Exendin-4 attenuates blast traumatic brain injury induced cognitive impairments, losses of synaptophysin and in vitro TBI-induced hippocampal cellular degeneration.

Mild blast traumatic brain injury (B-TBI) induced lasting cognitive impairments in novel object recognition and less severe deficits in Y-maze behaviors. B-TBI significantly reduced the levels of synaptophysin (SYP) protein staining in cortical (CTX) and hippocampal (HIPP) tissues. Treatment with exendin-4 (Ex-4) delivered by subcutaneous micro-osmotic pumps 48 hours prior to or 2 hours immediately after B-TBI prevented the induction of both cognitive deficits and B-TBI induced changes in SYP staining. The effects of a series of biaxial stretch injuries (BSI) on a neuronal derived cell line, HT22 cells, were assessed in an in vitro model of TBI. Biaxial stretch damage induced shrunken neurites and cell death. Treatment of HT22 cultures with Ex-4 (25 to 100 nM), prior to injury, attenuated the cytotoxic effects of BSI and preserved neurite length similar to sham treated cells. These data imply that treatment with Ex-4 may represent a viable option for the management of secondary events triggered by blast-induced, mild traumatic brain injury that is commonly observed in militarized zones.

Cognitive Impairments Induced by Concussive Mild Traumatic Brain Injury in Mouse Are Ameliorated by Treatment with Phenserine via Multiple Non-Cholinergic and Cholinergic Mechanisms.

Traumatic brain injury (TBI), often caused by a concussive impact to the head, affects an estimated 1.7 million Americans annually. With no approved drugs, its pharmacological treatment represents a significant and currently unmet medical need. In our prior development of the anti-cholinesterase compound phenserine for the treatment of neurodegenerative disorders, we recognized that it also possesses non-cholinergic actions with clinical potential. Here, we demonstrate neuroprotective actions of phenserine in neuronal cultures challenged with oxidative stress and glutamate excitotoxicity, two insults of relevance to TBI. These actions translated into amelioration of spatial and visual memory impairments in a mouse model of closed head mild TBI (mTBI) two days following cessation of clinically translatable dosing with phenserine (2.5 and 5.0 mg/kg BID x 5 days initiated post mTBI) in the absence of anti-cholinesterase activity. mTBI elevated levels of thiobarbituric acid reactive substances (TBARS), a marker of oxidative stress. Phenserine counteracted this by augmenting homeostatic mechanisms to mitigate oxidative stress, including superoxide dismutase [SOD] 1 and 2, and glutathione peroxidase [GPx], the activity and protein levels of which were measured by specific assays. Microarray analysis of hippocampal gene expression established that large numbers of genes were exclusively regulated by each individual treatment with a substantial number of them co-regulated between groups. Molecular pathways associated with lipid peroxidation were found to be regulated by mTBI, and treatment of mTBI animals with phenserine effectively reversed injury-induced regulations in the 'Blalock Alzheimer's Disease Up' pathway. Together these data suggest that multiple phenserine-associated actions underpin this compound's ability to ameliorate cognitive deficits caused by mTBI, and support the further evaluation of the compound as a therapeutic for TBI.

Blast traumatic brain injury-induced cognitive deficits are attenuated by preinjury or postinjury treatment with the glucagon-like peptide-1 receptor agonist, exendin-4.

INTRODUCTION: Blast traumatic brain injury (B-TBI) affects military and civilian personnel. Presently, there are no approved drugs for blast brain injury. METHODS: Exendin-4 (Ex-4), administered subcutaneously, was evaluated as a pretreatment (48 hours) and postinjury treatment (2 hours) on neurodegeneration, behaviors, and gene expressions in a murine open field model of blast injury. RESULTS: B-TBI induced neurodegeneration, changes in cognition, and genes expressions linked to dementia disorders. Ex-4, administered preinjury or postinjury, ameliorated B-TBI-induced neurodegeneration at 72 hours, memory deficits from days 7-14, and attenuated genes regulated by blast at day 14 postinjury. DISCUSSION: The present data suggest shared pathologic processes between concussive and B-TBI, with end points amenable to beneficial therapeutic manipulation by Ex-4. B-TBI-induced dementia-related gene pathways and cognitive deficits in mice somewhat parallel epidemiologic studies of Barnes et al. who identified a greater risk in US military veterans who experienced diverse TBIs, for dementia in later life.

