- Email: [email protected]
A NOVEL GABAAA5 NEGATIVE ALLOSTERIC MODULATOR ENHANCES LONG-TERM POTENTIATION AND IMPROVES COGNITION IN PRECLINICAL MODELS
P1016
Poster Presentations: Wednesday, July 27, 2016
were significant associations between memory test score and e4, age, education level, and gender. There was a significant e4 x age interaction (p¼0.03). We found no associations between e4+ and processing speed or attention, or between family history of e4+ and cognitive test scores. There were significant associations between self-reported memory problems and both e4+ (p<0.001) and family history of e4+ (p<0.001). We identified 2032 “likely prodromal” and 6227 “likely preclinical” participants for AD trials. Of those likely eligible participants who reported their e4 genotype (n¼1650), 16% of prodromal and 4% of preclinical were e4+. Conclusions: In a large, novel, internet-based cohort, self-reported e4 is associated with online memory test scores in younger participants, and cognitively-normal participants. In the general cohort, age eclipses the effects of e4 genotype on memory scores. Self-reported memory problems are strongly associated with e4 even when controlling for other variables. BHR participants identified as likely prodromal have four times greater prevalence of e4+ than older adults with normal cognitive test scores. Self-report of e4 in BHR can be used to prescreen for AD trials requiring e4, facilitate AD trials, and accelerate development of new AD treatments.
Platform uses electronic source data collection with real-time clinical guidance to standardize outcome measurements and improve data quality. In the present study, we compared paper-based administrations against administration with Virgil to determine the extent to which the use of electronic assessments (eCOA) minimized scoring errors in primary and co-primary endpoints of AD trials. Methods: Paper-based assessments from a recent clinical trial of mild cognitive impairment (MCI) were compared against eCOA administrations of the same scales in separate MCI trials. All studies are phase II/III multinational trials. Score discrepancies in CDR, ADAS-Cog, ADCS-ADL-MCI, and MMSE were identified via review of audio recordings and worksheets by the same cohort of expert calibrated reviewers. For each scale, discrepancies were compared between paper-based and Virgil administrations. Results: Percentages of reviews with at least one scoring discrepancy, as well as two or more discrepancies, were significantly and substantially lower in Virgil administrations compared to paper-based for all scales. Conclusions: Paper-based administrations create unnecessary variability around endpoint measurements, which can contribute to inconclusive results. The Virgil eCOA platform with real-time clinical guidance, auto-calculation of scores, and prompts for missing data and out-of-range errors can help standardize scale administration and scoring, thereby substantially reducing error variance and improving signal detection. P4-007
A NOVEL GABAAA5 NEGATIVE ALLOSTERIC MODULATOR ENHANCES LONG-TERM POTENTIATION AND IMPROVES COGNITION IN PRECLINICAL MODELS
Miki Nakanishi1, Soichi Kawaharada1, Nobuto Nakanishi1, Keisuke Hazama1, Masato Higashino1, Arwel Lewis2, Gary S. Clark2, Mark S. Chambers2, Kim L. Hirst2, Scott A. Maidment2, Grant Wishart2, Tetsuya Yasuhiro1, Seishi Katsumata1, 1Ono Pharmaceutical Co., Ltd., Osaka, Japan; 2BioFocus, Saffron Walden, United Kingdom. Contact e-mail: [email protected] Figure 1. Association between age and online OCL scores in e4+ and e4BHR participants. Scatter plot showing the effect of age on online OCL scores in a cohort of BHR participants who self-report that they are e4 negative (black circles) and e4 positive (red circle). Linear regression analyses (solid red and black lines) show a significant age x e4 interaction (p ¼ 0.03). N¼1403.
