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Introduction: The approved amyloid antibodies for early Alzheimer's disease (AD) carry a boxed warning about the risk of amyloid-related imaging abnormalities (ARIAs) that are highest in apolipoprotein E ( APOE ) ε4/ε4 homozygotes. ALZ-801/valiltramiprosate, an oral brain-penetrant amyloid beta oligomer inhibitor is being evaluated in APOE ε4/ε4 homozygotes with early AD., Methods: This Phase 3 randomized, double-blind, placebo-controlled, 78-week study of ALZ-801 administered as 265 mg twice per day tablets, enrolled 50- to 80-year-old homozygotes with Mini-Mental State Examination (MMSE) ≥ 22 and Clinical Dementia Rating-Global Score 0.5 or 1.0. The study is powered to detect a 2.0 to 2.5 drug-placebo difference on the Alzheimer's Disease Assessment Scale 13-item Cognitive subscale primary outcome with 150 subjects/arm. The key secondary outcomes are Clinical Dementia Rating-Sum of Boxes and Instrumental Activities of Daily Living; volumetric magnetic resonance imaging and fluid biomarkers are additional outcomes., Results: The APOLLOE4 Phase 3 trial enrolled 325 subjects with a mean age of 69 years, 51% female, MMSE 25.6, and 65% mild cognitive impairment. Topline results are expected in 2024., Discussion: APOLLOE4 is the first disease-modification AD trial focused on APOE ε4/ε4 homozygotes. Oral ALZ-801 has the potential to be the first effective and safe anti-amyloid treatment for the high-risk APOE ε4/ε4 population., Highlights: The APOLLOE4 Phase 3, placebo-controlled, 78-week study is designed to evaluate the efficacy and safety of ALZ-801 265 mg twice per day in early Alzheimer's disease (AD) subjects with the apolipoprotein E ( APOE ) ε4/ε4 genotype.The enrolled early AD population ( N  = 325) has 51% females, a mean age = 69 years, and a mean Mini-Mental State Examination = 25.6, with the majority being mild cognitive impairment subjects, a similar disease stage to the lecanemab Phase 3 AD trial (Clarity AD).The primary outcome is the cognitive Alzheimer's Disease Assessment Scale 13-item Cognitive subscale, with two functional measures as key secondary outcomes (Clinical Dementia Rating-Sum of Boxes, Amsterdam-Instrumental Activities of Daily Living), and with hippocampal volume and fluid biomarkers as additional outcomes.The study is unique in allowing a large number of microhemorrhages or siderosis at baseline magnetic resonance imaging, lesions that indicate concomitant cerebral amyloid angiopathy (CAA).At baseline, 32% of the enrolled population had at least 1 microhemorrhage, 24% had 1 to 4, and 8% had > 4 microhemorrhages; 10% had at least 1 siderosis lesion; with more males than females having microhemorrhages (63% vs. 37%) and siderosis (68% vs. 32%).Study results will become available in the second half of 2024 and, if positive, ALZ-801 may become the first oral drug to demonstrate a favorable benefit/risk profile in APOE ε4/ε4 AD subjects., Competing Interests: Drs. Susan Abushakra, Aidan Power, Earvin Liang, J. Patrick Kesslak, John A. Hey, Susan Flint, Rosalind McLaine, and Martin Tolar are employees and own stocks or stock options in Alzheon Inc.; Drs. Anton P. Porsteinsson, Marwan Sabbagh, and Merce Boada are advisors to several companies developing AD drugs. Drs. David Watson, Emer MacSweeney, and Merce Boada are investigators in the Phase 3 study and are active investigators in multiple AD clinical trials with various mechanisms of action. Author disclosures are available in the supporting information., (© 2024 Alzheon, Inc. and The Author(s). Alzheimer's & Dementia: Translational Research & Clinical Interventions published by Wiley Periodicals LLC on behalf of Alzheimer's Association.)

Introduction: ALZ-801/valiltramiprosate is a small-molecule oral inhibitor of beta amyloid (Aβ) aggregation and oligomer formation being studied in a phase 2 trial in APOE4 carriers with early Alzheimer's disease (AD) to evaluate treatment effects on fluid and imaging biomarkers and cognitive assessments., Methods: The single-arm, open-label phase 2 trial was designed to evaluate the effects of the ALZ-801 265 mg tablet taken twice daily (after 2 weeks once daily) on plasma fluid AD biomarkers, hippocampal volume (HV), and cognition over 104 weeks in APOE4 carriers. The study enrolled subjects aged 50-80 years, with early AD [Mini-Mental State Examination (MMSE) ≥ 22, Clinical Dementia Rating-Global (CDR-G) 0.5 or 1], apolipoprotein E4 (APOE4) genotypes including APOE4/4 and APOE3/4 genotypes, and positive cerebrospinal fluid (CSF) AD biomarkers or prior amyloid scans. The primary outcome was plasma p-tau 181 , HV evaluated by magnetic resonance imaging (MRI) was the key secondary outcome, and plasma Aβ42 and Aβ40 were the secondary biomarker outcomes. The cognitive outcomes were the Rey Auditory Verbal Learning Test and the Digit Symbol Substitution Test. Safety and tolerability evaluations included treatment-emergent adverse events and amyloid-related imaging abnormalities (ARIA). The study was designed and powered to detect 15% reduction from baseline in plasma p-tau 181 at the 104-week endpoint. A sample size of 80 subjects provided adequate power to detect this difference at a significance level of 0.05 using a two-sided paired t-test., Results: The enrolled population of 84 subjects (31 homozygotes and 53 heterozygotes) was 52% females, mean age 69 years, MMSE 25.7 [70% mild cognitive impairment (MCI), 30% mild AD] with 55% on cholinesterase inhibitors. Plasma p-tau 181 reduction from baseline was significant (31%, p = 0.045) at 104 weeks and all prior visits; HV atrophy was significantly reduced (p = 0.0014) compared with matched external controls from an observational Early AD study. Memory scores showed minimal decline from baseline over 104 weeks and correlated significantly with decreased HV atrophy (Spearman's 0.44, p = 0.002). Common adverse events were COVID infection and mild nausea, and no drug-related serious adverse events were reported. Of 14 early terminations, 6 were due to nonserious treatment-emergent adverse events and 1 death due to COVID. There was no vasogenic brain edema observed on MRI over 104 weeks., Conclusions: The effect of ALZ-801 on reducing plasma p-tau 181 over 2 years demonstrates target engagement and supports its anti-Aβ oligomer action that leads to a robust decrease in amyloid-induced brain neurodegeneration. The significant correlation between reduced HV atrophy and cognitive stability over 2 years suggests a disease-modifying effect of ALZ-801 treatment in patients with early AD. Together with the favorable safety profile with no events of vasogenic brain edema, these results support further evaluation of ALZ-801 in a broader population of APOE4 carriers, who represent two-thirds of patients with AD., Trial Registration: https://clinicaltrials.gov/study/NCT04693520 ., (© 2024. The Author(s).)

Introduction: ALZ-801/valiltramiprosate is an oral, small-molecule inhibitor of beta-amyloid (Aβ) aggregation and oligomer formation in late-stage development as a disease-modifying therapy for early Alzheimer's disease (AD). The present investigation provides a quantitative systems pharmacology (QSP) analysis of amyloid fluid biomarkers and cognitive results from a 2-year ALZ-801 Phase 2 trial in APOE4 carriers with early AD., Methods: The single-arm, open-label phase 2 study evaluated effects of ALZ-801 265 mg two times daily (BID) on cerebrospinal fluid (CSF) and plasma amyloid fluid biomarkers over 104 weeks in APOE4 carriers with early AD [Mini-Mental State Examination (MMSE) ≥ 22]. Subjects with positive CSF biomarkers for amyloid (Aβ 42 /Aβ 40 ) and tau pathology (p-tau 181 ) were enrolled, with serial CSF and plasma levels of Aβ 42 and Aβ 40 measured over 104 weeks. Longitudinal changes of CSF Aβ 42 , plasma Aβ 42 /Aβ 40 ratio, and cognitive Rey Auditory Verbal Learning Test (RAVLT) were compared with the established natural disease trajectories in AD using a QSP approach. The natural disease trajectory data for amyloid biomarkers and RAVLT were extracted from a QSP model and an Alzheimer's disease neuroimaging initiative population model, respectively. Analyses were stratified by disease severity and sex., Results: A total of 84 subjects were enrolled. Excluding one subject who withdrew at the early stage of the trial, data from 83 subjects were used for this analysis. The ALZ-801 treatment arrested the progressive decline in CSF Aβ 42 level and plasma Aβ 42 /Aβ 40 ratio, and stabilized RAVLT over 104 weeks. Both sexes showed comparable responses to ALZ-801, whereas mild cognitive impairment (MCI) subjects (MMSE ≥ 27) exhibited a larger biomarker response compared with more advanced mild AD subjects (MMSE 22-26)., Conclusions: In this genetically defined and biomarker-enriched early AD population, the QSP analysis demonstrated a positive therapeutic effect of oral ALZ-801 265 mg BID by arresting the natural decline of monomeric CSF and plasma amyloid biomarkers, consistent with the target engagement to prevent their aggregation into soluble neurotoxic oligomers and subsequently into insoluble fibrils and plaques over 104 weeks. Accompanying the amyloid biomarker changes, ALZ-801 also stabilized the natural trajectory decline of the RAVLT memory test, suggesting that the clinical benefits are consistent with its mechanism of action. This sequential effect arresting the disease progression on biomarkers and cognitive decline was more pronounced in the earlier symptomatic stages of AD. The QSP analysis provides fluid biomarker and clinical evidence for ALZ-801 as a first-in-class, oral small-molecule anti-Aβ oligomer agent with disease modification potential in AD., Trial Registry: https://clinicaltrials.gov/study/NCT04693520., (© 2024. The Author(s).)

