 forceps (5.01%) and 395 sequential instrumental deliveries (1.65%). The overall rate of OASI was 1.76%, with an upward trend seen in nulliparous mothers. Significant factors that increased the risk of OASI were nulliparity, Asian ethnicity, delivery by forceps or sequential instruments, and shoulder dystocia. Vacuum delivery did not significantly increase risk., Conclusion: Maternal age ≥ 35 years confers a protective effect after adjusting for parity, birth weight and mode of delivery. Given the context of an ageing reproductive population, additional research is required to investigate the impact of maternal age on anal sphincter injury.
- Published
- 2021
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3. Quantitative Analysis of 18 F-PF-06684511, a Novel PET Radioligand for Selective β-Secretase 1 Imaging, in Nonhuman Primate Brain.
- Author
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Takano A, Chen L, Nag S, Brodney MA, Arakawa R, Chang C, Amini N, Doran SD, Dutra JK, McCarthy TJ, Nolan CE, O'Neill BT, Villalobos A, Zhang L, and Halldin C
- Subjects
- Animals, Female, Kinetics, Ligands, Macaca fascicularis, Male, Models, Biological, Radiochemistry, Whole Body Imaging, Amyloid Precursor Protein Secretases metabolism, Brain diagnostic imaging, Brain metabolism, Fluorine Radioisotopes metabolism, Positron-Emission Tomography, Pyrazines metabolism, Thiazines metabolism
- Abstract
β-secretase 1 (BACE1) is a key enzyme in the generation of β-amyloid, which is accumulated in the brain of Alzheimer disease patients. PF-06684511 was identified as a candidate PET ligand for imaging BACE1 in the brain and showed high specific binding in an initial assessment in a nonhuman primate (NHP) PET study using
18 F-PF-06684511. In this effort, we aimed to quantitatively evaluate the regional brain distribution of18 F-PF-06684511 in NHPs under baseline and blocking conditions and to assess the target occupancy of BACE1 inhibitors. In addition, NHP whole-body PET measurements were performed to estimate the effective radiation dose. Methods: Initial brain PET measurements were performed at baseline and after oral administration of 5 mg/kg of LY2886721, a BACE1 inhibitor, in 2 cynomolgus monkeys. Kinetic analysis was performed with the radiometabolite-corrected plasma input function. In addition, a wide dose range of another BACE1 inhibitor, PF-06663195, was examined to investigate the relationship between the brain target occupancy and plasma concentration of the drug. Finally, the effective radiation dose of18 F-PF-06684511 was estimated on the basis of the whole-body PET measurements in NHPs. Results: Radiolabeling was accomplished successfully with an incorporation radiochemical yield of 4%-12% (decay-corrected) from18 F ion. The radiochemical purity was greater than 99%. The whole-brain uptake of18 F-PF-06684511 peaked (∼220% SUV) at approximately 20 min and decreased thereafter (∼100% SUV at 180 min). A 2-tissue-compartment model described the time-activity curves well. Pretreatment with LY2886721 reduced the total distribution volume of18 F-PF-06684511 by 48%-80% depending on the brain region, confirming its in vivo specificity. BACE1 occupancy of PF-06663195, estimated using the Lassen occupancy plot, showed a dose-dependent increase. The effective dose of18 F-PF-06684511 was 0.043 mSv/MBq for humans. Conclusion:18 F-PF-06684511 is the first successful PET radioligand for BACE1 brain imaging that demonstrates favorable in vivo binding and brain kinetics in NHPs., (© 2019 by the Society of Nuclear Medicine and Molecular Imaging.)- Published
- 2019
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4. Single prior caesarean section and risk of anal sphincter injury.
- Author
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O'Leary BD, Nolan CE, and Ciprike V
- Subjects
- Adult, Anesthesia, Epidural statistics & numerical data, Episiotomy statistics & numerical data, Extraction, Obstetrical instrumentation, Extraction, Obstetrical statistics & numerical data, Female, Fetal Macrosomia complications, Humans, Incidence, Infant, Newborn, Obstetrical Forceps statistics & numerical data, Parity, Parturition, Pregnancy, Protective Factors, Retrospective Studies, Risk Factors, Young Adult, Anal Canal injuries, Birth Weight, Lacerations epidemiology, Vaginal Birth after Cesarean statistics & numerical data
- Abstract
Introduction and Hypothesis: Injury to the anal sphincter at vaginal delivery remains the leading cause of faecal incontinence in women. Previous studies reported an increased incidence of obstetric anal sphincter injury (OASI) in women attempting vaginal birth after caesarean section (VBAC). The aim of the paper was to establish whether women in their second pregnancy, with one previous uterine scar, are at a higher risk of OASI compared with nulliparous women., Methods: All primiparous and secundiparous women with a previous caesarean section who delivered from 2008 to 2017 were analysed in a single-centre retrospective study. The primary endpoint was OASI. Labour characteristics in both groups were compared, and a multiple regression model was created., Results: There were 8573 vaginal deliveries of nulliparous women and 3453 deliveries of women in their second pregnancy with a previous caesarean section, of whom 550 had a successful VBAC. There was no significant difference in the rate of OASI between primiparous women and those who had a successful VBAC: 3.5% (297/8573) versus 3.1% (17/550), P = 0.730). Foetal macrosomia (>4 kg) and forceps delivery were risk factors for sphincter injury, while episiotomy and epidural anaesthesia were protective., Conclusions: VBAC does not confer an increased risk of OASI after a first delivery by caesarean section when compared with nulliparous women. The rate of successful VBAC may be contributory and suggests that the risk conferred by VBAC may be unit-specific. Unit and national-level audit is necessary to investigate this risk further.
- Published
- 2019
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5. Design and Synthesis of Clinical Candidate PF-06751979: A Potent, Brain Penetrant, β-Site Amyloid Precursor Protein Cleaving Enzyme 1 (BACE1) Inhibitor Lacking Hypopigmentation.
- Author
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O'Neill BT, Beck EM, Butler CR, Nolan CE, Gonzales C, Zhang L, Doran SD, Lapham K, Buzon LM, Dutra JK, Barreiro G, Hou X, Martinez-Alsina LA, Rogers BN, Villalobos A, Murray JC, Ogilvie K, LaChapelle EA, Chang C, Lanyon LF, Steppan CM, Robshaw A, Hales K, Boucher GG, Pandher K, Houle C, Ambroise CW, Karanian D, Riddell D, Bales KR, and Brodney MA
- Subjects
- Amyloid beta-Protein Precursor metabolism, Animals, Brain drug effects, Cells, Cultured, Dogs, Humans, Male, Melanocytes drug effects, Mice, Mice, Inbred C57BL, Models, Molecular, Molecular Structure, Protein Conformation, Amyloid Precursor Protein Secretases antagonists & inhibitors, Aspartic Acid Endopeptidases antagonists & inhibitors, Brain metabolism, Drug Design, Hypopigmentation chemically induced, Protease Inhibitors administration & dosage, Protease Inhibitors adverse effects, Protease Inhibitors chemistry, Pyrans administration & dosage, Pyrans adverse effects, Pyrans chemistry, Skin Pigmentation drug effects, Thiazines administration & dosage, Thiazines adverse effects, Thiazines chemistry, Thiazoles administration & dosage, Thiazoles adverse effects, Thiazoles chemistry
- Abstract
A major challenge in the development of β-site amyloid precursor protein cleaving enzyme 1 (BACE1) inhibitors for the treatment of Alzheimer's disease is the alignment of potency, drug-like properties, and selectivity over related aspartyl proteases such as Cathepsin D (CatD) and BACE2. The potential liabilities of inhibiting BACE2 chronically have only recently begun to emerge as BACE2 impacts the processing of the premelanosome protein (PMEL17) and disrupts melanosome morphology resulting in a depigmentation phenotype. Herein, we describe the identification of clinical candidate PF-06751979 (64), which displays excellent brain penetration, potent in vivo efficacy, and broad selectivity over related aspartyl proteases including BACE2. Chronic dosing of 64 for up to 9 months in dog did not reveal any observation of hair coat color (pigmentation) changes and suggests a key differentiator over current BACE1 inhibitors that are nonselective against BACE2 in later stage clinical development.
- Published
- 2018
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6. Identification of a Novel Positron Emission Tomography (PET) Ligand for Imaging β-Site Amyloid Precursor Protein Cleaving Enzyme 1 (BACE-1) in Brain.
- Author
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Zhang L, Chen L, Dutra JK, Beck EM, Nag S, Takano A, Amini N, Arakawa R, Brodney MA, Buzon LM, Doran SD, Lanyon LF, McCarthy TJ, Bales KR, Nolan CE, O'Neill BT, Schildknegt K, Halldin C, and Villalobos A
- Subjects
- Amyloid Precursor Protein Secretases antagonists & inhibitors, Animals, Aspartic Acid Endopeptidases antagonists & inhibitors, Fluorine Radioisotopes, Ligands, Male, Mice, Positron-Emission Tomography, Protease Inhibitors chemical synthesis, Protease Inhibitors chemistry, Protease Inhibitors pharmacokinetics, Pyrazines chemical synthesis, Pyrazines chemistry, Pyrazines pharmacokinetics, Radiopharmaceuticals chemical synthesis, Radiopharmaceuticals chemistry, Radiopharmaceuticals pharmacokinetics, Thiazines chemical synthesis, Thiazines chemistry, Thiazines pharmacokinetics, Amyloid Precursor Protein Secretases metabolism, Aspartic Acid Endopeptidases metabolism, Brain metabolism, Protease Inhibitors pharmacology, Pyrazines pharmacology, Radiopharmaceuticals pharmacology, Thiazines pharmacology
- Abstract
Alzheimer's disease (AD) is characterized by accumulation of β-amyloid (Aβ) plaques and neurofibrillary tau tangles in the brain. β-Site amyloid precursor protein cleaving enzyme 1 (BACE1) plays a key role in the generation of Aβ fragments via extracellular cleavage of the amyloid precursor protein (APP). We became interested in developing a BACE1 PET ligand to facilitate clinical assessment of BACE1 inhibitors and explore its potential in the profiling and selection of patients for AD trials. Using a set of PET ligand design parameters, compound 3 (PF-06684511) was rapidly identified as a lead with favorable in vitro attributes and structural handles for PET radiolabeling. Further evaluation in an LC-MS/MS "cold tracer" study in rodents revealed high specific binding to BACE1 in brain. Upon radiolabeling, [
18 F]3 demonstrated favorable brain uptake and high in vivo specificity in nonhuman primate (NHP), suggesting its potential for imaging BACE1 in humans.- Published
- 2018
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7. Aminomethyl-Derived Beta Secretase (BACE1) Inhibitors: Engaging Gly230 without an Anilide Functionality.