P4-006
VIRGIL ELECTRONIC CLINICAL OUTCOME ASSESSMENTS (ECOA) PLATFORM: IMPROVING SIGNAL DETECTION IN ALZHEIMER’S DISEASE CLINICAL TRIALS
Selam Negash1, Peter Boehm1, Shelly Steele1, Peter Sorantin1, Christopher Randolph1,2, 1MedAvante, Inc., Hamilton, NJ, USA; 2Loyola University Medical Center, Chicago, IL, USA. Contact e-mail: snegash@ medavante.com Background: The impact of Alzheimer’s disease (AD) globally is
astounding; the number of people living with the disease is predicted to quadruple by year 2050 to 106 million. Nonetheless, drug development for AD has had among the lowest success rate for any therapeutic area. The high failure rate in AD trials has, in part, been due to imprecision in endpoint measurements, which introduces noise. Clinical outcome assessments (COAs) that use traditional paper-based administrations are known to be cost- and time-inefficient, but they also are subject to human error, which creates variability in measurement. The Virgil Investigative Study
Background: GABAA receptors are pentameric membrane proteins
that gate chloride ions in response to binding of g-aminobutyric acid (GABA). GABAA ion channels containing the a5 subunit (GABAAa5) are highly expressed in hippocampus, the key area for cognitive processing. GABAAa5 negative allosteric modulators (NAMs) enhance cognition in rodents. Similar phenotypes are observed in animals with point mutations of GABAAa5. We identified a novel orally available GABAAa5 NAM and investigated the effects on long-term potentiation (LTP) and cognition in rats. Methods: HEK-293 cell lines stably expressing human GABAA receptors containing a1, 2, 3 or 5 were used in receptor binding assay and PatchXpress automated electrophysiology system. For LTP study, normal and Ab25-35/ibotenate-lesioned rats were used. Ab25-35 (4 mg) and ibotenate (0.6 mg) were injected bilateraly into hippocampus of rats. After 5 weeks, hippocampal slices were prepared and LTP was measured electrophysiologically using a multielectrode array system. Cognition was evaluated in passive avoidance test, 8-arm radial maze test and novel object recognition test in rats. Anxiogenic-like and proconvulsant effects associated with non-selective GABAANAM activity were evaluated in rat elevated plus maze test and mouse PTZ test, respectively. All experimental procedures were approved by the Animal Care and Use Committee of ONO pharmaceutical Co., Ltd. and conducted in accordance with Guidance for Animal Experiments. Results: We identified a GABAAa5NAM with high affinity (Ki value ¼ 7.9 nmol/L), NAM activity (EC50 ¼ 1.1 nmol/L, Emax ¼ -50%), and selectivity
Poster Presentations: Wednesday, July 27, 2016
for a5 (Emax for GABAAa1, 2 and 3 ¼ -2%, 5% and -6%, respectively). This compound (300 nmol/L) significantly enhanced LTP in hippocampal slices from normal rats. Hippocampal injection of Ab and ibotenate impaired LTP, and GABAAa5NAM rescued this effect. GABAAa5NAM (3, 10 and 20 mg/kg, p.o.) significantly reversed MK-801-induced cognitive deficit in rat passive avoidance test. This compound (10 mg/kg, p.o.) significantly reversed scopolamine/MK-801-induced cognitive deficit in rat 8-arm radial maze test. Moreover, this compound (10 mg/kg, p.o.) significantly enhanced cognition in normal rat novel object recognition test. This compound did not show anxiogenic-like or proconvulsant effects. Conclusions: GABAAa5 NAM enhanced LTP in normal and Ab/ibotenate-lesioned hippocampal slices. This compound improved cognition without anxiogenic or proconvulsant side effects.
P4-008
P1017
CADRD, IDPH-ADRF 63282003D, IHIA, and the Kenneth Stark Endowment for Alzheimer’s Research.
Figure 1. Representative traces (left) from local application (arrow) of saline (blue, middle), 0.1 mM Ab42 (black, top), and 0.1 mM Ab42 with 10.0 mM TTx (red, bottom) in 6-9 month old C57BL/6J mice. Glutamate release (right; mean 6 SEM) from varying concentrations of Ab42. One-way ANOVA with Fisher’s LSD post hoc; *p<0.05, **p<0.01, ***p<0.001 vs. saline; $$$$p<0.0001 vs. 0.1 mM Ab42; n¼9 per group.