New data suggest that the aggregation of misfolded native proteins initiates and drives the pathogenic cascade that leads to Alzheimer's disease (AD) and other age-related neurodegenerative disorders. We propose a unifying single toxin theory of brain neurodegeneration that identifies new targets and approaches to the development of disease-modifying treatments. An extensive body of genetic evidence suggests soluble aggregates of beta-amyloid (Aβ) as the primary neurotoxin in the pathogenesis of AD. New insights from fluid biomarkers, imaging, and clinical studies provide further evidence for the decisive impact of toxic Aβ species in the initiation and progression of AD. Understanding the distinct roles of soluble and insoluble amyloid aggregates on AD pathogenesis has been the key missing piece of the Alzheimer's puzzle. Data from clinical trials with anti-amyloid agents and recent advances in the diagnosis of AD demonstrate that the driving insult in biologically defined AD is the neurotoxicity of soluble Aβ aggregates, called oligomers and protofibrils, rather than the relatively inert insoluble mature fibrils and amyloid plaques. Amyloid oligomers appear to be the primary factor causing the synaptic impairment, neuronal stress, spreading of tau pathology, and eventual cell death that lead to the clinical syndrome of AD dementia. All other biochemical effects and neurodegenerative changes in the brain that are observed in AD are a response to or a downstream effect of this initial toxic insult by oligomers. Other neurodegenerative disorders follow a similar pattern of pathogenesis, in which normal brain proteins with important biological functions become trapped in the aging brain due to impaired clearance and then misfold and aggregate into neurotoxic species that exhibit prion-like behavior. These aggregates then spread through the brain and cause disease-specific neurodegeneration. Targeting the inhibition of this initial step in neurodegeneration by blocking the misfolding and aggregation of healthy proteins has the potential to slow or arrest disease progression, and if treatment is administered early in the course of AD and other neurodegenerative disorders, it may delay or prevent the onset of clinical symptoms.

Introduction: Hippocampal volume (HV) and cortical thickness are commonly used imaging biomarkers in Alzheimer's disease (AD) trials, and may have utility as selection criteria for enrichment strategies. Atrophy rates of these measures, in the high-risk apolipoprotein E (APOE) ε4/ε4 homozygous AD subjects are unknown., Methods: Data from Alzheimer's Disease Neuroimaging Initiative (ADNI-1) and a tramiprosate trial were analyzed in APOE ε4/ε4 and APOE ε3/ε3 subjects with mild cognitive impairment (MCI) or mild AD. Magnetic resonance imaging (MRI) data were centrally processed using FreeSurfer; total HV and composite average cortical thickness were derived and adjusted for age, head size, and education. Volumetric changes from baseline were assessed using Boundary Shift Integral, and correlated with cognitive changes., Results: APOE ε4/ε4 MCI subjects showed significantly higher % HV atrophy and cortical thinning at 12 months (4.4%, 3.1%, n = 29) compared to APOE ε3/ε3 subjects (2.8%, 1.8%, n = 93) and similarly in mild AD (7.4%, 4.7% n = 21 vs 5.4%, 3.3% n = 29). Differences were all significant at 24 months. Over 24 months, HV atrophy and cortical thinning correlated significantly with Alzheimer's Disease Assessment Scale-Cognitive subscale worsening in APOE ε4/ε4 MCI subjects, but not in mild AD., Discussion: Correlation of volumetric measures to cognitive change in APOE ε4/ε4 subjects with early AD supports their role as efficacy biomarkers. If confirmed in a Phase 3 trial with ALZ-801 (pro-drug of tramiprosate) in APOE ε4/ε4 early AD subjects, it may allow their use as surrogate outcomes in future treatment or prevention trials in AD., Competing Interests: Dr. Susan Abushakra serves as the chief medical officer of Alzheon, Inc. and holds stock and stock options of Alzheon, Inc. Dr. Martin Tolar serves as the founder, president, and chief executive officer of Alzheon, Inc. and holds stock and stock options of Alzheon, Inc. Dr. John A. Hey serves as the chief scientific officer of Alzheon, Inc. and holds stock and stock options of Alzheon, Inc. Dr. Aidan Power serves as a vice president at Alzheon, Inc. and holds stock and stock options of Alzheon, Inc. Dr. Anton Porsteinsson receives research support from the National Institutes of Health and U.S. Department of Defense. He also receives grants from AstraZeneca, Avanir, Biogen, Biohaven, Eisai, Eli Lilly, Genentech/Roche, Janssen, Novartis, Merck, and Toyama. He receives personal fees from Acadia Pharmaceuticals, Avanir, BioXcel, Eisai, Functional Neuromodulation, Grifols, Lundbeck, Merck, Neurim Pharmaceuticals, Pfizer, Tetra Discovery Partners, and Toyama. Dr. Marwan Sabbagh receives research support from the NIH and the Keep Memory Alive Foundation. He has consulting agreements with Allergan, Biogen, Bracket, Cortexyme, Grifols, Sanofi, Neurotrope, and Roche‐Genentech. He has ownership interest in Brain Health Inc., Versanum Inc., Neurotrope Inc., and uMethod Health. Drs. Joyce Suhy, Luc Bracoud, Joel Schaerer, and David Scott serve as full‐time employees of Bioclinica Inc. and have no competing interest in Alzheon Inc., (© 2020 The Authors. Alzheimer's & Dementia: Translational Research & Clinical Interventions published by Wiley Periodicals, Inc. on behalf of Alzheimer's Association.)

The body of evidence suggesting a causative, initiating role of beta amyloid (Aβ) in the pathogenesis of Alzheimer's disease (AD) is substantial. Yet, only a few anti-amyloid agents have shown meaningful efficacy in clinical trials. We evaluated the unifying characteristics of anti-amyloid agents with positive clinical or biomarker effects in long-duration trials and analyzed how pharmacological characteristics determine their clinical product profiles. Four agents with the potential for near term approval fulfill these criteria: the injectable antibodies, aducanumab, gantenerumab, and BAN2401, and a small molecule oral agent, ALZ-801. Aducanumab and BAN2401 showed significant efficacy on both clinical and biomarker outcomes; gantenerumab showed significant biomarker effects, with no clinical efficacy reported to date; and ALZ-801 showed significant clinical effects in the high-risk population of patients homozygous for the ε4 allele of apolipoprotein E gene (APOE4) and a dose-dependent preservation of hippocampal volume. We explored how the pharmacological properties of these agents, namely selectivity for Aβ oligomers, plasma half-life, brain penetration, and time to peak brain exposure, determine their clinical profiles. A crucial characteristic shared by these agents is their ability to engage neurotoxic soluble Aβ oligomers, albeit to various degrees. Aducanumab and gantenerumab partially target oligomers, while mostly clearing insoluble amyloid plaques; BAN2401 preferentially targets soluble protofibrils (large oligomers) over plaques; and ALZ-801 blocks the formation of oligomers without binding to plaques. The degree of selectivity for Aβ oligomers and brain exposure drive the magnitude and onset of clinical efficacy, while the clearance of plaques is associated with vasogenic brain edema. Only the highest doses of aducanumab and BAN2401 show modest efficacy, and higher dosing is limited by increased risk of vasogenic edema, especially in APOE4 carriers. These limitations can be avoided, and efficacy improved by small molecule agents that selectively inhibit the formation or block the toxicity of Aβ oligomers without clearing amyloid plaques. The most advanced selective anti-oligomer agent is ALZ-801, an optimized oral prodrug of tramiprosate, which demonstrated efficacy in homozygous APOE4/4 AD subjects. ALZ-801 selectively and fully inhibits the formation of Aβ42 oligomers at the clinical dose, without evidence of vasogenic edema, and will be evaluated in a phase 3 trial in homozygous APOE4/4 patients with early AD. In addition to clinical measures, the phase 3 trial will include cerebrospinal fluid, plasma, and imaging biomarkers to gain further insights into the role of soluble Aβ oligomers in the pathogenesis of AD and their impact on disease progression.