- Author
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Butler CR, Ogilvie K, Martinez-Alsina L, Barreiro G, Beck EM, Nolan CE, Atchison K, Benvenuti E, Buzon L, Doran S, Gonzales C, Helal CJ, Hou X, Hsu MH, Johnson EF, Lapham K, Lanyon L, Parris K, O'Neill BT, Riddell D, Robshaw A, Vajdos F, and Brodney MA
- Subjects
- Amino Acid Sequence, Amyloid Precursor Protein Secretases chemistry, Animals, Brain metabolism, Chromatography, High Pressure Liquid, Crystallization, Enzyme Inhibitors chemistry, Enzyme Inhibitors pharmacokinetics, Male, Mice, Patch-Clamp Techniques, Structure-Activity Relationship, Tandem Mass Spectrometry, Amyloid Precursor Protein Secretases antagonists & inhibitors, Anilides chemistry, Enzyme Inhibitors pharmacology, Glycine chemistry
- Abstract
A growing subset of β-secretase (BACE1) inhibitors for the treatment of Alzheimer's disease (AD) utilizes an anilide chemotype that engages a key residue (Gly230) in the BACE1 binding site. Although the anilide moiety affords excellent potency, it simultaneously introduces a third hydrogen bond donor that limits brain availability and provides a potential metabolic site leading to the formation of an aniline, a structural motif of prospective safety concern. We report herein an alternative aminomethyl linker that delivers similar potency and improved brain penetration relative to the amide moiety. Optimization of this series identified analogues with an excellent balance of ADME properties and potency; however, potential drug-drug interactions (DDI) were predicted based on CYP 2D6 affinities. Generation and analysis of key BACE1 and CYP 2D6 crystal structures identified strategies to obviate the DDI liability, leading to compound 16, which exhibits robust in vivo efficacy as a BACE1 inhibitor.
- Published
- 2017
- Full Text
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8. Chemoproteomic profiling reveals that cathepsin D off-target activity drives ocular toxicity of β-secretase inhibitors.
- Author
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Zuhl AM, Nolan CE, Brodney MA, Niessen S, Atchison K, Houle C, Karanian DA, Ambroise C, Brulet JW, Beck EM, Doran SD, O'Neill BT, Am Ende CW, Chang C, Geoghegan KF, West GM, Judkins JC, Hou X, Riddell DR, and Johnson DS
- Subjects
- Amyloid Precursor Protein Secretases metabolism, Animals, Cell Line, Enzyme Inhibitors chemistry, Enzyme Inhibitors pharmacology, Eye drug effects, Humans, Inhibitory Concentration 50, Mass Spectrometry, Mice, Knockout, Molecular Probes chemical synthesis, Molecular Probes chemistry, Peptides metabolism, Protein Binding, Rats, Wistar, Retinal Pigment Epithelium drug effects, Retinal Pigment Epithelium pathology, Staining and Labeling, Amyloid Precursor Protein Secretases antagonists & inhibitors, Cathepsin D metabolism, Enzyme Inhibitors toxicity, Eye pathology, Proteomics methods, Toxicity Tests
- Abstract
Inhibition of β-secretase BACE1 is considered one of the most promising approaches for treating Alzheimer's disease. Several structurally distinct BACE1 inhibitors have been withdrawn from development after inducing ocular toxicity in animal models, but the target mediating this toxicity has not been identified. Here we use a clickable photoaffinity probe to identify cathepsin D (CatD) as a principal off-target of BACE1 inhibitors in human cells. We find that several BACE1 inhibitors blocked CatD activity in cells with much greater potency than that displayed in cell-free assays with purified protein. Through a series of exploratory toxicology studies, we show that quantifying CatD target engagement in cells with the probe is predictive of ocular toxicity in vivo. Taken together, our findings designate off-target inhibition of CatD as a principal driver of ocular toxicity for BACE1 inhibitors and more generally underscore the power of chemical proteomics for discerning mechanisms of drug action.
- Published
- 2016
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9. Discovery of a Highly Selective Glycogen Synthase Kinase-3 Inhibitor (PF-04802367) That Modulates Tau Phosphorylation in the Brain: Translation for PET Neuroimaging.
- Author
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Liang SH, Chen JM, Normandin MD, Chang JS, Chang GC, Taylor CK, Trapa P, Plummer MS, Para KS, Conn EL, Lopresti-Morrow L, Lanyon LF, Cook JM, Richter KE, Nolan CE, Schachter JB, Janat F, Che Y, Shanmugasundaram V, Lefker BA, Enerson BE, Livni E, Wang L, Guehl NJ, Patnaik D, Wagner FF, Perlis R, Holson EB, Haggarty SJ, El Fakhri G, Kurumbail RG, and Vasdev N
- Subjects
- Brain metabolism, Crystallography, X-Ray, Dose-Response Relationship, Drug, Glycogen Synthase Kinase 3 antagonists & inhibitors, Glycogen Synthase Kinase 3 metabolism, Humans, Models, Molecular, Molecular Structure, Oxazoles chemical synthesis, Oxazoles chemistry, Phosphorylation drug effects, Protein Kinase Inhibitors chemical synthesis, Protein Kinase Inhibitors chemistry, Triazoles chemical synthesis, Triazoles chemistry, tau Proteins metabolism, Brain diagnostic imaging, Brain drug effects, Neuroimaging, Oxazoles pharmacology, Positron-Emission Tomography, Protein Kinase Inhibitors pharmacology, Triazoles pharmacology, tau Proteins antagonists & inhibitors
- Abstract
Glycogen synthase kinase-3 (GSK-3) regulates multiple cellular processes in diabetes, oncology, and neurology. N-(3-(1H-1,2,4-triazol-1-yl)propyl)-5-(3-chloro-4-methoxyphenyl)oxazole-4-carboxamide (PF-04802367 or PF-367) has been identified as a highly potent inhibitor, which is among the most selective antagonists of GSK-3 to date. Its efficacy was demonstrated in modulation of tau phosphorylation in vitro and in vivo. Whereas the kinetics of PF-367 binding in brain tissues are too fast for an effective therapeutic agent, the pharmacokinetic profile of PF-367 is ideal for discovery of radiopharmaceuticals for GSK-3 in the central nervous system. A (11) C-isotopologue of PF-367 was synthesized and preliminary PET imaging studies in non-human primates confirmed that we have overcome the two major obstacles for imaging GSK-3, namely, reasonable brain permeability and displaceable binding., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)
- Published
- 2016
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10. Utilizing structures of CYP2D6 and BACE1 complexes to reduce risk of drug-drug interactions with a novel series of centrally efficacious BACE1 inhibitors.
- Author
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Brodney MA, Beck EM, Butler CR, Barreiro G, Johnson EF, Riddell D, Parris K, Nolan CE, Fan Y, Atchison K, Gonzales C, Robshaw AE, Doran SD, Bundesmann MW, Buzon L, Dutra J, Henegar K, LaChapelle E, Hou X, Rogers BN, Pandit J, Lira R, Martinez-Alsina L, Mikochik P, Murray JC, Ogilvie K, Price L, Sakya SM, Yu A, Zhang Y, and O'Neill BT
- Subjects
- Amino Acid Sequence, Amyloidogenic Proteins metabolism, Animals, Crystallography, X-Ray, Cytochrome P-450 CYP2D6 genetics, Cytochrome P-450 CYP2D6 metabolism, Drug Design, ERG1 Potassium Channel, Ether-A-Go-Go Potassium Channels antagonists & inhibitors, Ether-A-Go-Go Potassium Channels metabolism, Humans, Inhibitory Concentration 50, Male, Mice, Inbred Strains, Models, Molecular, Molecular Sequence Data, Protease Inhibitors administration & dosage, Protease Inhibitors pharmacokinetics, Pyrazoles chemistry, Structure-Activity Relationship, Amyloid Precursor Protein Secretases antagonists & inhibitors, Amyloid Precursor Protein Secretases chemistry, Aspartic Acid Endopeptidases antagonists & inhibitors, Aspartic Acid Endopeptidases chemistry, Cytochrome P-450 CYP2D6 chemistry, Drug Interactions, Protease Inhibitors chemistry, Protease Inhibitors pharmacology
- Abstract
In recent years, the first generation of β-secretase (BACE1) inhibitors advanced into clinical development for the treatment of Alzheimer's disease (AD). However, the alignment of drug-like properties and selectivity remains a major challenge. Herein, we describe the discovery of a novel class of potent, low clearance, CNS penetrant BACE1 inhibitors represented by thioamidine 5. Further profiling suggested that a high fraction of the metabolism (>95%) was due to CYP2D6, increasing the potential risk for victim-based drug-drug interactions (DDI) and variable exposure in the clinic due to the polymorphic nature of this enzyme. To guide future design, we solved crystal structures of CYP2D6 complexes with substrate 5 and its corresponding metabolic product pyrazole 6, which provided insight into the binding mode and movements between substrate/inhibitor complexes. Guided by the BACE1 and CYP2D6 crystal structures, we designed and synthesized analogues with reduced risk for DDI, central efficacy, and improved hERG therapeutic margins.