Synthesis of the Alzheimer drug Posiphen into its primary metabolic products (+)-N1-norPosiphen, (+)-N8-norPosiphen and (+)-N1, N8-bisnorPosiphen, their inhibition of amyloid precursor protein, α-Synuclein synthesis, interleukin-1ß release, and cholinergic action.

A major pathological hallmark of Alzheimer disease (AD) is the appearance in the brain of senile plaques that are primarily composed of aggregated forms of ß-amyloid peptide (Aß) that derive from amyloid precursor protein (APP). Posiphen (1) tartrate is an experimental AD drug in current clinical trials that reduces Aß levels by lowering the rate of APP synthesis without toxicity. To support the clinical development of Posiphen (1) and elucidate its efficacy, its three major metabolic products, (+)-N1-norPosiphen (15), (+)-N8-norPosiphen (17) and (+)-N1, N8-bisnorPosiphen (11), were required in high chemical and optical purity. The efficient transformation of Posiphen (1) into these metabolic products, 15, 17 and 11, is described. The biological activity of these metabolites together with Posiphen (1) and its enantiomer, the AD drug candidate (-)-phenserine (2), was assessed against APP,α-synuclein and classical cholinergic targets. All the compounds potently inhibited the generation of APP and α-synuclein in neuronal cultures. In contrast, metabolites 11 and 15, and (-)-phenserine (2) but not Posiphen (1) or 17, possessed acetyl cholinesterase inhibitory action and no compounds bound either nicotinic or muscarinic receptors. As Posiphen (1) lowered CSF markers of inflammation in a recent clinical trial, the actions of 1 and 2 on proinflammatory cytokine interleukin (IL)-1ß release human peripheral blood mononuclear cells was evaluated, and found to be potently inhibited by both agents.

Exendin-4 induced glucagon-like peptide-1 receptor activation reverses behavioral impairments of mild traumatic brain injury in mice.

Mild traumatic brain injury (mTBI) represents a major and increasing public health concern and is both the most frequent cause of mortality and disability in young adults and a chief cause of morbidity in the elderly. Albeit mTBI patients do not show clear structural brain defects and, generally, do not require hospitalization, they frequently suffer from long-lasting cognitive, behavioral, and emotional problems. No effective pharmaceutical therapy is available, and existing treatment chiefly involves intensive care management after injury. The diffuse neural cell death evident after mTBI is considered mediated by oxidative stress and glutamate-induced excitotoxicity. Prior studies of the long-acting GLP-1 receptor agonist, exendin-4 (Ex-4), an incretin mimetic approved for type 2 diabetes mellitus treatment, demonstrated its neurotrophic/protective activity in cellular and animal models of stroke, Alzheimer's and Parkinson's diseases, and, consequent to commonalities in mechanisms underpinning these disorders, Ex-4 was assessed in a mouse mTBI model. In neuronal cultures in this study, Ex-4 ameliorated H2O2-induced oxidative stress and glutamate toxicity. To evaluate in vivo translation, we administered steady-state Ex-4 (3.5 pM/kg/min) or saline to control and mTBI mice over 7 days starting 48 h prior to or 1 h post-sham or mTBI (30 g weight drop under anesthesia). Ex-4 proved well-tolerated and fully ameliorated mTBI-induced deficits in novel object recognition 7 and 30 days post-trauma. Less mTBI-induced impairment was evident in Y-maze, elevated plus maze, and passive avoidance paradigms, but when impairment was apparent Ex-4 induced amelioration. Together, these results suggest that Ex-4 may act as a neurotrophic/neuroprotective drug to minimize mTBI impairment.