CAN ALZHEIMER’S-RELATED COGNITIVE DECLINE BE DELAYED THROUGH PRODROMAL TREATMENT? EVIDENCE FROM A MOUSE MODEL OF ALZHEIMER’S DISEASE
Kevin N. Hascup, Sarah O. Broderick, Erin R. Hascup, Southern Illinois University School of Medicine, Springfield, IL, USA. Contact e-mail: [email protected] Background: The pathophysiological cascade of Alzheimer’s dis-
ease (AD) postulates that amyloid-b (Ab)42 accumulation occurs years prior to cognitive decline. Evidence suggests that soluble Ab42 elicits glutamate release via a presynaptic mechanism. We sought to determine if hippocampal glutamatergic signaling is altered during the early stages of AD, and if so, can early intervention targeting the glutamatergic system alleviate cognitive decline. Methods: We utilized in vivo electrochemistry to selectively measure L-glutamate with an enzyme-based microelectrode array. Human Ab42 (pipette: 0.01 - 10.0 mM; recording site: 0.255-255.0 nM) dissolved in physiological saline (pH 7.4) was locally applied in the CA1 of isoflurane anesthetized 6-9 month male C57BL/6J mice and glutamate release was studied. Next, we examined 2-4, 6-8, and 1215 month AbPP/PS1 mice to understand how progressive accumulation of Ab42 alters glutamatergic neurotransmission. AbPP/PS1 and age matched C57BL/6J mice underwent an 8 day Morris water maze (MWM) paradigm to test spatial learning and memory. Following behavioral testing, mice were isoflurane anesthetized and basal and stimulus-evoked (70 mM KCl, isotonic, pH 7.4) glutamate release was measured in the CA1. Finally, LY379268 (a Group 2 metabotropic glutamate receptor agonist) was administered twice weekly (3.0 mg/kg i.p.) between 2-6 months of age, with MWM conducted at w11 months of age. Results: Local application of Ab42 elicited glutamate release in a dose-dependent manner that was blocked by tetrodotoxin (TTx; 10.0 mM; Figure 1). Evoked glutamate release was elevated in AbPP/PS1 mice at all age groups examined (Figure 2) and negatively correlated with memory as previously reported. Basal glutamate was elevated in AbPP/PS1 mice at 6-8 and 12-15 months, but not at 2-4 months (Figure 3). Overall, mice tolerated LY379268 injections and cognitive data will be presented. Conclusions: These data supports that Ab42 elicits glutamate release through a presynaptic mechanism that might lead to the elevated glutamate release and subsequent increased basal glutamate observed in AbPP/PS1 mice. Furthermore, targeting the glutamatergic system during pre-symptomatic AD may delay or prevent cognitive decline. Funding provided by
Figure 2. Local application of 70 mM KCl (100-200 nl) stimulated more glutamate release in the CA1 of AbPP/PS1 mice compared to C57BL/6J control mice at all ages examined. An age-related decrease in glutamate release in AbPP/PS1 mice was also observed. Two-way ANOVA with Fisher’s LSD post hoc; *p<0.05, ****p<0.001 vs. age-matched C57BL/ 6J mice; $$p<0.01 vs. 2-4 month AbPP/PS1 mice.
Figure 3. Basal glutamate was elevated in the CA1 of AbPP/PS1 mice at 6-8 and 12-15 months of age as compared to age-matched C57BL/6J control mice, but not at 2-4 months. Two-way ANOVA with Fisher’s LSD post hoc; *p<0.05.
Poster Presentations: Wednesday, July 27, 2016
were significant associations between memory test score and e4, age, education level, and gender. There was a significant e4 x age interaction (p¼0.03). We found no associations between e4+ and processing speed or attention, or between family history of e4+ and cognitive test scores. There were significant associations between self-reported memory problems and both e4+ (p<0.001) and family history of e4+ (p<0.001). We identified 2032 “likely prodromal” and 6227 “likely preclinical” participants for AD trials. Of those likely eligible participants who reported their e4 genotype (n¼1650), 16% of prodromal and 4% of preclinical were e4+. Conclusions: In a large, novel, internet-based cohort, self-reported e4 is associated with online memory test scores in younger participants, and cognitively-normal participants. In the general cohort, age eclipses the effects of e4 genotype on memory scores. Self-reported memory problems are strongly associated with e4 even when controlling for other variables. BHR participants identified as likely prodromal have four times greater prevalence of e4+ than older adults with normal cognitive test scores. Self-report of e4 in BHR can be used to prescreen for AD trials requiring e4, facilitate AD trials, and accelerate development of new AD treatments.