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Background: ALZ-801 is an oral, small-molecule inhibitor of beta amyloid (Aβ) oligomer formation in clinical development for Alzheimer's disease (AD). ALZ-801 is a prodrug of tramiprosate with improved pharmacokinetic properties and gastrointestinal tolerability. During clinical studies, we discovered that the primary metabolite of tramiprosate and its prodrug ALZ-801, 3-sulfopropanoic acid (3-SPA), is an endogenous molecule in the human brain and present in the cerebrospinal fluid (CSF) of patients with AD and other neurodegenerative brain diseases., Objective: The objectives of this research were to (1) identify and confirm the presence of 3-SPA in CSF samples from elderly, drug-naïve patients with memory deficits; (2) quantify the levels of 3-SPA in the CSF of patients with AD from tramiprosate phase III North American (NA) trial; (3) evaluate the in vitro anti-Aβ42 oligomer activity of 3-SPA; and (4) characterize the pharmacokinetics and brain-penetration properties of 3-SPA., Methods: Lumbar CSF samples from 64 drug-naïve patients with cognitive deficits (Mini-Mental State Examination [MMSE] score range 15-30) and six patients with AD treated with tramiprosate 150 mg twice daily in the phase III trial, at week 78, were analyzed. We used liquid chromatography-tandem mass spectrometry to confirm the structural molecular identity of endogenous 3-SPA with a 3-SPA reference standard and ion-mobility spectrometry-mass spectrometry with molecular dynamics to characterize interactions of 3-SPA with Aβ42 monomers, and the resultant conformational alterations. Rat studies using oral (30 mg/kg) and intravenous (10 mg/kg) doses were conducted to characterize the pharmacokinetic properties and brain penetration of 3-SPA., Results: We confirmed the presence of 3-SPA in the CSF of drug-naïve patients with cognitive deficits (mean concentration 11.7 ± 4.3 nM). The mean concentration of 3-SPA in patients with AD treated with tramiprosate was 135 ± 51 nM. In vitro studies revealed a multi-ligand interaction of 3-SPA with monomeric Aβ42 that inhibits the aggregation of Aβ42 into small oligomers. Comparisons of the molecular interactions of tramiprosate and 3-SPA with Aβ42 are also presented. Furthermore, in rat preclinical studies, 3-SPA displayed 100% oral bioavailability and 25% brain penetration, indicating that the metabolite is well absorbed and crosses the blood-brain barrier., Conclusions: We confirmed the endogenous presence of 3-SPA, the major metabolite of tramiprosate, in the CSF of drug-naïve elderly patients with memory deficits due to AD and a variety of other neurodegenerative disorders. The levels of 3-SPA were up to 12.6-fold greater in patients with AD receiving tramiprosate than in drug-naïve patients. In addition, we showed that 3-SPA has potent anti-Aβ oligomer activity, inhibiting aggregation of Aβ42 into small oligomers with efficacy comparable to that of tramiprosate. 3-SPA displays excellent oral availability and brain penetration in rats, suggesting that the higher CSF concentrations of 3-SPA in the human brain after oral administration of ALZ-801 or tramiprosate (and subsequent conversion to 3-SPA) result from the penetration of the metabolite into the central nervous system. These data suggest that 3-SPA is an endogenous agent with potential activity stabilizing the conformational flexibility of Aβ monomers that, in turn, inhibit Aβ misfolding and formation of soluble toxic Aβ oligomers in humans, thereby preventing the initial pathogenic step in the progression of AD. Clinical improvements observed in patients with AD carrying the ε4 allele of the apolipoprotein E gene in tramiprosate phase III studies may in part be explained by the therapeutic effects of excess levels of the metabolite in the brains of these patients. The potential protective role of 3-SPA in AD pathogenesis, as well as its therapeutic role in AD and other neurodegenerative disorders, warrants further investigation.

Background: ALZ-801 is an orally available, valine-conjugated prodrug of tramiprosate. Tramiprosate, the active agent, is a small-molecule β-amyloid (Aβ) anti-oligomer and aggregation inhibitor that was evaluated extensively in preclinical and clinical investigations for the treatment of Alzheimer's disease (AD). Tramiprosate has been found to inhibit β-amyloid oligomer formation by a multi-ligand enveloping mechanism of action that stabilizes Aβ42 monomers, resulting in the inhibition of formation of oligomers and subsequent aggregation. Although promising as an AD treatment, tramiprosate exhibited two limiting deficiencies: high intersubject pharmacokinetic (PK) variability likely due to extensive gastrointestinal metabolism, and mild-to-moderate incidence of nausea and vomiting. To address these, we developed an optimized prodrug, ALZ-801, which retains the favorable efficacy attributes of tramiprosate while improving oral PK variability and gastrointestinal tolerability. In this study, we summarize the phase I bridging program to evaluate the safety, tolerability and PK for ALZ-801 after single and multiple rising dose administration in healthy volunteers., Methods: Randomized, placebo-controlled, phase I studies in 127 healthy male and female adult and elderly volunteers included [1] a single ascending dose (SAD) study; [2] a 14-day multiple ascending dose (MAD) study; and [3] a single-dose tablet food-effect study. This program was conducted with both a loose-filled capsule and an immediate-release tablet formulation, under both fasted and fed conditions. Safety and tolerability were assessed, and plasma and urine were collected for liquid chromatography-mass spectrometry (LC-MS) determination and non-compartmental PK analysis. In addition, we defined the target dose of ALZ-801 that delivers a steady-state plasma area under the curve (AUC) exposure of tramiprosate equivalent to that studied in the tramiprosate phase III study., Results: ALZ-801 was well tolerated and there were no severe or serious adverse events (AEs) or laboratory findings. The most common AEs were transient mild nausea and some instances of vomiting, which were not dose-related and showed development of tolerance after continued use. ALZ-801 produced dose-dependent maximum plasma concentration (C max ) and AUC exposures of tramiprosate, which were equivalent to that after oral tramiprosate, but with a substantially reduced intersubject variability and a longer elimination half-life. Administration of ALZ-801 with food markedly reduced the incidence of gastrointestinal symptoms compared with the fasted state, without affecting plasma tramiprosate exposure. An immediate-release tablet formulation of ALZ-801 displayed plasma exposure and low variability similar to the loose-filled capsule. ALZ-801 also showed excellent dose-proportionality without accumulation or decrease in plasma exposure of tramiprosate over 14 days. Based on these data, 265 mg of ALZ-801 twice daily was found to achieve a steady-state AUC exposure of tramiprosate equivalent to 150 mg twice daily of oral tramiprosate in the previous phase III trials., Conclusions: ALZ-801, when administered in capsule and tablet forms, showed excellent oral safety and tolerability in healthy adults and elderly volunteers, with significantly improved PK characteristics over oral tramiprosate. A clinical dose of ALZ-801 (265 mg twice daily) was established that achieves the AUC exposure of 150 mg of tramiprosate twice daily, which showed positive cognitive and functional improvements in apolipoprotein E4/4 homozygous AD patients. These bridging data support the phase III development of ALZ-801in patients with AD.

Background: Amyloid beta (Aβ) oligomers play a critical role in the pathogenesis of Alzheimer's disease (AD) and represent a promising target for drug development. Tramiprosate is a small-molecule Aβ anti-aggregation agent that was evaluated in phase III clinical trials for AD but did not meet the primary efficacy endpoints; however, a pre-specified subgroup analysis revealed robust, sustained, and clinically meaningful cognitive and functional effects in patients with AD homozygous for the ε4 allele of apolipoprotein E4 (APOE4/4 homozygotes), who carry an increased risk for the disease. Therefore, to build on this important efficacy attribute and to further improve its pharmaceutical properties, we have developed a prodrug of tramiprosate ALZ-801 that is in advanced stages of clinical development. To elucidate how tramiprosate works, we investigated its molecular mechanism of action (MOA) and the translation to observed clinical outcomes., Objective: The two main objectives of this research were to (1) elucidate and characterize the MOA of tramiprosate via an integrated application of three independent molecular methodologies and (2) present an integrated translational analysis that links the MOA, conformation of the target, stoichiometry, and pharmacokinetic dose exposure to the observed clinical outcome in APOE4/4 homozygote subjects., Method: We used three molecular analytical methods-ion mobility spectrometry-mass spectrometry (IMS-MS), nuclear magnetic resonance (NMR), and molecular dynamics-to characterize the concentration-related interactions of tramiprosate versus Aβ42 monomers and the resultant conformational alterations affecting aggregation into oligomers. The molecular stoichiometry of the tramiprosate versus Aβ42 interaction was further analyzed in the context of clinical pharmacokinetic dose exposure and central nervous system Aβ42 levels (i.e., pharmacokinetic-pharmacodynamic translation in humans)., Results: We observed a multi-ligand interaction of tramiprosate with monomeric Aβ42, which differs from the traditional 1:1 binding. This resulted in the stabilization of Aβ42 monomers and inhibition of oligomer formation and elongation, as demonstrated by IMS-MS and molecular dynamics. Using NMR spectroscopy and molecular dynamics, we also showed that tramiprosate bound to Lys16, Lys28, and Asp23, the key amino acid side chains of Aβ42 that are responsible for both conformational seed formation and neuronal toxicity. The projected molar excess of tramiprosate versus Aβ42 in humans using the dose effective in patients with AD aligned with the molecular stoichiometry of the interaction, providing a clear clinical translation of the MOA. A consistent alignment of these preclinical-to-clinical elements describes a unique example of translational medicine and supports the efficacy seen in symptomatic patients with AD. This unique "enveloping mechanism" of tramiprosate also provides a potential basis for tramiprosate dose selection for patients with homozygous AD at earlier stages of disease., Conclusion: We have identified the molecular mechanism that may account for the observed clinical efficacy of tramiprosate in patients with APOE4/4 homozygous AD. In addition, the integrated application of the molecular methodologies (i.e., IMS-MS, NMR, and thermodynamics analysis) indicates that it is feasible to modulate and control the Aβ42 conformational dynamics landscape by a small molecule, resulting in a favorable Aβ42 conformational change that leads to a clinically relevant amyloid anti-aggregation effect and inhibition of oligomer formation. This novel enveloping MOA of tramiprosate has potential utility in the development of disease-modifying therapies for AD and other neurodegenerative diseases caused by misfolded proteins.