Tumor necrosis factor-α synthesis inhibitor 3,6'-dithiothalidomide attenuates markers of inflammation, Alzheimer pathology and behavioral deficits in animal models of neuroinflammation and Alzheimer's disease.

BACKGROUND: Neuroinflammation is associated with virtually all major neurodegenerative disorders, including Alzheimer's disease (AD). Although it remains unclear whether neuroinflammation is the driving force behind these disorders, compelling evidence implicates its role in exacerbating disease progression, with a key player being the potent proinflammatory cytokine TNF-α. Elevated TNF-α levels are commonly detected in the clinic and animal models of AD. METHODS: The potential benefits of a novel TNF-α-lowering agent, 3,6'-dithiothalidomide, were investigated in cellular and rodent models of neuroinflammation with a specific focus on AD. These included central and systemic inflammation induced by lipopolysaccharide (LPS) and Aß(1-42) challenge, and biochemical and behavioral assessment of 3xTg-AD mice following chronic 3,6'-dithiothaliodmide. RESULTS: 3,6'-Dithiothaliodmide lowered TNF-α, nitrite (an indicator of oxidative damage) and secreted amyloid precursor protein (sAPP) levels in LPS-activated macrophage-like cells (RAW 264.7 cells). This translated into reduced central and systemic TNF-α production in acute LPS-challenged rats, and to a reduction of neuroinflammatory markers and restoration of neuronal plasticity following chronic central challenge of LPS. In mice centrally challenged with A(ß1-42) peptide, prior systemic 3,6'-dithiothalidomide suppressed Aß-induced memory dysfunction, microglial activation and neuronal degeneration. Chronic 3,6'-dithiothalidomide administration to an elderly symptomatic cohort of 3xTg-AD mice reduced multiple hallmark features of AD, including phosphorylated tau protein, APP, Aß peptide and Aß-plaque number along with deficits in memory function to levels present in younger adult cognitively unimpaired 3xTg-AD mice. Levels of the synaptic proteins, SNAP25 and synaptophysin, were found to be elevated in older symptomatic drug-treated 3xTg-AD mice compared to vehicle-treated ones, indicative of a preservation of synaptic function during drug treatment. CONCLUSIONS: Our data suggest a strong beneficial effect of 3,6'-dithiothalidomide in the setting of neuroinflammation and AD, supporting a role for neuroinflammation and TNF-α in disease progression and their targeting as a means of clinical management.

Exendin-4 ameliorates motor neuron degeneration in cellular and animal models of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease characterized by a progressive loss of lower motor neurons in the spinal cord. The incretin hormone, glucagon-like peptide-1 (GLP-1), facilitates insulin signaling, and the long acting GLP-1 receptor agonist exendin-4 (Ex-4) is currently used as an anti-diabetic drug. GLP-1 receptors are widely expressed in the brain and spinal cord, and our prior studies have shown that Ex-4 is neuroprotective in several neurodegenerative disease rodent models, including stroke, Parkinson's disease and Alzheimer's disease. Here we hypothesized that Ex-4 may provide neuroprotective activity in ALS, and hence characterized Ex-4 actions in both cell culture (NSC-19 neuroblastoma cells) and in vivo (SOD1 G93A mutant mice) models of ALS. Ex-4 proved to be neurotrophic in NSC-19 cells, elevating choline acetyltransferase (ChAT) activity, as well as neuroprotective, protecting cells from hydrogen peroxide-induced oxidative stress and staurosporine-induced apoptosis. Additionally, in both wild-type SOD1 and mutant SOD1 (G37R) stably transfected NSC-19 cell lines, Ex-4 protected against trophic factor withdrawal-induced toxicity. To assess in vivo translation, SOD1 mutant mice were administered vehicle or Ex-4 at 6-weeks of age onwards to end-stage disease via subcutaneous osmotic pump to provide steady-state infusion. ALS mice treated with Ex-4 showed improved glucose tolerance and normalization of behavior, as assessed by running wheel, compared to control ALS mice. Furthermore, Ex-4 treatment attenuated neuronal cell death in the lumbar spinal cord; immunohistochemical analysis demonstrated the rescue of neuronal markers, such as ChAT, associated with motor neurons. Together, our results suggest that GLP-1 receptor agonists warrant further evaluation to assess whether their neuroprotective potential is of therapeutic relevance in ALS.