- Published
- 2015
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11. Discovery of a series of efficient, centrally efficacious BACE1 inhibitors through structure-based drug design.
- Author
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Butler CR, Brodney MA, Beck EM, Barreiro G, Nolan CE, Pan F, Vajdos F, Parris K, Varghese AH, Helal CJ, Lira R, Doran SD, Riddell DR, Buzon LM, Dutra JK, Martinez-Alsina LA, Ogilvie K, Murray JC, Young JM, Atchison K, Robshaw A, Gonzales C, Wang J, Zhang Y, and O'Neill BT
- Subjects
- Alzheimer Disease drug therapy, Amidines pharmacokinetics, Amidines pharmacology, Amyloid Precursor Protein Secretases metabolism, Amyloid beta-Peptides antagonists & inhibitors, Amyloid beta-Peptides metabolism, Animals, Aspartic Acid Endopeptidases metabolism, Brain metabolism, Brain pathology, Dogs, Drug Design, Enzyme Inhibitors pharmacokinetics, Enzyme Inhibitors pharmacology, Humans, Male, Mice, Models, Molecular, Plaque, Amyloid metabolism, Plaque, Amyloid pathology, Rats, Rats, Wistar, Sulfhydryl Compounds chemistry, Sulfhydryl Compounds pharmacokinetics, Sulfhydryl Compounds pharmacology, Sulfhydryl Compounds therapeutic use, Amidines chemistry, Amidines therapeutic use, Amyloid Precursor Protein Secretases antagonists & inhibitors, Aspartic Acid Endopeptidases antagonists & inhibitors, Brain drug effects, Enzyme Inhibitors chemistry, Enzyme Inhibitors therapeutic use, Plaque, Amyloid drug therapy
- Abstract
The identification of centrally efficacious β-secretase (BACE1) inhibitors for the treatment of Alzheimer's disease (AD) has historically been thwarted by an inability to maintain alignment of potency, brain availability, and desired absorption, distribution, metabolism, and excretion (ADME) properties. In this paper, we describe a series of truncated, fused thioamidines that are efficiently selective in garnering BACE1 activity without simultaneously inhibiting the closely related cathepsin D or negatively impacting brain penetration and ADME alignment, as exemplified by 36. Upon oral administration, these inhibitors exhibit robust brain availability and are efficacious in lowering central Amyloid β (Aβ) levels in mouse and dog. In addition, chronic treatment in aged PS1/APP mice effects a decrease in the number and size of Aβ-derived plaques. Most importantly, evaluation of 36 in a 2-week exploratory toxicology study revealed no accumulation of autofluorescent material in retinal pigment epithelium or histology findings in the eye, issues observed with earlier BACE1 inhibitors.
- Published
- 2015
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12. Effects of the γ-secretase inhibitor semagacestat on hippocampal neuronal network oscillation.
- Author
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Hajós M, Morozova E, Siok C, Atchison K, Nolan CE, Riddell D, Kiss T, and Hajós-Korcsok E
- Abstract
Neurological and psychiatric disorders are frequently associated with disruption of various cognitive functions, but development of effective drug treatments for these conditions has proven challenging. One of the main obstacles is the poor predictive validity of our preclinical animal models. In the present study the effects of the γ-secretase inhibitor semagacestat was evaluated in preclinical in vivo electrophysiological models. Recently disclosed Phase III findings on semagacestat indicated that Alzheimer's disease (AD) patients on this drug showed significantly worsened cognitive function compared to those treated with placebo. Since previous studies have shown that drugs impairing cognitive function (including scopolamine, NMDA (N-methyl-D-aspartate) receptor antagonists, and nociceptin receptor agonists) disrupt or decrease power of elicited theta oscillation in the hippocampus, we tested the effects of acute and sub-chronic administration of semagacestat in this assay. Field potentials were recorded across the hippocampal formation with NeuroNexus multi-site silicon probes in urethane anesthetized male C57BL/6 mice; hippocampal CA1 theta oscillation was elicited by electrical stimulation of the brainstem nucleus pontis oralis. Sub-chronic administration of semagacestat twice daily over 12 days at a dose known to reduce beta-amyloid peptide (Aβ) level [100 mg/kg, p.o. (per oral)] diminished power of elicited hippocampal theta oscillation. Acute, subcutaneous administration of semagacestat (100 mg/kg) produced a similar effect on hippocampal activity. We propose that the disruptive effect of semagacestat on hippocampal function could be one of the contributing mechanisms to its worsening of cognition in patients with AD. As it has been expected, both acute and sub-chronic administrations of semagacestat significantly decreased Aβ40 and Aβ42 levels but the current findings do not reveal the mode of action of semagacestat in disrupting hippocampal oscillation.
- Published
- 2013
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13. Cerebrospinal fluid β-Amyloid turnover in the mouse, dog, monkey and human evaluated by systematic quantitative analyses.
- Author
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Lu Y, Barton HA, Leung L, Zhang L, Hajos-Korcsok E, Nolan CE, Liu J, Becker SL, Wood KM, Robshaw AE, Taylor CK, O'Neill BT, Brodney MA, and Riddell D
- Subjects
- Amyloid Precursor Protein Secretases antagonists & inhibitors, Amyloid beta-Peptides blood, Animals, Dogs, Humans, Macaca fascicularis, Mice, Oligopeptides pharmacology, Oxadiazoles pharmacology, Pyrimidines pharmacology, Sulfonamides pharmacology, Thiazines pharmacology, Amyloid beta-Peptides cerebrospinal fluid
- Abstract
Background: Reducing brain β-amyloid (Aβ) via inhibition of β-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 Aβ 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 Aβ turnover process in these compartments., Objective: To understand Aβ turnover kinetics in preclinical species and humans., Methods: We collected CSF Aβ dynamic data after β- or γ-secretase inhibitor treatment from in-house experiments and the public domain, and analyzed the data using PK/PD modeling to obtain CSF Aβ turnover rates (kout) in the mouse, dog, monkey and human., Results: The kout for CSF Aβ40 follows allometry (kout = 0.395 × body weight(-0.351)). The kout for CSF Aβ40 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 Aβ40 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 Aβ in rodents may be different from those in the higher species. The understanding of Aβ turnover has considerable implications for the discovery and development of Aβ-lowering therapeutics, as illustrated from the perspectives of preclinical PK/PD characterization and preclinical-to-clinical translation., (Copyright © 2012 S. Karger AG, Basel.)
- Published
- 2013
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14. Spirocyclic sulfamides as β-secretase 1 (BACE-1) inhibitors for the treatment of Alzheimer's disease: utilization of structure based drug design, WaterMap, and CNS penetration studies to identify centrally efficacious inhibitors.
- Author
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Brodney MA, Barreiro G, Ogilvie K, Hajos-Korcsok E, Murray J, Vajdos F, Ambroise C, Christoffersen C, Fisher K, Lanyon L, Liu J, Nolan CE, Withka JM, Borzilleri KA, Efremov I, Oborski CE, Varghese A, and O'Neill BT
- Subjects
- ATP Binding Cassette Transporter, Subfamily B, Member 1 genetics, Amyloid Precursor Protein Secretases chemistry, Amyloid beta-Peptides metabolism, Animals, Aspartic Acid Endopeptidases chemistry, Aza Compounds pharmacokinetics, Aza Compounds pharmacology, Crystallography, X-Ray, Dogs, Drug Design, Female, Humans, Madin Darby Canine Kidney Cells, Male, Mice, Mice, Knockout, Microsomes, Liver metabolism, Models, Molecular, Molecular Structure, Permeability, Spiro Compounds pharmacokinetics, Spiro Compounds pharmacology, Stereoisomerism, Structure-Activity Relationship, Sulfonamides pharmacokinetics, Sulfonamides pharmacology, Transfection, Alzheimer Disease drug therapy, Amyloid Precursor Protein Secretases antagonists & inhibitors, Aspartic Acid Endopeptidases antagonists & inhibitors, Aza Compounds chemical synthesis, Brain metabolism, Spiro Compounds chemical synthesis, Sulfonamides chemical synthesis
- Abstract
β-Secretase 1 (BACE-1) is an attractive therapeutic target for the treatment and prevention of Alzheimer's disease (AD). Herein, we describe the discovery of a novel class of BACE-1 inhibitors represented by sulfamide 14g, using a medicinal chemistry strategy to optimize central nervous system (CNS) penetration by minimizing hydrogen bond donors (HBDs) and reducing P-glycoprotein (P-gp) mediated efflux. We have also taken advantage of the combination of structure based drug design (SBDD) to guide the optimization of the sulfamide analogues and the in silico tool WaterMap to explain the observed SAR. Compound 14g is a potent inhibitor of BACE-1 with excellent permeability and a moderate P-gp liability. Administration of 14g to mice produced a significant, dose-dependent reduction in central Aβ(X-40) levels at a free drug exposure equivalent to the whole cell IC(50) (100 nM). Furthermore, studies of the P-gp knockout mouse provided evidence that efflux transporters affected the amount of Aβ lowering versus that observed in wild-type (WT) mouse at an equivalent dose.
- Published
- 2012
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15. Cerebrospinal fluid amyloid-β (Aβ) as an effect biomarker for brain Aβ lowering verified by quantitative preclinical analyses.