Background: Alzheimer's Disease (AD) patients homozygous for the APOE4 allele (APOE4/4) have a distinct clinical and biological phenotype with high levels of beta amyloid (Aβ) pathology and toxic Aβ oligomers. Tramiprosate, an oral agent that inhibits Aβ monomer aggregation into toxic oligomers, was evaluated in two Phase 3 Mild to Moderate AD studies which did not show efficacy in the overall population. Re-analyses of these trials showed the most consistent clinical benefits in APOE4/4 patients. We analyzed efficacy in the APOE4/4 patients with Mild disease., Objectives: To determine the optimal stage of AD for future trials in APOE4/4 homozygotes., Design: Two randomized, double-blind, placebo-controlled parallel-arm multi-center studies of 78-weeks duration., Setting: Academic Alzheimer's disease centers, community-based memory clinics, and neuropsychiatric research sites., Participants: Participants included 2,025 AD patients with MMSE 16-26. Approximately 13-15% had APOE4/4 genotype (N= 147 and 110 per study), mean age 71.1 years, 56% females. Almost all were on stable symptomatic drugs., Intervention: Randomized subjects received oral placebo, 100mg BID, or 150mg BID of tramiprosate., Measurements: Co-primary outcomes were change from baseline in the ADAS-cog11 and CDR-SB. Disability assessment for dementia (DAD) was a secondary outcome., Results: In APOE4/4 homozygotes receiving 150mg BID tramiprosate, efficacy in the traditional Mild AD patients (MMSE 20-26) was higher than the overall group (MMSE 16-26) and efficacy in the Mild patients (MMSE 22-26) was highest. Tramiprosate benefits compared to placebo on ADAS-cog, CDR-SB, and DAD were 125%, 81% and 71%, respectively (p<0.02). The Mild subgroup (MMSE 22-26) showed cognitive stabilization with no decline over 78 weeks, both ADAS-cog and DAD effects increased over time. Tramiprosate safety in APOE4/4 patients was favorable. Most common adverse events were nausea, vomiting, depression and decreased weight., Conclusions: The Mild subgroup of APOE4/4 AD patients (MMSE 22-26) showed larger benefits on the high dose of tramiprosate than the overall Mild and Moderate group. Consistent with its preclinical effects on Aβ oligomers, tramiprosate seemed to stabilize cognitive performance, supporting its disease modification potential. Confirmatory studies using ALZ-801, an improved pro-drug formulation of tramiprosate, will target APOE4/4 patients with Mild AD., Competing Interests: Drs. Abushakra, Hey, Power and Tolar are employees of Alzheon, Inc. and hold stock or stock options of Alzheon, Inc. Drs. Porsteinsson, Scheltens, Sadowsky, Cummings, Gauthier, Vellas were investigators in the tramiprosate Phase 3 program and serve as advisors to Alzheon, Inc., and receive advisory fees and/or stock options of Alzheon. Drs. Shen and Mr. Wang serve as statistical consultants to Alzheon.

Background: Tramiprosate is an oral amyloid anti-aggregation agent that reduces amyloid oligomer toxicity in preclinical studies and was evaluated in two 78-week trials in North America and Western Europe that enrolled 2,025 patients with Mild to Moderate Alzheimer's Disease. The completed North American study did not achieve its efficacy objectives, but a pre-specified subgroup analysis suggested potential efficacy in apolipoprotein E4 (APOE4) carriers. To further explore this observation, we analyzed tramiprosate Phase 3 clinical data based on the number of APOE4 alleles., Objectives: To analyze tramiprosate efficacy, safety, and occurrence of vasogenic edema in the three APOE4 subgroups: homozygous, heterozygous and non-carriers., Design: Randomized, double-blind, placebo-controlled parallel-arm multi-center studies., Setting: Academic Alzheimer's disease and dementia centers, community-based dementia and memory clinics, and neuropsychiatric clinical research sites., Participants: Subjects included 2,025 patients, 50 years of age or older, with approximately 60% having APOE4 carrier status (10-15% homozygotes and 45-50% heterozygotes), and mild to moderate disease. All subjects were on stable symptomatic drugs., Intervention: Randomized subjects received placebo, 100 mg BID, or 150 mg BID of tramiprosate., Measurements: Co-primary outcomes in both studies were change from baseline in the ADAS-cog11 and CDR-SB assessment scales., Results: Highest efficacy was observed in APOE4/4 homozygotes receiving 150 mg BID of tramiprosate, showing statistically significant effects on ADAS-cog and positive trends on CDR-SB (respectively, 40-66% and 25-45% benefit compared to placebo). APOE4 heterozygotes showed intermediate efficacy, and non-carriers showed no benefit. In 426 patients with MRI scans, no cases of treatment-emergent vasogenic edema were observed. In the three subgroups, the most common adverse events were nausea, vomiting, and decreased weight., Conclusions: The "APOE4 Gene-Dose effect" is likely explained by the high prevalence of amyloid pathology in symptomatic APOE4 carriers. In APOE4/4 Alzheimer's disease patients, the high dose of tramiprosate showed favorable safety and clinically meaningful efficacy in addition to standard of care.

Nasal congestion is one of the most troublesome symptoms of many upper airways diseases. We characterized the effect of selective α2c-adrenergic agonists in animal models of nasal congestion. In porcine mucosa tissue, compound A and compound B contracted nasal veins with only modest effects on arteries. In in vivo experiments, we examined the nasal decongestant dose-response characteristics, pharmacokinetic/pharmacodynamic relationship, duration of action, potential development of tolerance, and topical efficacy of α2c-adrenergic agonists. Acoustic rhinometry was used to determine nasal cavity dimensions following intranasal compound 48/80 (1%, 75 µl). In feline experiments, compound 48/80 decreased nasal cavity volume and minimum cross-sectional areas by 77% and 40%, respectively. Oral administration of compound A (0.1-3.0 mg/kg), compound B (0.3-5.0 mg/kg), and d-pseudoephedrine (0.3 and 1.0 mg/kg) produced dose-dependent decongestion. Unlike d-pseudoephedrine, compounds A and B did not alter systolic blood pressure. The plasma exposure of compound A to produce a robust decongestion (EC(80)) was 500 nM, which related well to the duration of action of approximately 4.0 hours. No tolerance to the decongestant effect of compound A (1.0 mg/kg p.o.) was observed. To study the topical efficacies of compounds A and B, the drugs were given topically 30 minutes after compound 48/80 (a therapeutic paradigm) where both agents reversed nasal congestion. Finally, nasal-decongestive activity was confirmed in the dog. We demonstrate that α2c-adrenergic agonists behave as nasal decongestants without cardiovascular actions in animal models of upper airway congestion.

A novel series of benzimidazolone-containing histamine H3-receptor antagonists were prepared and their structure-activity relationship was explored. These benzimidazolone analogs demonstrate potent H3-receptor binding affinities, no P450 enzyme inhibition, and strong H3 functional activity. Compound 1o exhibits the best overall profile with H3Ki=0.95nM and rat AUC=12.9μMh., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

We define the pharmacological and pharmacokinetic profiles of a novel α(2C)-adrenoceptor agonist, compound A [N-[3,4-dihydro-4-(1H-imidazol-4-ylmethyl)-2H-1,4-benzoxazin-6-yl]-N-ethyl-N'-methylurea]. This compound has high affinity (K(i)) for the human α(2C)-adrenoceptor (K(i) = 12 nM), and 190- to 260-fold selectivity over the α(2A)- and α(2B)-adrenoceptor subtypes. In cell-based functional assays, compound A produced good agonist (EC(50) = 166 nM) and efficacy (E(max) = 64%) responses at the α(2C)-adrenoceptor, much lower potency and efficacy at the α(2A)-adrenoceptor (EC(50) = 1525 nM; E(max) = 8%) and α(2B)-adrenoceptor (EC(50) = 5814 nM; E(max) = 21%) subtypes, and low or no affinity and functional activity at the α(1A)-, α(1B)-, and α(1D)-adrenoceptor subtypes. In the human saphenous vein postjunctional α(2C)-adrenoceptor bioassay, compound A functions as a potent agonist (pD(2) = 6.3). In a real-time contraction bioassay of pig nasal mucosa, compound A preferentially constricted the veins (EC(50) = 108 nM), and the magnitude of arteriolar contraction reached only 50% of the maximum venular responses. Compound A exhibited no effect on locomotor activity, sedation, and body temperature in mice (up to 100 mg/kg) and did not cause hypertension and mydriasis (30 mg/kg) in conscious rats. Compound A is orally bioavailable (24%) with good plasma exposure. This compound is a substrate for the efflux P-glycoprotein transporter, resulting in very low central nervous system (CNS) penetration. In summary, compound A is a highly selective, orally active, and non-CNS-penetrating α(2C)-adrenoceptor agonist with desirable in vitro and in vivo pharmacological properties suitable for the treatment of nasal congestion.

A structure-activity relationship study of the lead piperazinylcarbonylpiperidine compound 3 resulted in the identification of 4-benzimidazolyl-piperidinylcarbonyl-piperidine 6h as a histamine-3 (H(3)) receptor antagonist. Additional optimization of 6h led to the identification of compounds 11i-k with K(i)

We describe the pharmacological and pharmacokinetic profiles of SCH 486757, a nociceptin/orphanin FQ peptide (NOP) receptor agonist that has recently entered human clinical trials for cough. SCH 486757 selectively binds human NOP receptor (K(i)=4.6+/-0.61nM) over classical opioid receptors. In a guinea pig capsaicin cough model, SCH 486757 (0.01-1mg/kg) suppressed cough at 2, 4, and 6h post oral administration with a maximum efficacy occurring at 4h equivalent to codeine, hydrocodone, dextromethorphan and baclofen. The antitussive effects of SCH 486757 (3.0mg/kg, p.o.) was blocked by the NOP receptor antagonist J113397 (12mg/kg, i.p.) but not by naltrexone (10mg/kg, p.o.). SCH 486757 does not produce tolerance to its antitussive activity after a 5-day BID dosing regimen. After acute and chronic dosing paradigms, SCH 486757 (1mg/kg) inhibited capsaicin-evoked coughing by 46+/-9% and 40+/-11%, respectively. In a feline mechanically-evoked cough model, SCH 486757 produces a maximum inhibition of cough and expiratory abdominal electromyogram amplitude of 59 and 61%, respectively. SCH 486757 did not significantly affect inspiratory electromyogram amplitude. We examined the abuse potential of SCH 486757 (10mg/kg, p.o.) in a rat conditioned place preference procedure which is sensitive to classical drugs of abuse, such as amphetamine and morphine. SCH 486757 was without effect in this model. Finally, SCH 486757 displays a good oral pharmacokinetic profile in the guinea pig, rat and dog. We conclude that SCH 486757 has a favorable antitussive profile in preclinical animal models.

