- Email: [email protected]
In vivo drug discovery using a nontransgenic model of Alzheimer's disease with face, construct and predictive validity
Poster Presentations P1 expression. Furthermore, treatment with the phosphatase inhibitor okadaic acid or the tubulin-binding toxin colchicine leads to hyperphosphorylation of tau and neurite degeneration. Conclusions: We conclude that the LUHMES system is well applicable for various pharmacological and biological assays and that it can be used to study the pathomechanisms underlying AD directly on a human neuronal background. P1-127
IN VIVO DRUG DISCOVERY USING A NONTRANSGENIC MODEL OF ALZHEIMER’S DISEASE WITH FACE, CONSTRUCT AND PREDICTIVE VALIDITY
Vanessa Villard1, Laurent Givalois2, Tangui Maurice2, Franc¸ois J. Roman3, 1 Amylgen, Montpellier Cedex 05, France; 2INSERM U. 710, Montpellier, France; 3FR Consulting, Nivelles, Belgium. Contact e-mail: vanessa. [email protected] Background: Relevant non-transgenic model of Alzheimer’s disease (AD) could be achieved by intracerebroventricular (ICV) infusion of oligomeric preparations of amyloid peptides (Ab). We based our model on the ICV injection of oligomeric Ab25-35 in mice or rats, prepared according to an original and proprietary procedure. Ab25-35 toxicity mimics, within days to weeks, numerous physiopathological features of AD, including synaptic and cell loss, induction of oxidative stress and apoptosis, neuroinflammation and activation of microglia, seeding of endogenous Ab40/42, activation of GSK-3b and other kinases involved in the hyper- and abnormal phosphorylation of Tau. Ab25-35 injected rats and mice also rapidly develop learning and memory deficits particularly affecting short-term memory processes. Anti-amnesic and/or neuroprotective activity of new compounds could therefore be evaluated following a large-scale in vivo drug screening strategy, based on validated analyses of morphological or molecular markers and behavioral responses. Methods: The predictive validity of the model was analyzed using pre-ICV or repeated post-ICV injections of reference AChE inhibitors, donepezil, rivastigmine and galantamine, or the neuroprotectant memantine. The behavioral deficits were analyzed in terms of spatial working memory (alternation in the Y maze), contextual long-term memory (passive avoidance) and episodic-like memory (object recognition). Morphological alteration were mainly examined in terms of cholinergic cell loss (VAChT immunolabeling). Results: All drugs prevented the morphological alterations and behavioral deficits after repeated post-ICV treatment. After pre-ICV treatment, differential efficacies were observed, and particularly, donepezil but not rivastigmine and galantamine was effective. Conclusions: These data confirmed the predictive validity of this acute nontransgenic model of AD, allowing rapid screening of drug activity with appropriate controls. P1-128
PHARMACOKINETIC/PHARMACODYNAMIC EVALUATION OF ACETYLCHOLINE MODULATION BY DONEPEZIL IN RATS AND PROJECTION TO HUMAN
Adam Ogden, Rouba Kozak, Xiao-Dan Ren, Jenny Zhang, Elaine Tseng, Emily Miller, Aarti Sawant, Pfizer Global Research & Development, Groton, CT, USA. Contact e-mail: [email protected] Background: Donepezil hydrochloride (AriceptÒ), a reversible inhibitor of acetylcholinesterase, is approved for the symptomatic treatment of mild to moderate Alzheimer’s disease. The increase in acetylcholine (ACh) concentrations in the brain is believed to be the major contributory factor in the therapeutic efficacy of donepezil. However, due to technical limitations of quantitating ACh concentrations in human matrices, the degree of modulation of ACh in human following donepezil administration is not known. The purpose of this study was to project ACh modulation by donepezil in human using a pharmacokinetic/pharmacodynamic (PK/PD) model developed in rats. Methods: The effects of subcutaneous (SC) doses of donepezil at 0.1 to 1 mg/kg on extracellular ACh levels in the prefrontal cortex were evaluated in freely-moving rats using microdialysis. Donepezil pharmacokinetics were determined in a satellite group of rats. ACh and donepezil concentrations were determined by LC-MS/MS. PK/PD data were modeled using an indirect response model assuming inhibition of ACh elimination. The time-