Design, synthesis and biological assessment of novel N-substituted 3-(phthalimidin-2-yl)-2,6-dioxopiperidines and 3-substituted 2,6-dioxopiperidines for TNF-α inhibitory activity.

Eight novel 2-(2,6-dioxopiperidin-3-yl)phthalimidine EM-12 dithiocarbamates 9 and 10, N-substituted 3-(phthalimidin-2-yl)-2,6-dioxopiperidines 11-14 and 3-substituted 2,6-dioxopiperidines 16 and 18 were synthesized as tumor necrosis factor-α (TNF-α) synthesis inhibitors. Synthesis involved utilization of a novel condensation approach, a one-pot reaction involving addition, iminium rearrangement and elimination, to generate the phthalimidine ring required for the creation of compounds 9-14. Agents were, thereafter, quantitatively assessed for their ability to suppress the synthesis on TNF-α in a lipopolysaccharide (LPS)-challenged mouse macrophage-like cellular screen, utilizing cultured RAW 264.7 cells. Whereas compounds 9, 14 and 16 exhibited potent TNF-α lowering activity, reducing TNF-α by up to 48% at 30 µM, compounds 12, 17 and 18 presented moderate TNF-α inhibitory action. The TNF-α lowering properties of these analogs proved more potent than that of revlimid (3) and thalidomide (1). In particular, N-dithiophthalimidomethyl-3-(phthalimidin-2-yl)-2,6-dioxopiperidine 14 not only possessed the greatest potency of the analogs to reduce TNF-α synthesis, but achieved this with minor cellular toxicity at 30 µM. The pharmacological focus of the presented compounds is towards the development of well-tolerated agents to ameliorate the neuroinflammation, that is, commonly associated with neurodegenerative disorders, epitomized by Alzheimer's disease and Parkinson's disease.

Preparation and Characterization of Tetrabenazine Enantiomers against Vesicular Monoamine Transporter 2.

As a clinical medication for the treatment of hyperkinetic movement disorders, in conditions such as Huntington's disease, tetrabenazine (TBZ) has been always used in its racemic form. To establish whether or not its beneficial therapeutic actions are enantiospecific, a practical total synthetic route was developed to yield each enantiomeric form to allow their chemical and pharmacological characterization. We briefly summarize the total synthesis of TBZ and report a detailed procedure for resolution of TBZ into its enantiomers, (+)-TBZ and (-)-TBZ. This allowed determination of the optical rotation and absolute configurations of each TBZ enantiomer, based on X-ray crystallographic analysis, together with characterization of their inhibitory action at the vesicular monoamine transporter 2, where (+)-TBZ proved three-fold more active than (-)-TBZ.

Long-acting anticholinesterases for myasthenia gravis: synthesis and activities of quaternary phenylcarbamates of neostigmine, pyridostigmine and physostigmine.

The N-monophenylcarbamate analogues of neostigmine methyl sulfate (6) and pyridostigmine bromide (8) together with their precursors (5), (7), and the N(1)-methylammonium analogues of (-)-phenserine (12), (-)-tolserine (14), (-)-cymserine (16) and (-)-phenethylcymserine (18) were synthesized to produce long-acting peripheral inhibitors of acetylcholinesterase or butyrylcholinesterase. Evaluation of their cholinesterase inhibition against human enzyme ex vivo demonstrated that, whereas compounds 5-8 possessed only marginal activity, 12, 14, 16 and 18 proved to be potent anticholinesterases. An extended duration of cholinesterase inhibition was determined in rodent, making them of potential interest as long-acting agents for myasthenia gravis.

A synthetic uric acid analog accelerates cutaneous wound healing in mice.