A series of 3-pyridinyl-tropane analogues based on previously reported compound 1 have been synthesized and shown to bind to the nociceptin receptor with high affinity. From the SAR study and our lead optimization efforts, compound 10 was found to possess potent oral antitussive activity in the capsaicin-induced guinea pig model. The rationale for compound selection and the biological profile of the optimized lead (10) are disclosed.

In the late 1980's reports linking the non-sedating antihistamines terfenadine and astemizole with torsades de pointes, a form of ventricular tachyarrhythmia that can degenerate into ventricular fibrillation and sudden death, appeared in the clinical literature. A substantial body of evidence demonstrates that the arrhythmogenic effect of these cardiotoxic antihistamines, as well as a number of structurally related compounds, results from prolongation of the QT interval due to suppression of specific delayed rectifier ventricular K+ currents via blockade of the hERG-IKr channel. In order to better understand the structural requirements for hERG and H(1) binding for terfenadine, a series of analogs of terfenadine has been prepared and studied in both in vitro and in vivo hERG and H(1) assays.

Background: Previous studies have demonstrated that nociceptin/orphanin FQ (N/OFQ), the endogenous peptide ligand for the G-protein-coupled NOP receptor, inhibits cough in experimental models. SCH 225288 is a nonpeptide, orally active NOP agonist that may provide the foundation for the development of novel treatments for cough., Methods: First we characterized the selectivity of SCH 225288 in human receptor binding assays. Afterwards, the antitussive activity of SCH 225288 was studied in three mechanistically distinct cough models. Specifically, we observed the cough-suppressant effect of SCH 225288 in a guinea pig capsaicin irritant-evoked cough model, a feline mechanically induced cough model and finally in a canine Bordetella bronchiseptica disease model., Results: SCH 225288 selectively binds human NOP receptor (K(i) = 0.38 +/- 0.02 nmol/l) over classical opioid receptors (COR). In a guinea pig capsaicin cough model, SCH 225288 (0.1-1 mg/kg) suppressed cough at 2, 4, and 6 h after oral administration. The antitussive effect of SCH 225288 (3.0 mg/kg, p.o.) was blocked by the NOP antagonist J113397 (12 mg/kg, i.p.) but not by the classical opioid receptor (COR) antagonist, naltrexone (3.0 mg/kg, i.p.). In the anesthetized cat, we evaluated the effects of SCH 225288 given either intravenously or via the intravertebral artery against the increases in cough number and respiratory expiratory and inspiratory muscle (rectus abdominis and parasternal) electromyographic (EMG) activities due to perturbations of the intrathoracic trachea. SCH 225288 (0.03-3.0 mg/kg, i.v.) inhibited both cough number and abdominal EMG amplitudes. Similarly, SCH 225288 (0.001-0.3 mg/kg) administered intra-arterially also diminished cough number and abdominal EMG amplitudes. No significant effect of the drug was noted on parasternal EMG activity. Finally, we studied the antitussive actions of SCH 225288 (1.0 mg/kg) in a canine B. bronchiseptica disease model. In this model, dogs were challenged intranasally with B. bronchiseptica. Comparisons were made between a vehicle group, an SCH 225288 (1.0 mg/kg, p.o., q.d.) and a butorphanol (0.6 mg/kg, p.o., b.i.d.) group on the mean change in cough scores from baseline values and days 6-9 after B. bronchiseptica challenge. SCH 225288 (1.0 mg/kg, p.o., q.d.) displayed a positive antitussive tendency (p = 0.06) to inhibit B. bronchiseptica cough whereas butorphanol (0.6 mg/kg, p.o., b.i.d.) was devoid of antitussive activity., Conclusions: Taken together, the present data show that SCH 225288 is a potent and effective antitussive agent in animal models of cough. Furthermore, these findings indicate that NOP agonists represent a promising new therapeutic approach for the treatment of cough without the side effect liabilities associated with opioid antitussives., (Copyright 2009 S. Karger AG, Basel.)

The vascular bed in nasal mucosa of different species, including human, is highly vascularized and an extensive sinusoidal network of large capacitance vessels is present deep within the submucosa. When this network of venous sinusoids is engorged with blood, the swollen mucosa reduces the size of the airway lumen and congestion ensues. Nasal vasculature tone is strongly influenced by the sympathetic nervous system and the only drugs approved specifically to relieve vascular nasal obstruction are alpha-adrenoceptor sympathomimetic agents. Due to their vasoconstrictor action, the sympathomimetic decongestants oppose vasodilation, reducing nasal airway resistance and thus facilitating nose breathing. However, standard decongestants that are non-selective alpha-adrenoceptor agonists are associated with the potential for side-effect liabilities including hypertension, stroke, insomnia and nervousness. We propose than a selective alpha 2-adrenoceptor agonist, by acting preferentially on nasal venous capacitance vessels, will elicit decongestion with a reduced side-effect liability. In the present study, we evaluated the effects of the selective alpha 2-adrenoceptor agonist BHT-920 in a real-time tissue contractility assay using isolated pig nasal explants and in an in vivo cat model of congestion. The vasoconstrictor and decongestant effects of BHT-920 were compared to the non-selective alpha-adrenoceptor agonist epinephrine and the standard decongestant oxymetazoline. Our results showed that the alpha 2-adrenoceptor agonist BHT-920 preferentially contracts venous sinusoids confirming previous observations [Corboz MR, Varty LM, Rivelli MA, Mutter JC, Mingo G, McLeod R, et al. Effects of an alpha 2-adrenoceptor agonist in nasal mucosa. Arch Physiol Biochem 2003;11: 335-6, Corboz MR, Rivelli MA, Varty LM, Mutter J, Cartwright M, Rizzo CA, et al. Pharmacological characterization of postjunctional alpha-adrenoceptor in human nasal mucosa. Am J Rhinol 2005;19: 495-502] and displays decongestion without affecting blood pressure. Therefore, an alpha 2-adrenoceptor agonist, by causing constriction in the capacitance vessels of nasal mucosa, can produce nasal decongestion without the effects on blood pressure observed with the standard selective alpha 1-adrenoceptor and non-selective alpha-adrenoceptor sympathomimetic decongestants.

In vivo anesthetized guinea pigs were used to investigate the effect of tachykinin NK1- and NK2-receptor antagonists, as a single dose or in combination, against hyperventilation-induced bronchoconstriction (HIB). Guinea pigs were ventilated with a rodent ventilator and placed in a whole-body plethysmograph. Hyperventilation was induced by increasing the respiratory rate from 50 to 185 breaths/min for 10 min that produced a 177+/-45% increase in pulmonary resistance (RL) and a 68+/-7% decrease in lung compliance (CDyn). Intravenous (0.03-0.3mg/kg) and oral (0.3-10mg/kg) pretreatments with the tachykinin NK2-antagonist SR 48968 produced a dose-dependent inhibition of HIB whereas pretreatments with the tachykinin NK1-antagonist CP 99994 (1mg/kg intravenously and 30 mg/kg orally) had no effect on HIB. Intravenous and oral combinations of inactive and low doses of CP 99994 and SR 48968 produced a greater inhibition of HIB than SR 48968 alone. Also, the tachykinin NK3-antagonist SB 223412 (1-3mg/kg intravenously and 30 mg/kg orally) did not significantly reduce HIB although a trend was observed at the highest dose tested intravenously (3mg/kg). We conclude that HIB in the guinea pig is mostly mediated by the tachykinin NK2-receptors and to a lesser extent by the tachykinin NK1-receptors. Because the hyperventilation response in guinea pigs may be a surrogate for exercise-induced obstructive airway disease in human, these results suggest that combined use of dual tachykinin NK1- and NK2-receptor antagonists may provide greater benefit than treatment with single activity tachykinin NK-receptor antagonist.