Platform uses electronic source data collection with real-time clinical guidance to standardize outcome measurements and improve data quality. In the present study, we compared paper-based administrations against administration with Virgil to determine the extent to which the use of electronic assessments (eCOA) minimized scoring errors in primary and co-primary endpoints of AD trials. Methods: Paper-based assessments from a recent clinical trial of mild cognitive impairment (MCI) were compared against eCOA administrations of the same scales in separate MCI trials. All studies are phase II/III multinational trials. Score discrepancies in CDR, ADAS-Cog, ADCS-ADL-MCI, and MMSE were identified via review of audio recordings and worksheets by the same cohort of expert calibrated reviewers. For each scale, discrepancies were compared between paper-based and Virgil administrations. Results: Percentages of reviews with at least one scoring discrepancy, as well as two or more discrepancies, were significantly and substantially lower in Virgil administrations compared to paper-based for all scales. Conclusions: Paper-based administrations create unnecessary variability around endpoint measurements, which can contribute to inconclusive results. The Virgil eCOA platform with real-time clinical guidance, auto-calculation of scores, and prompts for missing data and out-of-range errors can help standardize scale administration and scoring, thereby substantially reducing error variance and improving signal detection. P4-007
A NOVEL GABAAA5 NEGATIVE ALLOSTERIC MODULATOR ENHANCES LONG-TERM POTENTIATION AND IMPROVES COGNITION IN PRECLINICAL MODELS
Miki Nakanishi1, Soichi Kawaharada1, Nobuto Nakanishi1, Keisuke Hazama1, Masato Higashino1, Arwel Lewis2, Gary S. Clark2, Mark S. Chambers2, Kim L. Hirst2, Scott A. Maidment2, Grant Wishart2, Tetsuya Yasuhiro1, Seishi Katsumata1, 1Ono Pharmaceutical Co., Ltd., Osaka, Japan; 2BioFocus, Saffron Walden, United Kingdom. Contact e-mail: [email protected] Figure 1. Association between age and online OCL scores in e4+ and e4BHR participants. Scatter plot showing the effect of age on online OCL scores in a cohort of BHR participants who self-report that they are e4 negative (black circles) and e4 positive (red circle). Linear regression analyses (solid red and black lines) show a significant age x e4 interaction (p ¼ 0.03). N¼1403.
P4-006
VIRGIL ELECTRONIC CLINICAL OUTCOME ASSESSMENTS (ECOA) PLATFORM: IMPROVING SIGNAL DETECTION IN ALZHEIMER’S DISEASE CLINICAL TRIALS
Selam Negash1, Peter Boehm1, Shelly Steele1, Peter Sorantin1, Christopher Randolph1,2, 1MedAvante, Inc., Hamilton, NJ, USA; 2Loyola University Medical Center, Chicago, IL, USA. Contact e-mail: snegash@ medavante.com Background: The impact of Alzheimer’s disease (AD) globally is
astounding; the number of people living with the disease is predicted to quadruple by year 2050 to 106 million. Nonetheless, drug development for AD has had among the lowest success rate for any therapeutic area. The high failure rate in AD trials has, in part, been due to imprecision in endpoint measurements, which introduces noise. Clinical outcome assessments (COAs) that use traditional paper-based administrations are known to be cost- and time-inefficient, but they also are subject to human error, which creates variability in measurement. The Virgil Investigative Study
Background: GABAA receptors are pentameric membrane proteins
that gate chloride ions in response to binding of g-aminobutyric acid (GABA). GABAA ion channels containing the a5 subunit (GABAAa5) are highly expressed in hippocampus, the key area for cognitive processing. GABAAa5 negative allosteric modulators (NAMs) enhance cognition in rodents. Similar phenotypes are observed in animals with point mutations of GABAAa5. We identified a novel orally available GABAAa5 NAM and investigated the effects on long-term potentiation (LTP) and cognition in rats. Methods: HEK-293 cell lines stably expressing human GABAA receptors containing a1, 2, 3 or 5 were used in receptor binding assay and PatchXpress automated electrophysiology system. For LTP study, normal and Ab25-35/ibotenate-lesioned rats were used. Ab25-35 (4 mg) and ibotenate (0.6 mg) were injected bilateraly into hippocampus of rats. After 5 weeks, hippocampal slices were prepared and LTP was measured electrophysiologically using a multielectrode array system. Cognition was evaluated in passive avoidance test, 8-arm radial maze test and novel object recognition test in rats. Anxiogenic-like and proconvulsant effects associated with non-selective GABAANAM activity were evaluated in rat elevated plus maze test and mouse PTZ test, respectively. All experimental procedures were approved by the Animal Care and Use Committee of ONO pharmaceutical Co., Ltd. and conducted in accordance with Guidance for Animal Experiments. Results: We identified a GABAAa5NAM with high affinity (Ki value ¼ 7.9 nmol/L), NAM activity (EC50 ¼ 1.1 nmol/L, Emax ¼ -50%), and selectivity
Poster Presentations: Wednesday, July 27, 2016
for a5 (Emax for GABAAa1, 2 and 3 ¼ -2%, 5% and -6%, respectively). This compound (300 nmol/L) significantly enhanced LTP in hippocampal slices from normal rats. Hippocampal injection of Ab and ibotenate impaired LTP, and GABAAa5NAM rescued this effect. GABAAa5NAM (3, 10 and 20 mg/kg, p.o.) significantly reversed MK-801-induced cognitive deficit in rat passive avoidance test. This compound (10 mg/kg, p.o.) significantly reversed scopolamine/MK-801-induced cognitive deficit in rat 8-arm radial maze test. Moreover, this compound (10 mg/kg, p.o.) significantly enhanced cognition in normal rat novel object recognition test. This compound did not show anxiogenic-like or proconvulsant effects. Conclusions: GABAAa5 NAM enhanced LTP in normal and Ab/ibotenate-lesioned hippocampal slices. This compound improved cognition without anxiogenic or proconvulsant side effects.