Wound healing is a complex process involving intrinsic dermal and epidermal cells, and infiltrating macrophages and leukocytes. Excessive oxidative stress and associated inflammatory processes can impair wound healing, and antioxidants have been reported to improve wound healing in animal models and human subjects. Uric acid (UA) is an efficient free radical scavenger, but has a very low solubility and poor tissue penetrability. We recently developed novel UA analogs with increased solubility and excellent free radical-scavenging properties and demonstrated their ability to protect neural cells against oxidative damage. Here we show that the uric acid analog (6, 8 dithio-UA, but not equimolar concentrations of UA or 1, 7 dimethyl-UA) modified the behaviors of cultured vascular endothelial cells, keratinocytes and fibroblasts in ways consistent with enhancement of the wound healing functions of all three cell types. We further show that 6, 8 dithio-UA significantly accelerates the wound healing process when applied topically (once daily) to full-thickness wounds in mice. Levels of Cu/Zn superoxide dismutase were increased in wound tissue from mice treated with 6, 8 dithio-UA compared to vehicle-treated mice, suggesting that the UA analog enhances endogenous cellular antioxidant defenses. These results support an adverse role for oxidative stress in wound healing and tissue repair, and provide a rationale for the development of UA analogs in the treatment of wounds and for modulation of angiogenesis in other pathological conditions.

Enhancing the GLP-1 receptor signaling pathway leads to proliferation and neuroprotection in human neuroblastoma cells.

Increasing evidence suggests that glucagon-like peptide-1 (GLP-1), an incretin hormone of current interest in type 2 diabetes, is neuroprotective in both cell culture and animal models. To characterize the neuroprotective properties of GLP-1 and associated underlying mechanisms, we over-expressed the GLP-1 receptor (GLP-1R) on human neuroblastoma SH-SY5Y cells to generate a neuronal culture system featuring enhanced GLP-1R signaling. In GLP-1R over-expressing SH-SY5Y (SH-hGLP-1R#9) cells, GLP-1 and the long-acting agonist exendin-4 stimulated cell proliferation and increased cell viability by 2-fold at 24 h at physiologically relevant concentrations. This GLP-1R-dependent action was mediated via the protein kinase A and phosphoinositide 3-kinase signaling pathways, with the MAPK pathway playing a minor role. GLP-1 and exendin-4 pretreatment dose-dependently protected SH-hGLP-1R#9 cells from hydrogen peroxide (H(2)O(2))- and 6-hydroxydopamine-induced cell death. This involved amelioration of elevated caspase 3 activity, down-regulation of pro-apoptotic Bax and up-regulation of anti-apoptotic Bcl-2 protein. In the presence of 6-hydroxydopamine, GLP-1's ability to lower caspase-3 activity was abolished with the phosphoinositide 3-kinase inhibitor, LY2940002, and partly reduced with the protein kinase A inhibitor, H89. Hence, GLP-1R mediated neurotrophic and anti-apoptotic actions co-contribute to the neuroprotective property of GLP-1 in neuronal cell cultures, and reinforce the potential therapeutic value of GLP-1R agonists in neurodegenerative disorders involving oxidative stress.

GLP-1 receptor stimulation reduces amyloid-beta peptide accumulation and cytotoxicity in cellular and animal models of Alzheimer's disease.

Type 2 (T2) diabetes mellitus (DM) has been associated with an increased incidence of neurodegenerative disorders, including Alzheimer's disease (AD). Several pathological features are shared between diabetes and AD, including dysfunctional insulin signaling and a dysregulation of glucose metabolism. It has therefore been suggested that not only may the two conditions share specific molecular mechanisms but also that agents with proven efficacy in one may be useful against the other. Hence, the present study characterized the effects of a clinically approved long-acting analogue, exendin-4 (Ex-4), of the endogenous insulin releasing incretin, glucagon-like peptide-1 (GLP-1), on stress-induced toxicity in neuronal cultures and on amyloid-beta protein (Abeta) and tau levels in triple transgenic AD (3xTg-AD) mice with and without streptozocin (STZ)-induced diabetes. Ex-4 ameliorated the toxicity of Abeta and oxidative challenge in primary neuronal cultures and human SH-SY5Y cells in a concentration-dependent manner. When 11 to 12.5 month old female 3xTg AD mice were challenged with STZ or saline, and thereafter treated with a continuous subcutaneous infusion of Ex-4 or vehicle, Ex-4 ameliorated the diabetic effects of STZ in 3xTg-AD mice, elevating plasma insulin and lowering both plasma glucose and hemoglobin A1c (HbA1c) levels. Furthermore, brain levels of Abeta protein precursor and Abeta, which were elevated in STZ 3xTg-AD mice, were significantly reduced in Ex-4 treated mice. Brain tau levels were unaffected following STZ challenge, but showed a trend toward elevation that was absent following Ex-4 treatment. Together, these results suggest a potential value of Ex-4 in AD, particularly when associated with T2DM or glucose intolerance.