- Author
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Lu Y, Riddell D, Hajos-Korcsok E, Bales K, Wood KM, Nolan CE, Robshaw AE, Zhang L, Leung L, Becker SL, Tseng E, Barricklow J, Miller EH, Osgood S, O'Neill BT, Brodney MA, Johnson DS, and Pettersson M
- Subjects
- Alzheimer Disease cerebrospinal fluid, Alzheimer Disease drug therapy, Alzheimer Disease metabolism, Amyloid Precursor Protein Secretases antagonists & inhibitors, Amyloid Precursor Protein Secretases cerebrospinal fluid, Amyloid Precursor Protein Secretases metabolism, Amyloid beta-Peptides metabolism, Animals, Biomarkers cerebrospinal fluid, Biomarkers metabolism, Cerebrospinal Fluid chemistry, Guinea Pigs, Male, Mice, Mice, 129 Strain, Protease Inhibitors pharmacology, Rats, Rats, Sprague-Dawley, Amyloid beta-Peptides cerebrospinal fluid, Brain metabolism
- Abstract
Reducing the generation of amyloid-β (Aβ) in the brain via inhibition of β-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 β-secretase inhibitors (BACEi), γ-secretase inhibitors (GSI), and γ-secretase modulators (GSM), Aβ in cerebrospinal fluid (CSF) has been presumed to be an effect biomarker for Aβ lowering in the brain. However, this presumption is challenged by the lack of quantitative understanding of the relationship between brain and CSF Aβ lowering. In this study, we strived to elucidate how the intrinsic pharmacokinetic (PK)/pharmacodynamic (PD) relationship for CSF Aβ lowering is related to that for brain Aβ 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 Aβ as an effect biomarker for brain Aβ 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 Aβ lowering in the brain.
- Published
- 2012
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16. Phase II study of single-agent navitoclax (ABT-263) and biomarker correlates in patients with relapsed small cell lung cancer.
- Author
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Rudin CM, Hann CL, Garon EB, Ribeiro de Oliveira M, Bonomi PD, Camidge DR, Chu Q, Giaccone G, Khaira D, Ramalingam SS, Ranson MR, Dive C, McKeegan EM, Chyla BJ, Dowell BL, Chakravartty A, Nolan CE, Rudersdorf N, Busman TA, Mabry MH, Krivoshik AP, Humerickhouse RA, Shapiro GI, and Gandhi L
- Subjects
- Aged, Aniline Compounds administration & dosage, Aniline Compounds adverse effects, Antineoplastic Agents adverse effects, Disease-Free Survival, Female, Humans, Male, Middle Aged, Recurrence, Sulfonamides administration & dosage, Sulfonamides adverse effects, Thrombocytopenia chemically induced, Aniline Compounds therapeutic use, Antineoplastic Agents therapeutic use, Lung Neoplasms drug therapy, Proto-Oncogene Proteins c-bcl-2 antagonists & inhibitors, Small Cell Lung Carcinoma drug therapy, Sulfonamides therapeutic use
- Abstract
Purpose: Bcl-2 is a critical regulator of apoptosis that is overexpressed in the majority of small cell lung cancers (SCLC). Nativoclax (ABT-263) is a potent and selective inhibitor of Bcl-2 and Bcl-x(L). The primary objectives of this phase IIa study included safety at the recommended phase II dose and preliminary, exploratory efficacy assessment in patients with recurrent and progressive SCLC after at least one prior therapy., Experimental Design: Thirty-nine patients received navitoclax 325 mg daily, following an initial lead-in of 150 mg daily for 7 days. Study endpoints included safety and toxicity assessment, response rate, progression-free and overall survival (PFS and OS), as well as exploratory pharmacodynamic correlates., Results: The most common toxicity associated with navitoclax was thrombocytopenia, which reached grade III-IV in 41% of patients. Partial response was observed in one (2.6%) patient and stable disease in 9 (23%) patients. Median PFS was 1.5 months and median OS was 3.2 months. A strong association between plasma pro-gastrin-releasing peptide (pro-GRP) level and tumor Bcl-2 copy number (R = 0.93) was confirmed. Exploratory analyses revealed baseline levels of cytokeratin 19 fragment antigen 21-1, neuron-specific enolase, pro-GRP, and circulating tumor cell number as correlates of clinical benefit., Conclusion: Bcl-2 targeting by navitoclax shows limited single-agent activity against advanced and recurrent SCLC. Correlative analyses suggest several putative biomarkers of clinical benefit. Preclinical models support that navitoclax may enhance sensitivity of SCLC and other solid tumors to standard cytotoxics. Future studies will focus on combination therapies.
- Published
- 2012
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17. Application of the bicyclo[1.1.1]pentane motif as a nonclassical phenyl ring bioisostere in the design of a potent and orally active γ-secretase inhibitor.
- Author
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Stepan AF, Subramanyam C, Efremov IV, Dutra JK, O'Sullivan TJ, DiRico KJ, McDonald WS, Won A, Dorff PH, Nolan CE, Becker SL, Pustilnik LR, Riddell DR, Kauffman GW, Kormos BL, Zhang L, Lu Y, Capetta SH, Green ME, Karki K, Sibley E, Atchison KP, Hallgren AJ, Oborski CE, Robshaw AE, Sneed B, and O'Donnell CJ
- Subjects
- Administration, Oral, Animals, Biological Availability, Brain metabolism, Bridged Bicyclo Compounds pharmacokinetics, Bridged Bicyclo Compounds pharmacology, Cell Line, Dogs, Female, Humans, Mice, Microsomes, Liver metabolism, Oxadiazoles pharmacokinetics, Oxadiazoles pharmacology, Pentanes pharmacokinetics, Pentanes pharmacology, Rats, Stereoisomerism, Structure-Activity Relationship, Sulfonamides pharmacokinetics, Sulfonamides pharmacology, Tissue Distribution, Amyloid Precursor Protein Secretases antagonists & inhibitors, Bridged Bicyclo Compounds chemical synthesis, Oxadiazoles chemical synthesis, Pentanes chemical synthesis, Sulfonamides chemical synthesis
- Abstract
Replacement of the central, para-substituted fluorophenyl ring in the γ-secretase inhibitor 1 (BMS-708,163) with the bicyclo[1.1.1]pentane motif led to the discovery of compound 3, an equipotent enzyme inhibitor with significant improvements in passive permeability and aqueous solubility. The modified biopharmaceutical properties of 3 translated into excellent oral absorption characteristics (~4-fold ↑ C(max) and AUC values relative to 1) in a mouse model of γ-secretase inhibition. In addition, SAR studies into other fluorophenyl replacements indicate the intrinsic advantages of the bicyclo[1.1.1]pentane moiety over conventional phenyl ring replacements with respect to achieving an optimal balance of properties (e.g., γ-secretase inhibition, aqueous solubility/permeability, in vitro metabolic stability). Overall, this work enhances the scope of the [1.1.1]-bicycle beyond that of a mere "spacer" unit and presents a compelling case for its broader application as a phenyl group replacement in scenarios where the aromatic ring count impacts physicochemical parameters and overall drug-likeness., (© 2012 American Chemical Society)
- Published
- 2012
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18. Quantitative pharmacokinetic/pharmacodynamic analyses suggest that the 129/SVE mouse is a suitable preclinical pharmacology model for identifying small-molecule γ-secretase inhibitors.
- Author
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Lu Y, Zhang L, Nolan CE, Becker SL, Atchison K, Robshaw AE, Pustilnik LR, Osgood SM, Miller EH, Stepan AF, Subramanyam C, Efremov I, Hallgren AJ, and Riddell D
- Subjects
- Alanine blood, Alanine pharmacokinetics, Alanine pharmacology, Amyloid Precursor Protein Secretases metabolism, Amyloid beta-Peptides blood, Amyloid beta-Protein Precursor metabolism, Animals, Azepines blood, Azepines pharmacokinetics, Brain drug effects, Brain enzymology, Drug Evaluation, Preclinical, Enzyme Inhibitors blood, Enzyme Inhibitors pharmacokinetics, Humans, Mice, Mice, 129 Strain, Mice, Transgenic, Models, Animal, Oxadiazoles blood, Oxadiazoles pharmacokinetics, Small Molecule Libraries, Sulfonamides blood, Sulfonamides pharmacokinetics, Alanine analogs & derivatives, Amyloid Precursor Protein Secretases antagonists & inhibitors, Amyloid beta-Peptides metabolism, Azepines pharmacology, Drug Discovery, Enzyme Inhibitors pharmacology, Oxadiazoles pharmacology, Sulfonamides pharmacology
- Abstract
Alzheimer's disease (AD) poses a serious public health threat to the United States. Disease-modifying drugs slowing AD progression are in urgent need, but they are still unavailable. According to the amyloid cascade hypothesis, inhibition of β- or γ-secretase, key enzymes for the production of amyloid β (Aβ), may be viable mechanisms for the treatment of AD. For the discovery of γ-secretase inhibitors (GSIs), the APP-overexpressing Tg2576 mouse has been the preclinical model of choice, in part because of the ease of detection of Aβ species in its brain, plasma, and cerebrospinal fluid (CSF). Some biological observations and practical considerations, however, argue against the use of the Tg2576 mouse. We reasoned that an animal model would be suitable for GSI discovery if the pharmacokinetic (PK)/pharmacodynamic (PD) relationship of a compound for Aβ lowering in this model is predictive of that in human. In this study, we assessed whether the background 129/SVE strain is a suitable preclinical pharmacology model for identifying new GSIs by evaluating the translatability of the intrinsic PK/PD relationships for brain and CSF Aβ across the Tg2576 and 129/SVE mouse and human. Using semimechanistically based PK/PD modeling, our analyses indicated that the intrinsic PK/PD relationship for brain Aβx-42 and CSF Aβx-40 in the 129/SVE mouse is indicative of that for human CSF Aβ. This result, in conjunction with practical considerations, strongly suggests that the 129/SVE mouse is a suitable model for GSI discovery. Concurrently, the necessity and utilities of PK/PD modeling for rational interpretation of Aβ data are established.