Sch527123 [2-hydroxy-N,N-dimethyl-3-[[2-[[1(R)-(5-methyl-2-furanyl)propyl]amino]-3,4-dioxo-1-cyclobuten-1-yl]amino]ben-zamide] is a potent, selective antagonist of the human CXCR1 and CXCR2 receptors (Gonsiorek et al., 2007). Here we describe its pharmacologic properties at rodent CXCR2 and at the CXCR1 and CXCR2 receptors in the cynomolgus monkey, as well as its in vivo activity in models demonstrating prominent pulmonary neutrophilia, goblet cell hyperplasia, and mucus production. Sch527123 bound with high affinity to the CXCR2 receptors of mouse (K(d) = 0.20 nM), rat (K(d) = 0.20 nM), and cynomolgus monkey (K(d) = 0.08 nM) and was a potent antagonist of CXCR2-mediated chemotaxis (IC(50) approximately 3-6 nM). In contrast, Sch527123 bound to cynomolgus CXCR1 with lesser affinity (K(d) = 41 nM) and weakly inhibited cynomolgus CXCR1-mediated chemotaxis (IC(50) approximately 1000 nM). Oral treatment with Sch527123 blocked pulmonary neutrophilia (ED(50) = 1.2 mg/kg) and goblet cell hyperplasia (32-38% inhibition at 1-3 mg/kg) in mice following the intranasal lipopolysaccharide (LPS) administration. In rats, Sch527123 suppressed the pulmonary neutrophilia (ED(50) = 1.8 mg/kg) and increase in bronchoalveolar lavage (BAL) mucin content (ED(50) =<0.1 mg/kg) induced by intratracheal (i.t.) LPS. Sch527123 also suppressed the pulmonary neutrophilia (ED(50) = 1.3 mg/kg), goblet cell hyperplasia (ED(50) = 0.7 mg/kg), and increase in BAL mucin content (ED(50) = <1 mg/kg) in rats after i.t. administration of vanadium pentoxide. In cynomolgus monkeys, Sch527123 reduced the pulmonary neutrophilia induced by repeat bronchoscopy and lavage (ED(50) = 0.3 mg/kg). Therefore, Sch527123 may offer benefit for the treatment of inflammatory lung disorders in which pulmonary neutrophilia and mucus hypersecretion are important components of the underlying disease pathology.

The objective of the present experiments was to study the effects of pulmonary inflammation induced by subacute Sulfur-dioxide (SO(2)) exposure on capsaicin-induced responses in isolated primary vagal sensory neurons and cough. Additionally, we examined the effects of SO(2) exposure on respiratory function and lung histology. All experiments were conducted 24 h after 4 days of subacute SO(2) (1000 ppm, 3 h/day for 4 days) exposure. In in vitro experiments, intracellular Ca(2+) concentrations were measured in single nodose ganglia cells isolated from SO(2) treated and control guinea pigs, using a fluorescence-based methodology. In nodose ganglia cells from SO(2)-exposed animals, intracellular Ca(2+) responses evoked by capsaicin (1 x 10(-7) and 1 x 10(-6) M) were significantly augmented (87% and 59%, respectively) compared to nodose ganglia from control animals. In vivo experiments, cough responses induced by a submaximal dose of aerosolized capsaicin (30 microM) were increased approximately 50% in SO(2) exposed animals compared to control animals. The enhanced cough response produced by SO(2) was inhibited by the corticosteroid, dexamethasone (10 mg/kg, p.o. b.i.d for 4 days and 10 mg/kg, p.o. once on day 5). In separate experiments, guinea pigs exposed to SO(2) displayed a decrease in respiratory frequency and minute ventilation and an increase in enhanced pause (PenH), a surrogate measure for pulmonary obstruction. Associated with the SO(2)-induced increase in cough and changes in respiratory parameters was an increase in BAL neutrophils. BAL neutrophil counts were 5+/-4 and 691+/-141 cells x 10(3)/ml for air and SO(2)-exposed animals, respectively. The neutrophillic inflammation induced by SO(2) was attenuated by dexamethasone treatment. Finally, staining for collagen, smooth muscle and goblet cells showed inflammation, remodeling and goblet cell metaphasia in the SO(2)-exposed animals. Our results demonstrate that SO(2) exposure enhances TRPV1 receptor function at the level of the nodose ganglia. This effect occurs in parallel with an increase sensitivity of the cough response to capsaicin.

We have shown previously in normal subjects that a sensory measure, the Urge-to-Cough rating, increases at concentrations of inhaled capsaicin that are lower than those necessary to elicit reflex cough. This finding suggests that the Urge-to-Cough may represent an index of the cough response. Research on cough in the human has most often employed challenge with inhaled capsaicin to induce reflex cough. Current measures of cough sensitivity in the human provide no information regarding the intensity of cough. The influence of codeine on cough perceptual sensitivity and the relationship to cough intensity with capsaicin-induced cough in normal subjects has not been evaluated. This study determined the effect of codeine on capsaicin-induced cough perceptual sensitivity and motor response in normal subjects in a double-blind, placebo-controlled, crossover study. This approach investigated the relevance of cough sensitivity, intensity, and sensory modalities in the assessment of cough suppression in humans. This study consisted of three experimental trials: administration of placebo, 30 mg codeine and 60 mg codeine. The study was double-blinded. The order of the three trials was randomized. Respiratory motor pattern was recorded with EMGs from the rectus abdominis, lateral abdominal muscles and eighth intercostal space. The subjects leaned into a fume hood to inspire deeply for 2 s once through a mouthpiece connected to the nebulizer. A modified Borg scale was used to estimate their Urge-to-Cough. The experimental trial consisted of eight test solutions of 0-200 microM capsaicin. Each solution was presented three times in a randomized block order for a total of 24 presentations. The lowest capsaicin concentration to elicit a cough was determined. The lowest capsaicin concentration to elicit an Urge-to-Cough greater than zero was identified. The Urge-to-Cough sensitivity was determined from the log-log slope. For placebo, the Urge-to-Cough was zero with inhalation of the vehicle and no coughs were observed. The threshold capsaicin concentration for subjects to report an Urge-to-Cough was 15.6 microM (+/-2.6 SEM). The capsaicin concentration threshold for eliciting a cough was significantly greater, 39.3 microM (+/-5.6 SEM). As the capsaicin concentration increased, the magnitude estimation of the Urge to-Cough increased. The slope of the log-log relationship for the Urge-to-Cough was 0.94 (+/-0.07 SEM). As the capsaicin concentration increased, the number and intensity of the coughs increased. The administration of 30 and 60 mg codeine had no significant effect on the threshold capsaicin concentration for the Urge-to-Cough. There was also no significant codeine effect on the slope of the log-log Urge-to-Cough relationship. Thirty and sixty milligram codeine had no significant effect on the relationship between the capsaicin concentration and the number and intensity of the coughs. The results of this study demonstrate that the threshold for a subject to perceive an Urge-to-Cough was less than the capsaicin concentration that elicits the cough motor response. There was a direct relationship between the sensory intensity (magnitude estimation of the Urge-to-Cough) and the cough number and intensity. Thus, as the sense of an Urge-to-Cough increased the cough motor response increased. Neither the 30 nor 60 mg codeine affected the perceptual or motor sensitivity to capsaicin-induced cough. These results showed that the initial threshold for responding to capsaicin-induced cough is the perception of an Urge-to-Cough, followed by a motor cough response if the capsaicin is increased above the perceptual threshold. As the capsaicin concentration increases, both the perceptual need to cough and the cough motor response increase. The response of subjects to inhalation of capsaicin consisted of both a sensory component leading to perception of an Urge-to-Cough and motor cough behavior.

Nasal congestion, one of the major disease features of rhinitis, is induced by the filling of venous sinusoids causing mucosal engorgement with resultant obstruction of nasal airflow. The only available drugs that directly target the underlying vascular features driving nasal obstruction are the sympathomimetic alpha-adrenoceptor agonists due to their vasoconstrictor action. However, standard decongestants are nonselective alpha-adrenoceptor agonists, which have the potential for side-effects liabilities such as hypertension, stroke, insomnia and nervousness. In the present study, the effects of nonsubtype selective alpha(2)-adrenoceptor agonists BHT-920 and PGE-6201204 were evaluated in several isolated nasal mucosa contractile bioassays including dog, pig and monkey, and in a real-time tissue contractility assay using isolated pig nasal explants for BHT-920. The decongestant activity of PGE-6201204 was evaluated in vivo in a cat model of experimental congestion. Our results showed that alpha(2)-adrenoceptor agonists (1) contract nasal mucosa of different species, (2) exert a preferential vasoconstrictor effect on the capacitance vessels (veins and sinusoids), and (3) elicit decongestion. In conclusion, a selective alpha(2)-adrenoceptor agonist causing constriction preferentially in the large venous sinusoids and veins of nasal mucosa and producing nasal decongestion is expected to show efficacy in the treatment of nasal congestion without the characteristic arterio-constrictor action of the standard nonselective sympathomimetic decongestants.

The airway defensive response to tussive agents, such as capsaicin, is frequently assessed by counting the number of cough sounds, or expulsive events. This method does not identify or differentiate important respiratory events that occur in the respiratory muscles and lungs, which are critical in assessing airway defensive responses. The purpose of this study was to characterize the airway defensive behaviours (cough and expiration reflex) to capsaicin exposure in humans. We observed complex motor behaviours in response to capsaicin exposure. These behaviours were defined as cough reacceleration (CRn) and expiration reflex (ERn), where n is the number of expulsive events with and without a preceding inspiratory phase, respectively. Airway defensive responses were defined in terms of frequency (number of expulsive events), strength (activation of abdominal muscles) and behaviour type (CRn vs. ERn). Thirty-six subjects (15 females, 24+/-4 yr) were instrumented with EMG electrodes placed over the rectus abdominis (RA), external abdominal oblique (EO) and the 8th intercostal space (IC8). A custom-designed mouth pneumotachograph was used to assess the airflow acceleration, plateau velocity and phase duration of the expulsive phase. Subjects inhaled seven concentrations of capsaicin (5-200 microM) in a randomized block order. The total number of expulsive events (frequency) and the sum of integrated EMG for the IC8, RA and EO (strength) increased in a curvilinear fashion. Differentiating the airway defense responses into type demonstrated predominately CR1 and CR2 (i.e. inspiration followed by one and two expulsive events, respectively) with very few ER's at <50 microM capsaicin. At higher concentrations (>50 microM) ER's with one or more expulsive events (ER1) appeared, and the number of CR's with three or more expulsive events (CR3) increased. The decrease in EMG activation and airflow measurements with each successive expulsive event suggests a decline in power and shear force as the number of expulsive events increased. Therefore, the airway defensive response to capsaicin is a complex motor pattern that functions to coordinate ER's and CR's with differing numbers of expulsive events possibly to prevent aspirations and keep air moving to promote clearance.