A cellular model of inflammation for identifying TNF-alpha synthesis inhibitors.

Neuroinflammation is a common facet of both acute and chronic neurodegenerative conditions, exemplified by stroke and by Alzheimer's and Parkinson's disease, and the presence of elevated levels of the proinflammatory cytokine, tumor necrosis factor-alpha (TNF-alpha), has been documented in each. Although initial TNF-alpha generation is associated with a protective compensatory response, its unregulated chronic elevation is generally detrimental and can drive the disease process. In such circumstances, therapeutic strategies that can both gain access to the brain and target the production of TNF-alpha are predicted to be of clinical benefit. An in vitro mouse macrophage-like cellular screen, utilizing RAW 264.7 cells, was hence developed to identify novel TNF-alpha lowering agents incorporating lipophilic physicochemical characteristics predicted to allow penetration of the blood-brain barrier. Cultured RAW 264.7 cells exposed to lipopolysaccharide (LPS) induced a rapid, marked and concentration-dependent cellular release of TNF-alpha into the cell culture media, which was readily detected by enzyme linked immunosorbent assay (ELISA). The effects of four characterized thalidomide-based TNF-alpha lowering agents were assessed alongside 10 novel uncharacterized compounds synthesized on the same backbone. One of these new analogs possessed activity of sufficient magnitude to warrant further investigation. Activity determined in the cellular model translated to an in vivo rodent model of acute LPS-induced TNF-alpha elevation. The utility of the TNF-alpha cellular assay lies in its simplicity and robust nature, providing a tool for initial pharmacological screening to allow for the rapid identification novel TNF-alpha lowering agents.

GLP-1 receptor stimulation preserves primary cortical and dopaminergic neurons in cellular and rodent models of stroke and Parkinsonism.

Glucagon-like peptide-1 (GLP-1) is an endogenous insulinotropic peptide secreted from the gastrointestinal tract in response to food intake. It enhances pancreatic islet beta-cell proliferation and glucose-dependent insulin secretion, and lowers blood glucose and food intake in patients with type 2 diabetes mellitus (T2DM). A long-acting GLP-1 receptor (GLP-1R) agonist, exendin-4 (Ex-4), is the first of this new class of antihyperglycemia drugs approved to treat T2DM. GLP-1Rs are coupled to the cAMP second messenger pathway and, along with pancreatic cells, are expressed within the nervous system of rodents and humans, where receptor activation elicits neurotrophic actions. We detected GLP-1R mRNA expression in both cultured embryonic primary cerebral cortical and ventral mesencephalic (dopaminergic) neurons. These cells are vulnerable to hypoxia- and 6-hydroxydopamine-induced cell death, respectively. We found that GLP-1 and Ex-4 conferred protection in these cells, but not in cells from Glp1r knockout (-/-) mice. Administration of Ex-4 reduced brain damage and improved functional outcome in a transient middle cerebral artery occlusion stroke model. Ex-4 treatment also protected dopaminergic neurons against degeneration, preserved dopamine levels, and improved motor function in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of Parkinson's disease (PD). Our findings demonstrate that Ex-4 can protect neurons against metabolic and oxidative insults, and they provide preclinical support for the therapeutic potential for Ex-4 in the treatment of stroke and PD.