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course of ACh in human following therapeutic doses of donepezil (5 and 10 mg) was simulated using the PK/PD model developed in rats and the observed human pharmacokinetics of donepezil (AriceptÒ package insert, Nov 2006). Human-equivalent doses of donepezil in rats were determined based on the average effect on ACh using area under the effect curve (AUEC) calculations. Results: Donepezil produced dose-dependent ACh increases in rats. The maximum inhibition of ACh elimination was estimated to be 96.9 6 0.9%, and the IC50 based on total plasma concentrations was 3.19 6 0.56 ng/mL. Human steady-state simulations predicted that baseline ACh levels were increased w3-5x following therapeutic doses of donepezil. Conclusions: AUEC calculations indicate that human-equivalent doses of donepezil are 1-2 mg/kg SC in rats. These doses are projected to achieve the same average increase of ACh in rat and human. However, differential disposition of donepezil results in different peak-to-trough concentrations in rat and human producing a difference in maximum and minimum ACh modulation in rats as compared to humans. Nevertheless, the current PK/PD analysis may be applicable for defining research strategies for the preclinical detection and characterization of putative cognition enhancers. P1-129
INTRANASAL DEFEROXAMINE IMPROVES MEMORY AND DECREASES GSK3b ACTIVITY IN C57 MICE
Jared M. Fine1, Amanda M. Baillargeon1, Julian V. Tokarev1, Jacob M. Cooner1, Tyler J. Smith1, Aleta L. Svitak1, Katrina Krogh1, Lauren T. Thornton1, William H. Frey1,2, Leah R. Hanson1, 1HealthPartners Research Foundation, St. Paul, MN, USA; 2University of Minnesota, Minneapolis, MN, USA. Contact e-mail: [email protected] Background: Deferoxamine (DFO) is a metal chelator found to decrease cognitive decline in a clinical trial of Alzheimer’s patients. However, this trial included painful intramuscular injections and side-effects including nausea were reported. Recent pre-clinical studies have shown that when delivered intranasally, DFO decreased memory loss as measured by water maze in the APP/PS1 (amyloid) and P301L (tau) mouse models of Alzheimer’s disease. In this study, normal C57 mice, the background strain of both Alzheimer’s models, were treated with intranasal (IN) DFO to determine whether memory might be improved from baseline levels. Methods: Treatment groups (n ¼ 14 mice) consisted of IN DFO, IN saline (controls for IN delivery), intraperitoneal (IP) DFO (control for peripheral vs central effects), and IP saline (control for IP delivery). Mice were treated five days/week for four weeks with 2.4 mg of DFO or saline. Each day for four weeks, mice were subjected to daily behavior tests beginning 30 min. after dosing. At the end of the 4 weeks mice were dosed for a final time, euthanized after 30 min., and brain tissues collected and analyzed for biochemical changes. Results: Intranasal DFO treatment significantly improved both reference and working memory in the Morris and radial arm water maze tests (p < 0.05), whereas treatment with IP DFO did not. No differences were detected in the elevated plus, Y, or open field mazes, or rotarod and grip strength tests. Brain tissues of mice treated with IN DFO yielded a significant decrease in activity of GSK3b, a kinase that plays a significant role in Alzheimer’s disease (AD). There was also a decrease in oxidative stress in the brains of mice treated with IN DFO as measured with oxyblot. Conclusions: These results demonstrate that IN DFO can improve memory in normal mice. The fact that IN DFO improved memory and IP DFO did not suggests that targeting to the brain may be beneficial. The associated decreases in GSK3b activity and oxidative stress, which are both increased in Alzheimer’s disease, suggest that IN DFO has potential for the treatment of neurodegenerative diseases including Alzheimer’s. P1-130