We examined the molecular pharmacology and in vivo effects of a TRPV1 receptor antagonist, N-(4-Tertiarybutylphenyl)-4(3-cholorphyridin-2-yl)-tetrahydro-pyrazine1(2H) - carboxamide (BCTC) on the guinea pig TRPV1 cation channel. BCTC antagonized capsaicin-induced activation and PMA-mediated activation of guinea pig TRPV1 with IC50 values of 12.2 +/- 5.2 nM, and 0.85 +/- 0.10 nM, respectively. In addition, BCTC (100 nM) completely blocked the ability of heterologously expressed gpTRPV1 to respond to decreases in pH. Thus, BCTC is able to block polymodal activation of gpTRPV1. Furthermore, in nodose ganglia cells, capsaicin induced Ca2+ influx through TRPV1 channel was inhibited via BCTC in a concentration dependent manner. In in vivo studies capsaicin (10 - 300 muM) delivered by aerosol to the pulmonary system of non-sensitized guinea pigs produced an increase in cough frequency. In these studies, the tussigenic effects of capsaicin (300 muM) were blocked in a dose dependent fashion when BCTC (0.01-3.0 mg/kg, i.p.) was administered 30 minutes before challenge. The high dose of BCTC (3.0 mg/kg, i.p) produced a maximum inhibition of capsaicin-induced cough of 65%. We also studied the effects of BCTC (0.03 and 3.0) when administered 60 minutes before capsaicin. Under these conditions, BCTC (3.0 mg/kg, i.p) produced a maximum decrease in capsaicin-induced cough of 31%. In ovalbumin passively sensitized guinea pigs, we found that BCTC (1 and 3 mg/kg, i.p.) attenuated antigen ovalbumin (0.3%) cough responses by 27% and 60%, respectively. We conclude that TRPV1 channel activation may play role in cough mediated by antigen in sensitized guinea pigs. Our results supports increasing evidence that TRPV1 may play a role in the generation of the cough response.

The Brown-Norway rat is often used to study the allergic pulmonary response. However, relatively little is known about the delayed phase reactions after allergen challenge in this species. To evaluate the temporal changes in lung function and elucidate the mechanisms involved in the delayed phase response, Brown-Norway rats were sensitized and challenged to aerosolized ovalbumin and lung functions were measured by forced expiratory maneuvers and forced oscillation for up to 10 days after a single antigen challenge. Statistically significant (P < 0.05) reductions in inspiratory capacity, forced vital capacity, functional residual capacity, peak expiratory flow and maximum mid-expiratory flow and increases in respiratory system resistance and elastance were seen by 1 to 3 days after ovalbumin challenge that returned to baseline by 10 days. The reductions in lung function after ovalbumin challenge were blocked by the corticosteroid, betamethasone (1 mg/kg, p.o.). Histological evaluation of lung tissue of sensitized rats demonstrated evidence of interstitial pulmonary edema, an increase in tissue eosinophils and an increase in Periodic Acid Schiff-positive cells in the airway epithelium. Bronchoalveolar lavage fluid samples showed large numbers of eosinophils and increased mucin content up to 6 days after antigen challenge. There was also an increase in wet-to-dry lung weight ratio in the lungs of sensitized rats after antigen. These results demonstrate that prolonged reductions in lung function occur after a single antigen challenge in Brown-Norway rats that is probably due to inflammatory processes producing interstitial pulmonary edema, mucus secretion and cellular influx into the lungs.

Introduction: A method to measure the mucin concentration in bronchoalveolar lavage (BAL) fluid was developed to aid efforts to identify pharmacologically the mechanisms that modulate pathophysiological mucin secretion. Mucins are the major macromolecular components of mucus. In the airways, mucus is the first line of defense against inhaled microorganisms (infection) and particulates (irritation)., Methods: An enzyme-linked immunosorbent assay (ELISA) was developed, comparing two monoclonal anti-mucin antibodies (A10G5 and 45M1) raised to human mucin, to quantify the mucin in BAL fluid from animal models of pulmonary inflammation. To validate the ELISA method, rats were exposed to ovalbumin (OVA, in sensitized rats), lipopolysaccharide (LPS), vanadium pentoxide (V(2)O(5)), or saline. One hundred microliters of BAL fluid was analyzed for mucin concentration. Pooled BAL fluid from untreated rats was used as an internal "plate standard", as a standard mucin that cross-reacts with A10G5 was unavailable., Results: We found both antibodies reacted with rat, human, and guinea-pig mucin; where the 45M1 antibody also reacted with the mucin in porcine BAL, while A10G5 did not. We determined the mucin concentration in each BAL fluid sample relative to the standard, defined as a mucin concentration of 100 plate units. BAL fluid from LPS (218+/-25 plate units, n=5), OVA (386+/-31, n=3), V(2)O(5) (1208+/-450, n=6) challenged rats displayed significantly elevated mucin concentration over their saline controls (126+/-22, n=12). Subsequently, the 45M1 antibody displayed immunoreactivity with a commercially available crude preparation of porcine stomach mucin, allowing us to calculate the concentration of mucin directly compared to the known concentration of the porcine stomach mucin standard. Both the 45M1 and A10G5 based ELISA assays detected higher mucin content in the saline challenged rat than the saline challenged guinea pig BAL., Discussion: The recent availability of the 45M1 antibody and the use of the crude purification of porcine stomach mucin as a reference standard should allow for direct comparison of mucin concentration in BAL (and other fluids).

A novel series of histamine H3 receptor antagonists based on the 4-[(1H-imidazol-4-yl)methyl]piperidine template displaying low CYP2D6 and CYP3A4 inhibitory profiles has been identified. Structural features responsible for the reduction of P450 activity, a typical liability of 4-substituted imidazoles, have been established.

We report the discovery of novel histamine H(3) receptor antagonists based on 4-[(1H-imidazol-4-yl)methyl]piperidine. The most potent compounds in the series (e.g., 7) result from the attachment of a substituted aniline amide to the main pharmacophore piperidine via a two-methylene linker.

Background: Functional alpha1- and alpha2-adrenoreceptor subtype pharmacology was characterized in an in vitro human nasal mucosa contractile bioassay., Methods: Nasal mucosa was obtained from 49 donor patients and mucosal strips were placed in chambers filled with Krebs-Ringer solution and attached to isometric force transducers., Results: Nonselective a-adrenoreceptor agonists epinephrine, norepinephrine, and oxymetazoline produced concentration-dependent contractions of isolated human nasal mucosa (pD2 = 5.2, 4.9, and 6.5, respectively). The alpha2-adrenoreceptor agonist BHT-920 (10 microM)-induced contractions were blocked by yohimbine (0.01-1 microM) and prazosin (0.01-1 microM) inhibited the contractile response to the alpha1-adrenoreceptor agonist phenylephrine (10 microM). Histological analysis showed that phenylephrine and BHT-920 differentially contracted the arteries and veins of human nasal mucosa, respectively., Conclusion: Our results indicate that functional alpha1- and alpha2-adrenoceptors are present and functional in human nasal mucosa. The alpha2-adrenoceptors display a predominant role in contracting the veins and the alpha1-adrenoceptors appear to preferentially constrict the human nasal arteries.

It has been hypothesized that the destruction of lung tissue observed in smokers with chronic obstructive pulmonary disease and emphysema is mediated by neutrophils recruited to the lungs by smoke exposure. This study investigated the role of the chemokine receptor CXCR2 in mediating neutrophilic inflammation in the lungs of mice acutely exposed to cigarette smoke. Exposure to dilute mainstream cigarette smoke for 1 h, twice per day for 3 days, induced acute inflammation in the lungs of C57BL/6 mice, with increased neutrophils and the neutrophil chemotactic CXC chemokines macrophage inflammatory protein (MIP)-2 and KC. Treatment with SCH-N, an orally active small molecule inhibitor of CXCR2, reduced the influx of neutrophils into the bronchoalveolar lavage (BAL) fluid. Histological changes were seen, with drug treatment reducing perivascular inflammation and the number of tissue neutrophils. beta-Glucuronidase activity was reduced in the BAL fluid of mice treated with SCH-N, indicating that the reduction in neutrophils was associated with a reduction in tissue damaging enzymes. Interestingly, whereas MIP-2 and KC were significantly elevated in the BAL fluid of smoke exposed mice, they were further elevated in mice exposed to smoke and treated with drug. The increase in MIP-2 and KC with drug treatment may be due to the decrease in lung neutrophils that either are not present to bind these chemokines or fail to provide a feedback signal to other cells producing these chemokines. Overall, these results demonstrate that inhibiting CXCR2 reduces neutrophilic inflammation and associated lung tissue damage due to acute cigarette smoke exposure.