Tetrahydrofurobenzofuran cymserine, a potent butyrylcholinesterase inhibitor and experimental Alzheimer drug candidate, enzyme kinetic analysis.

Synaptic loss, particularly related to the forebrain cholinergic system, is considered to be an early event that leads to Alzheimer's disease (AD) and has led to the development of acetylcholinesterase inhibitors (AChE-Is) as the mainstay of treatment for several degenerative disorders that culminate in dementia. The primary dose-limiting toxicities of all clinically available AChE-Is are, similar to useful actions on cognition, cholinergically mediated and they ultimately limit the value of this drug class in achieving anything but symptomatic improvements. In addition, AChE levels in brain areas associated with AD decline with disease progression, which likely ultimately limits the therapeutic utility of this drug class. New research indicates that selective inhibition of butyrylcholinesterase (BuChE), a closely related enzyme that is markedly elevated in AD brain, increases acetylcholine (ACh) and augments cognition in rodents free of the characteristic undesirable actions of AChE-Is. BuChE inhibition hence represents an innovative treatment approach for AD, and agents are currently being synthesized to optimally achieve this. The novel compound, tetrahydrofurobenzofuran cymserine (THFBFC), is derived from our effort to produce a potent and BuChE-selective inhibitor as a candidate to test the hypothesis that BuChE-Is would be efficacious and better tolerated than AChE-Is in AD. Herein, we applied innovative enzyme kinetic analyses to characterize the quantitative interaction of THFBFC with human BuChE. These provided values for the agent's IC(50), together with specific new kinetic constants, such as K (T50), K (T1/2), R (I), (o)K (RT), (o)P(max), K(PT) and PT(1/2), to aid define target concentrations for clinical translation. Additional classical kinetic parameters, including K(i), K(m)or K(s), k(cat) or V(max) and V (mi) were also determined. THFBFC proved to be a potent competitive inhibitor of human BuChE and, like its isomer dihydrobenzodioxepine cymserine, is a potentially interesting AD drug candidate.

Kinetics of human serum butyrylcholinesterase inhibition by a novel experimental Alzheimer therapeutic, dihydrobenzodioxepine cymserine.

Cholinergic loss is the single most replicated neurotransmitter deficiency in Alzheimer's disease (AD) and has led to the use of acetylcholinesterase inhibitors (AChE-Is) and unselective cholinesterase inhibitors (ChE-Is) as the mainstay of treatment. AChE-Is and ChE-Is, however, induce dose-limiting adverse effects. Recent studies indicate that selective butyrylcholinesterase inhibitors (BuChE-Is) elevate acetylcholine (ACh) in brain, augment long-term potentiation, and improve cognitive performance in rodents without the classic adverse actions of AChE-Is and ChE-Is. BuChE-Is thereby represent a new strategy to ameliorate AD, particularly since AChE activity is depleted in AD brain, in line with ACh levels, whereas BuChE activity is elevated. Our studies have focused on the design and development of cymserine analogues to induce selective time-dependent brain BuChE inhibition, and on the application of innovative and quantitative enzyme kinetic analyses to aid selection of drug candidates. The quantitative interaction of the novel inhibitor, dihydrobenzodioxepine cymserine (DHBDC), with human BuChE was characterized. DHBDC demonstrated potent concentration-dependent binding with BuChE. The IC(50) and specific new kinetic constants, such as K(T50), P(PC), K(T1/2) and R(I), were determined at dual substrate concentrations of 0.10 and 0.60 mM butyrylthiocholine and reaction times, and are likely attainable in humans. Other classical kinetic parameters such as K(ia), K(ma), V(ma) and V(mi) were also determined. In synopsis, DHBDC proved to be a highly potent competitive inhibitor of human BuChE in comparison to its structural analogue, cymserine, and represents an interesting drug candidate for AD.

Soluble neuroprotective antioxidant uric acid analogs ameliorate ischemic brain injury in mice.