- Published
- 2011
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19. Metabolism-directed design of oxetane-containing arylsulfonamide derivatives as γ-secretase inhibitors.
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Stepan AF, Karki K, McDonald WS, Dorff PH, Dutra JK, Dirico KJ, Won A, Subramanyam C, Efremov IV, O'Donnell CJ, Nolan CE, Becker SL, Pustilnik LR, Sneed B, Sun H, Lu Y, Robshaw AE, Riddell D, O'Sullivan TJ, Sibley E, Capetta S, Atchison K, Hallgren AJ, Miller E, Wood A, and Obach RS
- Subjects
- Amyloid beta-Peptides cerebrospinal fluid, Amyloid beta-Peptides metabolism, Animals, Brain metabolism, Cell Line, Crystallography, X-Ray, Dogs, Drug Design, Ethers, Cyclic metabolism, Ethers, Cyclic pharmacology, Humans, In Vitro Techniques, Mice, Microsomes, Liver metabolism, Oxidation-Reduction, Receptors, Notch metabolism, Stereoisomerism, Structure-Activity Relationship, Sulfonamides metabolism, Sulfonamides pharmacology, Tissue Distribution, Amyloid Precursor Protein Secretases antagonists & inhibitors, Ethers, Cyclic chemical synthesis, Sulfonamides chemical synthesis
- Abstract
A metabolism-based approach toward the optimization of a series of N-arylsulfonamide-based γ-secretase inhibitors is reported. The lead cyclohexyl analogue 6 suffered from extensive oxidation on the cycloalkyl motif by cytochrome P450 3A4, translating into poor human liver microsomal stability. Knowledge of the metabolic pathways of 6 triggered a structure-activity relationship study aimed at lowering lipophilicity through the introduction of polarity. This effort led to several tetrahydropyran and tetrahydrofuran analogues, wherein the 3- and 4-substituted variants exhibited greater microsomal stability relative to their 2-substituted counterparts. Further reduction in lipophilicity led to the potent γ-secretase inhibitor and 3-substituted oxetane 1 with a reduced propensity toward oxidative metabolism, relative to its 2-substituted isomer. The slower rates of metabolism with 3-substituted cyclic ethers most likely originate from reductions in lipophilicity and/or unfavorable CYP active site interactions with the heteroatom. Preliminary animal pharmacology studies with a representative oxetane indicate that the series is generally capable of lowering Aβ in vivo. As such, the study also illustrates the improvement in druglikeness of molecules through the use of the oxetane motif.
- Published
- 2011
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20. Design, synthesis, and in vivo characterization of a novel series of tetralin amino imidazoles as γ-secretase inhibitors: discovery of PF-3084014.
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Brodney MA, Auperin DD, Becker SL, Bronk BS, Brown TM, Coffman KJ, Finley JE, Hicks CD, Karmilowicz MJ, Lanz TA, Liston D, Liu X, Martin BA, Nelson RB, Nolan CE, Oborski CE, Parker CP, Richter KE, Pozdnyakov N, Sahagan BG, Schachter JB, Sokolowski SA, Tate B, Wood DE, Wood KM, Van Deusen JW, and Zhang L
- Subjects
- Animals, Biological Assay, Drug Design, Enzyme Inhibitors chemistry, Guinea Pigs, Imidazoles chemical synthesis, Imidazoles chemistry, Imidazoles pharmacology, Inhibitory Concentration 50, Molecular Structure, Structure-Activity Relationship, Tetrahydronaphthalenes chemistry, Valine chemical synthesis, Valine chemistry, Valine pharmacology, Amyloid Precursor Protein Secretases antagonists & inhibitors, Enzyme Activation drug effects, Enzyme Inhibitors chemical synthesis, Enzyme Inhibitors pharmacology, Tetrahydronaphthalenes chemical synthesis, Tetrahydronaphthalenes pharmacology, Valine analogs & derivatives
- Abstract
A novel series of tetralin containing amino imidazoles, derived from modification of the corresponding phenyl acetic acid derivatives is described. Replacement of the amide led to identification of a potent series of tetralin-amino imidazoles with robust central efficacy. The reduction of brain Aβ in guinea pigs in the absence of changes in B-cells suggested a potential therapeutic index with respect to APP processing compared with biomarkers of notch related toxicity. Optimization of the FTOC to plasma concentrations at the brain Aβ EC(50) lead to the identification of compound 14f (PF-3084014) which was selected for clinical development., (Copyright © 2011 Elsevier Ltd. All rights reserved.)
- Published
- 2011
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21. Diamide amino-imidazoles: a novel series of γ-secretase inhibitors for the treatment of Alzheimer's disease.
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Brodney MA, Auperin DD, Becker SL, Bronk BS, Brown TM, Coffman KJ, Finley JE, Hicks CD, Karmilowicz MJ, Lanz TA, Liston D, Liu X, Martin BA, Nelson RB, Nolan CE, Oborski CE, Parker CP, Richter KE, Pozdnyakov N, Sahagan BG, Schachter JB, Sokolowski SA, Tate B, Van Deusen JW, Wood DE, and Wood KM
- Subjects
- Amination drug effects, Amyloid beta-Peptides blood, Amyloid beta-Peptides metabolism, Animals, Biological Assay, Diamide chemical synthesis, Diamide chemistry, Diamide pharmacology, Enzyme Inhibitors chemistry, Guinea Pigs, HeLa Cells, Humans, Imidazoles chemistry, Inhibitory Concentration 50, Molecular Structure, Structure-Activity Relationship, Alzheimer Disease, Amyloid Precursor Protein Secretases antagonists & inhibitors, Enzyme Activation drug effects, Enzyme Inhibitors chemical synthesis, Enzyme Inhibitors pharmacology, Imidazoles chemical synthesis, Imidazoles pharmacology
- Abstract
The synthesis and structure-activity relationship (SAR) of a novel series of di-substituted imidazoles, derived from modification of DAPT, are described. Subsequent optimization led to identification of a highly potent series of inhibitors that contain a β-amine in the imidazole side-chain resulting in a robust in vivo reduction of plasma and brain Aβ in guinea pigs. The therapeutic index between Aβ reductions and changes in B-cell populations were studied for compound 10 h., (Copyright © 2011 Elsevier Ltd. All rights reserved.)
- Published
- 2011
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22. Pharmacodynamics and pharmacokinetics of the gamma-secretase inhibitor PF-3084014.
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Lanz TA, Wood KM, Richter KE, Nolan CE, Becker SL, Pozdnyakov N, Martin BA, Du P, Oborski CE, Wood DE, Brown TM, Finley JE, Sokolowski SA, Hicks CD, Coffman KJ, Geoghegan KF, Brodney MA, Liston D, and Tate B
- Subjects
- Animals, B-Lymphocytes cytology, B-Lymphocytes drug effects, Brain drug effects, Brain enzymology, Cell Line, Dose-Response Relationship, Drug, Enzyme Inhibitors adverse effects, Enzyme Inhibitors chemistry, Escherichia coli genetics, Female, Guinea Pigs, Humans, Lymphocyte Count, Male, Mice, Mice, Inbred Strains, Molecular Structure, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Spleen cytology, Spleen drug effects, Tetrahydronaphthalenes adverse effects, Tetrahydronaphthalenes chemistry, Tissue Distribution, Transfection, Valine adverse effects, Valine chemistry, Valine pharmacokinetics, Valine pharmacology, Amyloid Precursor Protein Secretases antagonists & inhibitors, Enzyme Inhibitors pharmacokinetics, Enzyme Inhibitors pharmacology, Tetrahydronaphthalenes pharmacokinetics, Tetrahydronaphthalenes pharmacology, Valine analogs & derivatives
- Abstract
PF-3084014 [(S)-2-((S)-5,7-difluoro-1,2,3,4-tetrahydronaphthalen-3-ylamino)-N-(1-(2-methyl-1-(neopentylamino)propan-2-yl)-1H-imidazol-4-yl)pentanamide] is a novel gamma-secretase inhibitor that reduces amyloid-beta (Abeta) production with an in vitro IC(50) of 1.2 nM (whole-cell assay) to 6.2 nM (cell-free assay). This compound inhibits Notch-related T- and B-cell maturation in an in vitro thymocyte assay with an EC(50) of 2.1 microM. A single acute dose showed dose-dependent reduction in brain, cerebrospinal fluid (CSF), and plasma Abeta in Tg2576 mice as measured by enzyme-linked immunosorbent assay and immunoprecipitation (IP)/mass spectrometry (MS). Guinea pigs were dosed with PF-3084014 for 5 days via osmotic minipump at 0.03 to 3 mg/kg/day and exhibited dose-dependent reduction in brain, CSF, and plasma Abeta. To further characterize Abeta dynamics in brain, CSF, and plasma in relation to drug exposure and Notch-related toxicities, guinea pigs were dosed with 0.03 to 10 mg/kg PF-3084014, and tissues were collected at regular intervals from 0.75 to 30 h after dose. Brain, CSF, and plasma all exhibited dose-dependent reductions in Abeta, and the magnitude and duration of Abeta lowering exceeded those of the reductions in B-cell endpoints. Other gamma-secretase inhibitors have shown high potency at elevating Abeta in the conditioned media of whole cells and the plasma of multiple animal models and humans. Such potentiation was not observed with PF-3084014. IP/MS analysis, however, revealed dose-dependent increases in Abeta11-40 and Abeta1-43 at doses that potently inhibited Abeta1-40 and Abeta1-42. PF-3084014, like previously described gamma-secretase inhibitors, preferentially reduced Abeta1-40 relative to Abeta1-42. Potency at Abeta relative to Notch-related endpoints in vitro and in vivo suggests that a therapeutic index can be achieved with this compound.