PHYSIOPATHOLOGICAL CHANGES INDUCED AFTER CEREBRAL INJECTION OF AN AMYLOIDb FRAGMENT IN RATS: RELEVANCE TO ALZHEIMER’S DISEASE
Laurent Givalois, Charleine Zussy, Anthony Brureau, Brice Delair, Stephane Marchal, Emeline Keller, Tangui Maurice, Molecular Mechanisms in Neurodegenerative Dementia Lab., Montpellier, France. Contact e-mail: [email protected]
IN VIVO DRUG DISCOVERY USING A NONTRANSGENIC MODEL OF ALZHEIMER’S DISEASE WITH FACE, CONSTRUCT AND PREDICTIVE VALIDITY
Vanessa Villard1, Laurent Givalois2, Tangui Maurice2, Franc¸ois J. Roman3, 1 Amylgen, Montpellier Cedex 05, France; 2INSERM U. 710, Montpellier, France; 3FR Consulting, Nivelles, Belgium. Contact e-mail: vanessa. [email protected] Background: Relevant non-transgenic model of Alzheimer’s disease (AD) could be achieved by intracerebroventricular (ICV) infusion of oligomeric preparations of amyloid peptides (Ab). We based our model on the ICV injection of oligomeric Ab25-35 in mice or rats, prepared according to an original and proprietary procedure. Ab25-35 toxicity mimics, within days to weeks, numerous physiopathological features of AD, including synaptic and cell loss, induction of oxidative stress and apoptosis, neuroinflammation and activation of microglia, seeding of endogenous Ab40/42, activation of GSK-3b and other kinases involved in the hyper- and abnormal phosphorylation of Tau. Ab25-35 injected rats and mice also rapidly develop learning and memory deficits particularly affecting short-term memory processes. Anti-amnesic and/or neuroprotective activity of new compounds could therefore be evaluated following a large-scale in vivo drug screening strategy, based on validated analyses of morphological or molecular markers and behavioral responses. Methods: The predictive validity of the model was analyzed using pre-ICV or repeated post-ICV injections of reference AChE inhibitors, donepezil, rivastigmine and galantamine, or the neuroprotectant memantine. The behavioral deficits were analyzed in terms of spatial working memory (alternation in the Y maze), contextual long-term memory (passive avoidance) and episodic-like memory (object recognition). Morphological alteration were mainly examined in terms of cholinergic cell loss (VAChT immunolabeling). Results: All drugs prevented the morphological alterations and behavioral deficits after repeated post-ICV treatment. After pre-ICV treatment, differential efficacies were observed, and particularly, donepezil but not rivastigmine and galantamine was effective. Conclusions: These data confirmed the predictive validity of this acute nontransgenic model of AD, allowing rapid screening of drug activity with appropriate controls. P1-128
PHARMACOKINETIC/PHARMACODYNAMIC EVALUATION OF ACETYLCHOLINE MODULATION BY DONEPEZIL IN RATS AND PROJECTION TO HUMAN
Adam Ogden, Rouba Kozak, Xiao-Dan Ren, Jenny Zhang, Elaine Tseng, Emily Miller, Aarti Sawant, Pfizer Global Research & Development, Groton, CT, USA. Contact e-mail: [email protected] Background: Donepezil hydrochloride (AriceptÒ), a reversible inhibitor of acetylcholinesterase, is approved for the symptomatic treatment of mild to moderate Alzheimer’s disease. The increase in acetylcholine (ACh) concentrations in the brain is believed to be the major contributory factor in the therapeutic efficacy of donepezil. However, due to technical limitations of quantitating ACh concentrations in human matrices, the degree of modulation of ACh in human following donepezil administration is not known. The purpose of this study was to project ACh modulation by donepezil in human using a pharmacokinetic/pharmacodynamic (PK/PD) model developed in rats. Methods: The effects of subcutaneous (SC) doses of donepezil at 0.1 to 1 mg/kg on extracellular ACh levels in the prefrontal cortex were evaluated in freely-moving rats using microdialysis. Donepezil pharmacokinetics were determined in a satellite group of rats. ACh and donepezil concentrations were determined by LC-MS/MS. PK/PD data were modeled using an indirect response model assuming inhibition of ACh elimination. The time-
S211