Uric acid is a major antioxidant in the blood of humans that can protect cultured neurons against oxidative and metabolic insults. However, uric acid has a very low solubility which compromises its potential clinical use for neurodegenerative disorders. Here we describe the synthesis, characterization and preclinical development of neuroprotective methyl- and sulfur-containing analogs of uric acid with increased solubility. In vitro and cell culture screening identified 1,7-dimethyluric acid (mUA2) and 6,8-dithiouric acid (sUA2) as two analogs with high antioxidant and neuroprotective activities. When administered intravenously in mice, uric acid analogs mUA2 and sUA2 lessened damage to the brain and improved functional outcome in an ischemia-reperfusion mouse model of stroke. Analogs sUA2 and mUA2 were also effective in reducing damage to the cerebral cortex when administered up to 4 h after stroke onset in a permanent middle cerebral artery occlusion mouse model. These findings suggest a therapeutic potential for soluble analogs of uric acid in the treatment of stroke and related neurodegenerative conditions.

Preclinical investigation of the topical administration of phenserine: transdermal flux, cholinesterase inhibition, and cognitive efficacy.

Phenserine (PS) was designed as a selective acetylcholinesterase (AChE) inhibitor, with a tartrate form (PST) for oral administration in mild to moderate Alzheimer's disease (AD). Recent phase 3 trials of PST in Europe indicate that any clinically relevant activity of PST may be limited by its duration of action. Like many oral drugs, bioavailability and plasma concentrations of PST are regulated by hepatic and gastrointestinal first-pass effects. To minimize the kinetic limitations of first-pass metabolism, transdermal formulations of PS and PST (ointment/patch) were developed and characterized in vitro and in vivo. Initial in vitro kinetic characterization of PS or PST formulations used a diffusion cell chamber and skin samples isolated from hairless mice. Liquid paraffin and fatty alcohol/propylene glycol (FAPG) were found to be suitable vehicles for ointment formulation. Addition of a penetration enhancer, 1-[2-(decylthio)ethyl]-azacyclopentane-2-one (HPE-101), improved stratum corneum permeability. Application of the optimal formulation of PS/HPE-101/FAPG to the shaved back of rats resulted in significantly lowered plasma and brain AChE activities and improved cognitive performance in animals with scopolamine-induced cognitive impairment. These results suggest that the transdermal application of AChE inhibitors may represent an effective therapeutic strategy for AD. Particular benefits over oral therapies might include avoiding first-pass metabolic effects and improved dosing compliance.

Apoptotic and behavioral sequelae of mild brain trauma in mice.

Mild traumatic brain injury (mTBI) is a not uncommon event in adolescents and young adults. Although it does not result in clear morphological brain defects, it is associated with long-term cognitive, emotional, and behavioral problems. Herein, we characterized the biochemical and behavioral changes associated with experimental mTBI in mice that may act as either targets or surrogate markers for interventional therapy. Specifically, mTBI was induced by 30-g and 50-g weight drop, and at 8 and 72 hr thereafter markers of cellular apoptosis-caspase-3, Bax, apoptosis-inducing factor (AIF), and cytochrome-c (Cyt-c)-were quantified by Western blot analysis in hippocampus ipsilateral to the impact. Levels of amyloid-beta precursor protein (APP) were also measured, and specific behavioral tests-passive avoidance, open field, and forced swimming (Porsolt) paradigms-were undertaken to assess learning, emotionality, and emotional memory. In the absence of hemorrhage or infarcts, as assessed by triphenyltetrazolium chloride staining, procaspase-3 and Bax levels were markedly altered following mTBI at both times. No cleaved caspase-3 was detected, and levels of AIF and Cyt-c, but not APP, were significantly changed at 72 hr. Mice subjected to mTBI were indistinguishable from controls by neurological examination at 1 and 24 hr, and by passive avoidance/open field at 72 hr, but could be differentiated in the forced swimming paradigm. In general, this model mimics the diffuse effects of mTBI on brain function associated with the human condition and highlights specific apoptotic proteins and a behavioral paradigm as potential markers for prospective interventional strategies.