- Published
- 2010
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23. Anesthesia leads to tau hyperphosphorylation through inhibition of phosphatase activity by hypothermia.
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Planel E, Richter KE, Nolan CE, Finley JE, Liu L, Wen Y, Krishnamurthy P, Herman M, Wang L, Schachter JB, Nelson RB, Lau LF, and Duff KE
- Subjects
- Anesthetics administration & dosage, Anesthetics adverse effects, Animals, Hippocampus drug effects, Hippocampus enzymology, Hippocampus metabolism, Hypothermia enzymology, Male, Mice, Phosphorylation drug effects, Protein Phosphatase 2, Anesthesia adverse effects, Hypothermia metabolism, Phosphoprotein Phosphatases antagonists & inhibitors, Phosphoprotein Phosphatases metabolism, tau Proteins metabolism
- Abstract
Postoperative cognitive dysfunction, confusion, and delirium are common after general anesthesia in the elderly, with symptoms persisting for months or years in some patients. Even middle-aged patients are likely to have postoperative cognitive dysfunction for months after surgery, and Alzheimer's disease (AD) patients appear to be particularly at risk of deterioration after anesthesia. Several investigators have thus examined whether general anesthesia is associated with AD, with some studies suggesting that exposure to anesthetics may increase the risk of AD. However, little is known on the biochemical consequences of anesthesia on pathogenic pathways in vivo. Here, we investigated the effect of anesthesia on tau phosphorylation and amyloid precursor protein (APP) metabolism in mouse brain. We found that, regardless of the anesthetic used, anesthesia induced rapid and massive hyperphosphorylation of tau, rapid and prolonged hypothermia, inhibition of Ser/Thr PP2A (protein phosphatase 2A), but no changes in APP metabolism or Abeta (beta-amyloid peptide) accumulation. Reestablishing normothermia during anesthesia completely rescued tau phosphorylation to normal levels. Our results indicate that changes in tau phosphorylation were not a result of anesthesia per se, but a consequence of anesthesia-induced hypothermia, which led to inhibition of phosphatase activity and subsequent hyperphosphorylation of tau. These findings call for careful monitoring of core temperature during anesthesia in laboratory animals to avoid artifactual elevation of protein phosphorylation. Furthermore, a thorough examination of the effect of anesthesia-induced hypothermia on the risk and progression of AD is warranted.
- Published
- 2007
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24. Concentration-dependent modulation of amyloid-beta in vivo and in vitro using the gamma-secretase inhibitor, LY-450139.
- Author
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Lanz TA, Karmilowicz MJ, Wood KM, Pozdnyakov N, Du P, Piotrowski MA, Brown TM, Nolan CE, Richter KE, Finley JE, Fei Q, Ebbinghaus CF, Chen YL, Spracklin DK, Tate B, Geoghegan KF, Lau LF, Auperin DD, and Schachter JB
- Subjects
- Alanine pharmacology, Animals, Cells, Cultured, Dose-Response Relationship, Drug, Guinea Pigs, Male, Mice, Time Factors, Alanine analogs & derivatives, Amyloid Precursor Protein Secretases antagonists & inhibitors, Amyloid beta-Peptides blood, Azepines pharmacology, Enzyme Inhibitors pharmacology
- Abstract
LY-450139 is a gamma-secretase inhibitor shown to have efficacy in multiple cellular and animal models. Paradoxically, robust elevations of plasma amyloid-beta (Abeta) have been reported in dogs and humans after administration of subefficacious doses. The present study sought to further evaluate Abeta responses to LY-450139 in the guinea pig, a nontransgenic model that has an Abeta sequence identical to that of human. Male guinea pigs were treated with LY-450139 (0.2-60 mg/kg), and brain, cerebrospinal fluid, and plasma Abeta levels were characterized at 1, 3, 6, 9, and 14 h postdose. Low doses significantly elevated plasma Abeta levels at early time points, with return to baseline within hours. Higher doses inhibited Abeta levels in all compartments at early time points, but elevated plasma Abeta levels at later time points. To determine whether this phenomenon occurs under steady-state drug exposure, guinea pigs were implanted with subcutaneous minipumps delivering LY-450139 (0.3-30 mg/kg/day) for 5 days. Plasma Abeta was significantly inhibited at 10-30 mg/kg/day, but significantly elevated at 1 mg/kg/day. To further understand the mechanism of Abeta elevation by LY-450139, H4 cells overexpressing the Swedish mutant of amyloid-precursor protein and a mouse embryonic stem cell-derived neuronal cell line were studied. In both cellular models, elevated levels of secreted Abeta were observed at subefficacious concentrations, whereas dose-responsive inhibition was observed at higher concentrations. These results suggest that LY-450139 modulates the gamma-secretase complex, eliciting Abeta lowering at high concentrations but Abeta elevation at low concentrations.
- Published
- 2006
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25. Substituted 6-phenyl-pyridin-2-ylamines: selective and potent inhibitors of neuronal nitric oxide synthase.
- Author
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Nason DM, Heck SD, Bodenstein MS, Lowe JA 3rd, Nelson RB, Liston DR, Nolan CE, Lanyon LF, Ward KM, and Volkmann RA
- Subjects
- Amines pharmacology, Enzyme Inhibitors pharmacology, Humans, Neural Inhibition drug effects, Nitric Oxide Synthase Type I, Pyridines pharmacology, Amines chemistry, Enzyme Inhibitors chemistry, Nerve Tissue Proteins antagonists & inhibitors, Nitric Oxide Synthase antagonists & inhibitors, Pyridines chemistry
- Abstract
The synthesis and nNOS and eNOS activity of 6-(4-(dimethylaminoalkyl)-/6-(4-(dimethylaminoalkoxy)-5-ethyl-2-methoxyphenyl)-pyridin-2-ylamines and 6-(4-(dimethylaminoalkyl)-/6-(4-(dimethylaminoalkoxy)-2,5-dimethoxyphenyl)-pyridin-2-ylamines 1-8 are described. These compounds are potent inhibitors of the human nNOS isoform.
- Published
- 2004
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26. Telomere shortening and cellular senescence in a model of chronic renal allograft rejection.
- Author
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Joosten SA, van Ham V, Nolan CE, Borrias MC, Jardine AG, Shiels PG, van Kooten C, and Paul LC
- Subjects
- Animals, Cell Cycle genetics, Chronic Disease, Disease Models, Animal, Graft Rejection pathology, Ischemia, Kidney, Male, Rats, Rats, Inbred F344, Rats, Inbred Lew, Reperfusion, Transplantation, Homologous, Transplantation, Isogeneic, Cellular Senescence genetics, Graft Rejection genetics, Kidney Transplantation pathology, Telomere genetics
- Abstract
Cellular senescence has been suggested to play a role in the deterioration of renal graft function and has been linked to telomere shortening. We have investigated markers of cellular senescence in the F344 to LEW rat model of chronic renal transplant rejection. Syngeneic and LEW to F344 transplants were used as controls. Substantial telomere shortening was observed in all transplants, including allogeneic and syngeneic grafts from day 7 post-transplant onwards. Ischemia of native F344 kidneys was already sufficient to induce telomere shortening. It is known that shortened telomeres can activate cell cycle regulators, such as p21 and p16. Accordingly, all cases showed a transient p21 increase, with a maximum at day 7 and a sustained expression of p16. Importantly, senescence-associated beta-galactosidase staining, a cytological marker for senescence, was only observed in tubular epithelial cells of chronically rejecting F344 allografts from day 30 post-transplantation onwards. Long-term surviving LEW allografts or syngeneic F344 grafts were negative for senescence-associated beta-galactosidase. In conclusion, ischemia during transplantation results in telomere shortening and subsequent activation of p21 and p16, whereas senescence-associated beta-galactosidase staining is only present in chronically rejecting kidney grafts.
- Published
- 2003
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27. Proliferation in Adrenocortical Tumors: Correlation with Clinical Outcome and p53 Status.
- Author
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McNicol AM, Struthers AL, Nolan CE, Hermans J, and Haak HR
- Abstract
There appears to be a relationship between mitotic activity and malignant behavior in adrenocortical tumors, and carcinomas with a high mitotic index may have a poorer prognosis. This has been investigated further by quantifying and comparing the Ki-67 index using antibody MIB-1 in a series of 14 adrenocortical adenomas and 40 carcinomas. The levels have been correlated with survival and disease-free survival in carcinomas and with evidence of abnormal p53 expression as detected by immunohistochemistry. Nevertheless, many carcinomas have a low level of proliferation that may reflect varying abnormalities within the regulation of both cell division and apoptosis. Expression of bcl-2 protein, an inhibitor of apoptosis has therefore also been examined. The Ki-67 index in carcinomas was significantly higher than in adenomas, but below 4% there was overlap. There was no significant difference in survival between carcinomas with MIB-1 index <3% and those greater, but the lower group had significantly longer disease-free survival (p = 0.02). There was no significant difference between p53 immunopositive and p53 immunonegative carcinomas. No tumor showed immunopositivity for bcl-2 protein. It is concluded that MIB-1 index may contribute additional prognostic information in adrenocortical tumors. Inhibition of apoptosis by bcl-2 does not appear to play a role in tumor growth.
- Published
- 1997
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28. Expression of p53 in adrenocortical tumours: clinicopathological correlations.