course of ACh in human following therapeutic doses of donepezil (5 and 10 mg) was simulated using the PK/PD model developed in rats and the observed human pharmacokinetics of donepezil (AriceptÒ package insert, Nov 2006). Human-equivalent doses of donepezil in rats were determined based on the average effect on ACh using area under the effect curve (AUEC) calculations. Results: Donepezil produced dose-dependent ACh increases in rats. The maximum inhibition of ACh elimination was estimated to be 96.9 6 0.9%, and the IC50 based on total plasma concentrations was 3.19 6 0.56 ng/mL. Human steady-state simulations predicted that baseline ACh levels were increased w3-5x following therapeutic doses of donepezil. Conclusions: AUEC calculations indicate that human-equivalent doses of donepezil are 1-2 mg/kg SC in rats. These doses are projected to achieve the same average increase of ACh in rat and human. However, differential disposition of donepezil results in different peak-to-trough concentrations in rat and human producing a difference in maximum and minimum ACh modulation in rats as compared to humans. Nevertheless, the current PK/PD analysis may be applicable for defining research strategies for the preclinical detection and characterization of putative cognition enhancers. P1-129
INTRANASAL DEFEROXAMINE IMPROVES MEMORY AND DECREASES GSK3b ACTIVITY IN C57 MICE
Jared M. Fine1, Amanda M. Baillargeon1, Julian V. Tokarev1, Jacob M. Cooner1, Tyler J. Smith1, Aleta L. Svitak1, Katrina Krogh1, Lauren T. Thornton1, William H. Frey1,2, Leah R. Hanson1, 1HealthPartners Research Foundation, St. Paul, MN, USA; 2University of Minnesota, Minneapolis, MN, USA. Contact e-mail: [email protected] Background: Deferoxamine (DFO) is a metal chelator found to decrease cognitive decline in a clinical trial of Alzheimer’s patients. However, this trial included painful intramuscular injections and side-effects including nausea were reported. Recent pre-clinical studies have shown that when delivered intranasally, DFO decreased memory loss as measured by water maze in the APP/PS1 (amyloid) and P301L (tau) mouse models of Alzheimer’s disease. In this study, normal C57 mice, the background strain of both Alzheimer’s models, were treated with intranasal (IN) DFO to determine whether memory might be improved from baseline levels. Methods: Treatment groups (n ¼ 14 mice) consisted of IN DFO, IN saline (controls for IN delivery), intraperitoneal (IP) DFO (control for peripheral vs central effects), and IP saline (control for IP delivery). Mice were treated five days/week for four weeks with 2.4 mg of DFO or saline. Each day for four weeks, mice were subjected to daily behavior tests beginning 30 min. after dosing. At the end of the 4 weeks mice were dosed for a final time, euthanized after 30 min., and brain tissues collected and analyzed for biochemical changes. Results: Intranasal DFO treatment significantly improved both reference and working memory in the Morris and radial arm water maze tests (p < 0.05), whereas treatment with IP DFO did not. No differences were detected in the elevated plus, Y, or open field mazes, or rotarod and grip strength tests. Brain tissues of mice treated with IN DFO yielded a significant decrease in activity of GSK3b, a kinase that plays a significant role in Alzheimer’s disease (AD). There was also a decrease in oxidative stress in the brains of mice treated with IN DFO as measured with oxyblot. Conclusions: These results demonstrate that IN DFO can improve memory in normal mice. The fact that IN DFO improved memory and IP DFO did not suggests that targeting to the brain may be beneficial. The associated decreases in GSK3b activity and oxidative stress, which are both increased in Alzheimer’s disease, suggest that IN DFO has potential for the treatment of neurodegenerative diseases including Alzheimer’s. P1-130
PHYSIOPATHOLOGICAL CHANGES INDUCED AFTER CEREBRAL INJECTION OF AN AMYLOIDb FRAGMENT IN RATS: RELEVANCE TO ALZHEIMER’S DISEASE
Laurent Givalois, Charleine Zussy, Anthony Brureau, Brice Delair, Stephane Marchal, Emeline Keller, Tangui Maurice, Molecular Mechanisms in Neurodegenerative Dementia Lab., Montpellier, France. Contact e-mail: [email protected]