P4-008
P1017
CADRD, IDPH-ADRF 63282003D, IHIA, and the Kenneth Stark Endowment for Alzheimer’s Research.
Figure 1. Representative traces (left) from local application (arrow) of saline (blue, middle), 0.1 mM Ab42 (black, top), and 0.1 mM Ab42 with 10.0 mM TTx (red, bottom) in 6-9 month old C57BL/6J mice. Glutamate release (right; mean 6 SEM) from varying concentrations of Ab42. One-way ANOVA with Fisher’s LSD post hoc; *p<0.05, **p<0.01, ***p<0.001 vs. saline; $$$$p<0.0001 vs. 0.1 mM Ab42; n¼9 per group.
CAN ALZHEIMER’S-RELATED COGNITIVE DECLINE BE DELAYED THROUGH PRODROMAL TREATMENT? EVIDENCE FROM A MOUSE MODEL OF ALZHEIMER’S DISEASE
Kevin N. Hascup, Sarah O. Broderick, Erin R. Hascup, Southern Illinois University School of Medicine, Springfield, IL, USA. Contact e-mail: [email protected] Background: The pathophysiological cascade of Alzheimer’s dis-
ease (AD) postulates that amyloid-b (Ab)42 accumulation occurs years prior to cognitive decline. Evidence suggests that soluble Ab42 elicits glutamate release via a presynaptic mechanism. We sought to determine if hippocampal glutamatergic signaling is altered during the early stages of AD, and if so, can early intervention targeting the glutamatergic system alleviate cognitive decline. Methods: We utilized in vivo electrochemistry to selectively measure L-glutamate with an enzyme-based microelectrode array. Human Ab42 (pipette: 0.01 - 10.0 mM; recording site: 0.255-255.0 nM) dissolved in physiological saline (pH 7.4) was locally applied in the CA1 of isoflurane anesthetized 6-9 month male C57BL/6J mice and glutamate release was studied. Next, we examined 2-4, 6-8, and 1215 month AbPP/PS1 mice to understand how progressive accumulation of Ab42 alters glutamatergic neurotransmission. AbPP/PS1 and age matched C57BL/6J mice underwent an 8 day Morris water maze (MWM) paradigm to test spatial learning and memory. Following behavioral testing, mice were isoflurane anesthetized and basal and stimulus-evoked (70 mM KCl, isotonic, pH 7.4) glutamate release was measured in the CA1. Finally, LY379268 (a Group 2 metabotropic glutamate receptor agonist) was administered twice weekly (3.0 mg/kg i.p.) between 2-6 months of age, with MWM conducted at w11 months of age. Results: Local application of Ab42 elicited glutamate release in a dose-dependent manner that was blocked by tetrodotoxin (TTx; 10.0 mM; Figure 1). Evoked glutamate release was elevated in AbPP/PS1 mice at all age groups examined (Figure 2) and negatively correlated with memory as previously reported. Basal glutamate was elevated in AbPP/PS1 mice at 6-8 and 12-15 months, but not at 2-4 months (Figure 3). Overall, mice tolerated LY379268 injections and cognitive data will be presented. Conclusions: These data supports that Ab42 elicits glutamate release through a presynaptic mechanism that might lead to the elevated glutamate release and subsequent increased basal glutamate observed in AbPP/PS1 mice. Furthermore, targeting the glutamatergic system during pre-symptomatic AD may delay or prevent cognitive decline. Funding provided by
Figure 2. Local application of 70 mM KCl (100-200 nl) stimulated more glutamate release in the CA1 of AbPP/PS1 mice compared to C57BL/6J control mice at all ages examined. An age-related decrease in glutamate release in AbPP/PS1 mice was also observed. Two-way ANOVA with Fisher’s LSD post hoc; *p<0.05, ****p<0.001 vs. age-matched C57BL/ 6J mice; $$p<0.01 vs. 2-4 month AbPP/PS1 mice.
Figure 3. Basal glutamate was elevated in the CA1 of AbPP/PS1 mice at 6-8 and 12-15 months of age as compared to age-matched C57BL/6J control mice, but not at 2-4 months. Two-way ANOVA with Fisher’s LSD post hoc; *p<0.05.