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McNicol AM, Nolan CE, Struthers AJ, Farquharson MA, Hermans J, and Haak HR
- Subjects
- Adrenal Cortex Neoplasms genetics, Adrenal Cortex Neoplasms surgery, Adrenocortical Adenoma genetics, Adrenocortical Adenoma surgery, Adrenocortical Carcinoma genetics, Adult, Female, Genes, p53, Humans, Immunohistochemistry, Male, Middle Aged, Mutation, Neoplasm Proteins metabolism, Ploidies, Polymerase Chain Reaction, Polymorphism, Single-Stranded Conformational, Survival Rate, Adrenal Cortex Neoplasms metabolism, Adrenocortical Adenoma metabolism, Adrenocortical Carcinoma metabolism, Biomarkers, Tumor metabolism, Tumor Suppressor Protein p53 metabolism
- Abstract
There are limited data to suggest that abnormalities of p53 expression may be a late event in the development of adrenocortical tumours. This has been investigated further by examining a series of adrenocortical adenomas and carcinomas by immunohistochemistry for p53 expression and a subset for evidence of mutation in exons 5-8 of the p53 gene using polymerase chain reaction/single strand conformational polymorphism (PCR/SSCP). In carcinomas, the findings have been correlated with survival data and with tumour ploidy. Immunopositivity for p53 was seen in 4 of 34 adenomas and 22 of 42 carcinomas. Mobility shifts were identified in 1 of 4 adenomas and 10 of 21 carcinomas. There was no correlation between immunostaining pattern or PCR/SSCP evidence of mutation and either survival or disease-free survival in carcinoma. There was also no correlation between p53 status and tumour ploidy. While these findings support a role for p53 in tumour progression, abnormal p53 expression does not appear to have any significant prognostic effect in carcinoma.
- Published
- 1997
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29. Identification of membrane-bound carbonic anhydrase in white matter coated vesicles: the fate of carbonic anhydrase and other white matter coated vesicle proteins in triethyl tin-induced leukoencephalopathy.
- Author
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Sapirstein VS, Durrie R, Nolan CE, and Marks N
- Subjects
- Animals, Blotting, Western, Brain drug effects, Brain pathology, Carbonic Anhydrases analysis, Cell Membrane enzymology, Coated Pits, Cell-Membrane drug effects, Electrophoresis, Polyacrylamide Gel, Membrane Proteins analysis, Myelin Sheath drug effects, Nerve Tissue Proteins analysis, Rats, Rats, Sprague-Dawley, Receptor, IGF Type 2 metabolism, Synaptophysin analysis, Brain enzymology, Carbonic Anhydrases metabolism, Coated Pits, Cell-Membrane enzymology, Membrane Proteins metabolism, Myelin Sheath enzymology, Nerve Tissue Proteins metabolism, Triethyltin Compounds toxicity
- Abstract
We have extended our studies on the content of white matter derived coated vesicles (WMCVs) to show that they are enriched in membrane-bound carbonic anhydrase. Within the myelin complex membrane-bound carbonic anhydrase is concentrated in the periaxolemmal domain; however, this protein is enriched almost sevenfold in the bilayer of coated vesicles even relative to this myelin membrane region. These data suggest that some vesicles are derived from a site at which this enzyme is highly localized. The enrichment observed for membrane-bound carbonic anhydrase is unique since other periaxolemmal proteins such as CNPase and plasmolipin are only present in equal amounts in periaxolemmal-myelin fractions and WMCVs. Based on their known localization, the presence of CNPase coupled with the absence of MAG in WMCVs suggest that these vesicles are derived from the paranodal region. The identification in WMCVs of periaxolemmal-myelin proteins associated with ion and fluid movement, such as carbonic anhydrase, Na+,K+ ATPase, and the putative K+ channel protein plasmolipin, prompted us to examine the status of these vesicles in triethyl tin (TET)-induced myelin edema. Coated vesicles and other membrane fractions were isolated from whole brains of control and TET-treated rats. Whole brains were used so we could compare the effects of TET on WMCV proteins with the effect on proteins enriched in gray matter coated vesicles. The results indicated that TET had no detectable effect on compact or periaxolemmal-myelin, however, Western blot analysis showed that WMCV proteins, such as carbonic anhydrase, CNPase, and plasmolipin, were virtually absent or greatly diminished from the whole brain coated vesicle fraction.(ABSTRACT TRUNCATED AT 250 WORDS)
- Published
- 1993
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30. Identification of plasmolipin as a major constituent of white matter clathrin-coated vesicles.
- Author
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Sapirstein VS, Nolan CE, Stern R, Gray-Board G, and Beard ME
- Subjects
- Animals, Blotting, Western, Brain ultrastructure, Cattle, Cerebral Cortex metabolism, Coated Pits, Cell-Membrane ultrastructure, Corpus Callosum metabolism, Electrophoresis, Lipid Metabolism, Microscopy, Electron, Myelin Proteins metabolism, Myelin Sheath metabolism, Nerve Tissue Proteins metabolism, Periaqueductal Gray metabolism, Brain metabolism, Clathrin metabolism, Coated Pits, Cell-Membrane metabolism, Proteolipids metabolism
- Abstract
We have isolated and characterized coated vesicles from bovine white matter and compared them to those isolated from gray matter. The virtual absence of synaptic vesicle antigens in the white matter coated vesicles indicates they are distinct from those found in gray matter and from vesicles derived from synaptic membranes. The white matter coated vesicles also lack compact myelin components, e.g., the myelin proteolipid, galactocerebroside, and sulfatides, as well as the periaxolemmal myelin marker myelin-associated glycoprotein. On the other hand, these vesicles contain 2',3'-cyclic nucleotide phosphohydrolase. The vesicles also contain high levels of plasmolipin, a protein present in myelin and oligodendrocytes. Plasmolipin was found to be four to five times higher in white matter coated vesicles than in gray matter coated vesicles. Based on western blot quantitation, the concentration of plasmolipin in white matter coated vesicles is 3-4% of the vesicle bilayer protein. These studies indicate that a significant proportion of coated vesicles from white matter may be derived from unique membrane domains of the myelin complex or oligodendroglial membrane, which are enriched in plasmolipin.
- Published
- 1992
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31. Expression of plasmolipin in the developing rat brain.
- Author
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Sapirstein VS, Nolan CE, Stadler II, and Fischer I
- Subjects
- Animals, Animals, Newborn, Antibody Specificity, Brain embryology, Brain growth & development, Cells, Cultured, Dogs, GAP-43 Protein, Gene Expression Regulation, Kidney Tubules metabolism, Membrane Glycoproteins analysis, Myelin and Lymphocyte-Associated Proteolipid Proteins, Nerve Tissue Proteins analysis, Neurons chemistry, Oligodendroglia metabolism, Rats, Swine, Brain metabolism, Fetal Proteins biosynthesis, Membrane Lipids biosynthesis, Membrane Proteins biosynthesis, Nerve Tissue Proteins biosynthesis, Proteolipids biosynthesis
- Abstract
Plasmolipin is an hydrophobic plasma membrane proteolipid present in both kidney and brain. The protein consists of two subunits of 17-18.5 kD, which together form K+ selective voltage-dependent channels. In this report, we define the embryonic and postnatal expression of plasmolipin in the developing rat brain. Plasmolipin was found to be essentially restricted to the postnatal period increasing eight-fold between the first to fourth week after birth. A fetal plasmolipin immunoreactive protein (FPIP) was identified in embryonic brain and also during the early postnatal development of the cerebellum. The expression of FPIP was biphasic with an initial transient increase between E15-E20 followed by a decrease in its levels. FPIP was not detected in the developed rat CNS. FPIP was found in a variety of dividing and immature cells including cultured astrocytes and embryonic neurons, neuroblastoma cells, and rat thymus. In contrast, plasmolipin was restricted to oligodendrocytes of the neural cells tested and to renal tubular epithelial cells.
- Published
- 1992
- Full Text
- View/download PDF
32. The phylogenic expression of plasmolipin in the vertebrate nervous system.
- Author
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Sapirstein VS, Nolan CE, Fischer I, Cochary E, Blau S, and Flynn CJ
- Subjects
- Amphibians, Animals, Birds, Blotting, Western, Brain Chemistry, Cattle, Cell Membrane chemistry, Fishes, Humans, Mammals, Myelin Sheath chemistry, Myelin and Lymphocyte-Associated Proteolipid Proteins, Peripheral Nerves chemistry, Rats, Reptiles, Spinal Cord chemistry, Tissue Distribution, Membrane Proteins, Nerve Tissue Proteins, Nervous System chemistry, Phylogeny, Proteolipids analysis
- Abstract
Plasmolipin is a plasma membrane proteolipid is a major myelin membrane component (Cochary et al., 1990). In this study we report the phylogenic expression of plasmolipin in the vertebrate nervous system. Using Western blot analysis with polyclonal antibodies, we have analyzed membrane fractions, including myelin, from elasmobranchs, teleosts, amphibians, reptiles, birds and mammals. On the basis of immune detection, plasmolipin appears to be restricted to the mammalian nervous system. Comparison of the central and peripheral nervous systems of mammals showed only minor differences in the level of plasmolipin in these two regions. Within mammals, little quantitative differences were observed when rat, human and bovine membrane fractions were compared. The late evolutionary expression of plasmolipin which results in its restriction to mammals makes it unique among the (major) myelin proteins. The potential physiologic significance of these data are discussed.
- Published
- 1991
- Full Text
- View/download PDF
33. Presence of the plasma membrane proteolipid (plasmolipin) in myelin.
- Author
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Cochary EF, Bizzozero OA, Sapirstein VS, Nolan CE, and Fischer I
- Subjects
- Animals, Apoproteins analysis, Brain Chemistry, Cattle, Cell Membrane analysis, Centrifugation, Density Gradient, Humans, Immunoblotting, Immunohistochemistry, Kidney analysis, Mice, Myelin Basic Protein analysis, Myelin Proteins analysis, Myelin and Lymphocyte-Associated Proteolipid Proteins, Oligodendroglia analysis, Rats, Sheep, Sodium-Potassium-Exchanging ATPase metabolism, Tissue Distribution, Membrane Proteins, Myelin Proteolipid Protein, Myelin Sheath analysis, Nerve Tissue Proteins, Proteolipids analysis
- Abstract
Plasma membrane proteolipid (plasmolipin), which was originally isolated from kidney membranes, has also been shown to be present in brain. In this study, we examined the distribution of plasmolipin in brain regions, myelin, and oligodendroglial membranes. Immunoblot analysis of different brain regions revealed that plasmolipin levels were higher in regions rich in white matter. Plasmolipin was also detected in myelin, myelin subfractions, and oligodendroglial membranes. Immunocytochemical analysis of the cerebellum revealed that plasmolipin was localized in the myelinated tracts. Plasmolipin levels in myelin were enriched during five successive cycles of myelin purification, similar to the enrichment of myelin proteolipid apoprotein (PLP) and myelin basic protein (MBP). In contrast, levels of Na+,K(+)-ATPase and a 70-kDa protein were decreased. When myelin or white matter was extracted with chloroform/methanol, it contained, in addition to PLP, a significant amount of plasmolipin. Quantitative immunoblot analysis suggested that plasmolipin constitutes in the range of 2.2-4.8% of total myelin protein. Plasmolipin, purified from kidney membranes, was detected by silver stain on gels at 18 kDa and did not show immunological cross-reactivity with either PLP or MBP. Thus, it is concluded that plasmolipin is present in myelin, possibly as a component of the oligodendroglial plasma membrane, but is structurally and immunologically different from the previously characterized myelin proteolipids.
- Published
- 1990
- Full Text
- View/download PDF
34. Composition and metabolism of gangliosides in rat peripheral nervous system during development.
- Author
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Chou KH, Nolan CE, and Jungalwala FB
- Subjects
- Animals, Chickens, Dogs, G(M3) Ganglioside metabolism, Galactosyltransferases metabolism, Mice, Neuraminidase metabolism, Rabbits, Rats, Rats, Inbred Strains, Schwann Cells metabolism, Sialyltransferases metabolism, Species Specificity, Trigeminal Nerve metabolism, Gangliosides metabolism, N-Acetylgalactosaminyltransferases, Trigeminal Nerve growth & development
- Published
- 1982
- Full Text
- View/download PDF
35. A clinical, epidemiologic, serologic, and virologic study of influenza C virus infection.
- Author
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Dykes AC, Cherry JD, and Nolan CE
- Subjects
- Adolescent, Adult, Antibodies, Viral immunology, Child, Child, Preschool, Hemagglutination Inhibition Tests, Humans, Influenza A virus isolation & purification, Orthomyxoviridae isolation & purification, Influenza, Human epidemiology, Influenza, Human immunology, Influenza, Human microbiology
- Abstract
During a study of influenza-like illness in employees in the Pediatric Clinic at UCLA Hospital and Clinics in late November 1978, an influenza C viral strain was recovered from one employee, one person had more than a fourfold hemagglutination inhibition antibody titer rise to influenza C, and one person had specific influenza C IgM antibody. A survey of 334 children and young adults noted a seropositivity rate to influenza C of 64% for children up to 5 years old; 96% for 6- to 10-year-olds; 100% for 11- to 15-year-olds; and 98% for those over 16 years old. The 64% seropositivity of those children 5 years old and younger indicates that infection with influenza C early in life is common. The increasing seropositivity rates with age suggest that circulation and reinfection with influenza C commonly occurs.
- Published
- 1980
36. Pre-packed reverse phase columns for isolation of complex lipids synthesized from radioactive precursors.
- Author
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Figlewicz DA, Nolan CE, Singh IN, and Jungalwala FB
- Subjects
- Animals, Brain metabolism, Carbon Radioisotopes, Cerebrosides isolation & purification, Chromatography, High Pressure Liquid methods, Gangliosides isolation & purification, In Vitro Techniques, Isotope Labeling, Lipids biosynthesis, Male, Phosphatidylcholines isolation & purification, Rats, Rats, Inbred Strains, Sulfoglycosphingolipids isolation & purification, Sulfur Radioisotopes, Lipids isolation & purification
- Abstract
Pre-packed reverse phase columns (Bond Elut) were used for the separation of complex lipids, such as phosphatidylcholine, cerebrosides, sulfatides, and gangliosides, from their respective water-soluble radioactive precursors after their in vitro biosynthesis. After an incubation in vitro, the entire reaction mixture is passed through the column, where complex lipids are retained and the hydrophilic radioactive precursors are washed away from the column. The retained lipids are then eluted with a more nonpolar organic solvent. The procedure is shown to be simpler and more efficient than the normally used Folch partitioning method or other procedures.
- Published
- 1985
37. Subcellular fractionation of rat sciatic nerve and specific localization of ganglioside LM1 in rat nerve myelin.
- Author
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Chou KH, Nolan CE, and Jungalwala FB
- Subjects
- Animals, Cell Membrane analysis, Chromatography, High Pressure Liquid, Chromatography, Thin Layer, Electrophoresis, Polyacrylamide Gel, Female, Glycolipids analysis, Male, Membrane Proteins analysis, Microscopy, Electron, Rats, Rats, Inbred Strains, Sciatic Nerve ultrastructure, Subcellular Fractions analysis, Gangliosides analysis, Myelin Sheath analysis, Sciatic Nerve analysis
- Abstract
Subcellular fractionation of rat sciatic nerve was developed to determine the specific localization of gangliosides in the nerve membrane fractions. Myelin, microsomal, and a plasma membrane-like fraction were isolated and purified by sucrose density gradient centrifugation. These subfractions were characterized by electron microscopy, marker enzyme assays, and their protein and lipid profile. In rat sciatic nerve myelin, 90 mol% of the total gangliosides were monosialogangliosides. LM1 (sialosyl-lactoneotetraosylceramide) (61 mol%) and GM3 (21%) were the major gangliosides of the rat nerve myelin. Two other neolacto series of gangliosides, viz., sialosyl-lactoneonorhexaosylceramide and sialosyl-lactoneooctaosylceramide, were also localized mostly in the myelin fraction. GM1 was only a minor (less than 2%) ganglioside in myelin. The ganglioside patterns of the microsomal and plasma membrane-like fractions were similar with minor quantitative differences and were entirely different from that of myelin. Monosialogangliosides were approximately 70-75 mol% of the total in these fractions. The major gangliosides of the microsomal and plasma membrane-like fractions were GM3 (approximately 40%) and GM1 (approximately 20%). LM1 in these fractions was minimal (less than approximately 5%). Significant amounts of GM3 with N-glycolylneuraminic acid (approximately 10%) and GM1b (4-14%) were also identified in the microsomal and plasma membrane-like fractions but not in myelin. These and the higher lactoneo series of gangliosides have not been previously reported to be present in the rat nervous system. Almost exclusive localization of LM1 in myelin in rat peripheral nervous system is consistent with our previous observation that deposition of LM1 in the nerve with age was very similar to that of myelin marker lipids cerebrosides and sulfatides.
- Published
- 1985
- Full Text
- View/download PDF
38. Liver ornithine decarboxylase during phenobarbital promotion of nitrosamine carcinogenesis.
- Author
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Farwell DC, Nolan CE, and Herbst EJ
- Subjects
- Animals, Liver Neoplasms chemically induced, Male, Neoplasms, Experimental metabolism, Rats, Spermidine metabolism, Spermine metabolism, Carboxy-Lyases metabolism, Diethylnitrosamine, Liver enzymology, Liver Neoplasms metabolism, Nitrosamines, Ornithine Decarboxylase metabolism, Phenobarbital toxicity
- Abstract
The incidence of liver tumors induced in rats by N-diethylnitrosamine was increased by the feeding of phenobarbital. Liver ornithine decarboxylase activity did not increase in animals receiving phenobarbital in the diet, and the concentration of polyamines in the liver was similarly unchanged. No relationship between the promotion of N-nitrosamine-induced liver tumors by phenobarbital and the ornithine decarboxylase activity of the liver was indicated by these experiments.
- Published
- 1978
- Full Text
- View/download PDF
39. Effects of retinoic acid on expression of the transformed phenotype in C6 glioma cells.
- Author
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Fischer I, Nolan CE, and Shea TB
- Subjects
- Animals, Cell Line, Glycoside Hydrolases metabolism, Kinetics, Lysosomes enzymology, Ornithine Decarboxylase Inhibitors, Phenotype, Rats, Cell Division drug effects, Glioma pathology, Tretinoin pharmacology
- Abstract
Retinoic acid (RA) inhibited the growth and induced morphological changes in C6 rat glioma cells. The effects of RA on growth rate became apparent after 48 hr and were concentration-dependent and reversible. There was a 60% inhibition of growth using 10(-5) RA, which increased at low serum concentration to over 90% inhibition and was minimized at high concentration of serum. RA did not change the saturation density of the cells. The morphology of C6 cells, was altered from its normal pattern of randomly oriented spindle shaped cells, to cells which aligned to form palisades of fibroblast-like cells. Biochemical analysis of the cells showed no significant change in the activities of several lysosomal hydrolyses or the level of total protein in RA-treated cells compared to control cells. There was, however, a significant decrease in the activity of ornithine decarboxylase early during the treatment with RA, and an increase in the levels of fibronectin secreted into the media by the RA-treated cell. These results suggest that RA can suppress the expression of the transformed phenotype of glioma cells.
- Published
- 1987
- Full Text
- View/download PDF
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