Background: Airway closure is frequently observed in human asthma. However, limited information exists on the factors that cause this condition. In this study, an allergic cynomolgus monkey model was used to characterize the condition of airway closure and assess the contribution of histamine H1 receptors to this response., Methods: Oscillatory lung mechanics, arterial blood gases during ventilation on 100% O2 and functional residual capacity (FRC) assessed by helium dilution were measured before and then 10 min and 24 h after Ascaris aerosol challenge in 12 male Ascaris-sensitive cynomolgus monkeys. The monkeys were pretreated with intravenous saline or chlorpheniramine maleate (0.3 mg/kg) in a randomized crossover design., Results: Ascaris challenge produced a large increase in airway resistance, an increase in lung tissue damping (G) that measures ventilation inhomogeneity in the lung, a reduction in arterial oxygen tension (PaO2) during ventilation on 100% O2 and a reduction in FRC. These effects were seen 10 min after the Ascaris challenge, but by 24 h, these parameters had returned close to the baseline values. Chlorpheniramine maleate (0.3 mg/kg, i.v.) produced a 12-fold shift in the histamine bronchoconstrictor dose-response curve. Pretreatment of monkeys with chlorpheniramine maleate (0.3 mg/kg, i.v.) attenuated the increase in airway resistance induced by Ascaris challenge, but had only a small effect on the increase in G and the reductions in PaO2 and FRC after antigen., Conclusions: These results demonstrate that airway closure occurs immediately after the antigen challenge in allergic cynomolgus monkeys and that histamine H1 receptors contribute very minimally to this response., (Copyright 2005 S. Karger AG, Basel)

The TRPV1 channel is mainly expressed in sensory nerves. Activation of the channel induces neuropeptide release from central and peripheral sensory nerve terminals, resulting in the sensation of pain, neurogenic inflammation, smooth muscle contraction and cough. The TRPV1 channel can be activated by vanilloids such as capsaicin, as well as endogenous stimulators including H(+), heat, lipoxygenase products and anandamide. TRPV1 channel function is upregulated by several endogenous mediators present in inflammatory conditions, which decreases the threshold for activation of the channel. Under these conditions, TRPV1 can be activated by physiological body temperature, slight acidification or lower concentration of TRPV1 agonists. There is evidence that TRPV1 plays a role in the development of pathophysiological changes and symptoms in several diseases. In this review, we discuss TRPV1 channel activation and regulation in normal and diseased conditions, the role of TRPV1 in pain, cough, asthma and urinary incontinence, and the potential use of TRPV1 antagonists as a novel therapy for these diseases.

The pharmacological consequences of combining a histamine H1 receptor antagonist with a H3 antagonist on cutaneous microvascular permeability due to intradermal (i.d.) injections of compound 48/80, a mast cell liberator of histamine, was studied in the anesthetized guinea pig. Compound 48/80 (0.0003, 0.001, 0.003 and 0.01%) induced permeability responses were attenuated, as determined by Evans blue extravasation, in animals pretreated with the H1 antagonist, chlorpheniramine (CTM; 1.0 mg/kg, i.v.) by 17 +/- 4, 31 +/- 4, 32 +/- 4 and 37 +/- 4%, respectively. Combination treatment with an H1 and H3 antagonist displayed greater inhibitory efficacy against the effects elicited by compound 48/80. Specifically, combined treatment with CTM (1.0 mg/kg, i.v.) and the H3 antagonist, thioperamide (THIO 1.0 mg/kg,i.v.) inhibited the skin responses of i.d. compound 48/80 (0.0003, 0.001, 0.003 and 0.01%) by 36 +/- 4, 45 +/- 4, 49 +/- 4 and 54 +/- 4%. A second H3 antagonist, clobenpropit (CLOB; 0.3 mg/kg, i.v.) plus CTM (1.0 mg/kg, i.v.) also inhibited Evans blue extravasation. Treatment with THIO (1.0 mg/kg, i.v.) and CLOB (0.3 mg/kg, i.v.) administered alone had no effect on compound 48/80-induced skin responses. We conclude that combination administration of a H1 and a H3 histamine receptor antagonist produces greater inhibitory effect on cutaneous microvascular permeability produced by released mast cell-derived histamine than either a H1 or H3 antagonist administered separately. In addition, the antiallergy activity of combining a H3 antihistamine with a H3 antagonist activity might provide a novel approach for the treatment of allergic skin diseases such as urticaria.

Forced oscillation is a technique that has been used to measure airway and lung tissue impedance. To evaluate airway and lung tissue impedance in a colony of cynomolgus monkeys housed at Schering-Plough Research Institute, a forced oscillation technique was used to measure Newtonian resistance (R(N)), tissue damping (G), tissue elastance (H) and lung hysteresivity (eta). Functional residual capacity (FRC) was also measured to correlate the lung impedance data with FRC. There was no difference in R(N), G, H and eta between Ascaris sensitive allergic monkeys (n=25) and a small cohort (n=5) of non-allergic monkeys under baseline conditions. However, a highly significant (p<0.0001) negative correlation (r=0.71) was found between FRC and H. Significant correlations were also found between FRC and G (r=0.53) and FRC and R(N) (r=0.50). Bronchoprovocation with aerosolized histamine increased R(N), G, H and eta and reduced FRC by 29+/-3% (n=30) from baseline. In monkeys that were hyperreactive to the histamine challenge, an exaggerated increase in lung tissue damping was seen whereas monkeys that were less reactive to the histamine showed greater increases in R(N). Aerosolized albuterol (0.003-3mg/ml) produced a concentration-dependent reversal of the increases in R(N), G, H and eta induced by histamine with the greatest reversal seen on R(N). Deep inspiration, performed after the aerosolized albuterol exposure, also reversed the histamine-induced changes in R(N), G, and H with the complete reversal seen on the increase in H. These results demonstrate that significant correlations exist between airway and lung tissue impedance and FRC and that airway and lung tissue mechanics contribute significantly to inherent bronchoconstrictor reactivity and to the bronchodilator response to a beta-adrenergic agonist and deep inspiration in cynomolgus monkeys.

It has been suggested that the rat is relatively more susceptible to toxicity induced by inhibitors for type 4 cAMP-specific phosphodiesterase (PDE4). In this study designed to elucidate possible biochemical basis for the higher susceptibility, we compared PDE4 expression levels and their functional relevance among rats, monkeys and humans. In several toxicologically relevant tissues and blood leukocytes, the mRNA expression levels of PDEs 4A, 4B, 4C and 4D were significantly higher in rats than in humans. We confirmed that higher PDE4 expression levels were correlated with a higher enzyme activity level in rat leukocytes. The PDE4 enzyme activity level of leukocytes in monkeys fell between that of rats and humans. Functionally, the potencies of the PDE4 inhibitors rolipram, SB 207499 and SCH 351591 in inhibiting tumor necrosis factor production from leukocytes were in the following order: rat > monkey > human. In addition, rolipram was about 10-fold more potent in rats than in humans in inhibiting phenylephrine-induced contraction of renal artery. These inhibitors were confirmed to be highly selective for PDE4 in comparison to all other PDE families, and to inhibit rat and human PDE4s with identical potencies. Taken together, these results suggest that the higher susceptibility of rats to PDE4 inhibitor-induced toxicity might be due to their higher expression levels of PDE4, and that PDE4 inhibitors may be safer in humans than in monkeys and, particularly, rats.

Tachykinins are important in the development of pulmonary inflammation in mice but the tachykinin receptor subtype mediating this response has not been defined. To elucidate the role of tachykinin NK3-receptors on allergen-induced pulmonary inflammation, studies were performed on ovalbumin (OVA) sensitized and challenged mice with genetic disruption of the tachykinin NK3-receptor (NK3-/-). Aerosol OVA (0.5%) challenge produced eosinophil influx into the bronchoalveolar lavage fluid and lung tissue, goblet cell hyperplasia and damage to the airway epithelium of both NK3-/- mice and in wild type control mice (NK3+/+). There was no difference in the magnitude of these allergic inflammatory pulmonary responses between NK3-/- and NK3+/+ mice. These results find no role for tachykinin NK3-receptors on the pulmonary eosinophilia and lung damage after antigen challenge in mice.

Hypotonic stimulation induces airway constriction in normal and asthmatic airways. However, the osmolarity sensor in the airway has not been characterized. TRPV4 (also known as VR-OAC, VRL-2, TRP12, OTRPC4), an osmotic-sensitive cation channel in the transient receptor potential (TRP) channel family, was recently cloned. In the present study, we show that TRPV4 mRNA was expressed in cultured human airway smooth muscle cells as analyzed by RT-PCR. Hypotonic stimulation induced Ca(2+) influx in human airway smooth muscle cells in an osmolarity-dependent manner, consistent with the reported biological activity of TRPV4 in transfected cells. In cultured muscle cells, 4alpha-phorbol 12,13-didecanoate (4-alphaPDD), a TRPV4 ligand, increased intracellular Ca(2+) level only when Ca(2+) was present in the extracellular solution. The 4-alphaPDD-induced Ca(2+) response was inhibited by ruthenium red (1 microM), a known TRPV4 inhibitor, but not by capsazepine (1 microM), a TRPV1 antagonist, indicating that 4-alphaPDD-induced Ca(2+) response is mediated by TRPV4. Verapamil (10 microM), an L-type voltage-gated Ca(2+) channel inhibitor, had no effect on the 4-alphaPDD-induced Ca(2+) response, excluding the involvement of L-type Ca(2+) channels. Furthermore, hypotonic stimulation elicited smooth muscle contraction through a mechanism dependent on membrane Ca(2+) channels in both isolated human and guinea pig airways. Hypotonicity-induced airway contraction was not inhibited by the L-type Ca(2+) channel inhibitor nifedipine (1 microM) or by the TRPV1 inhibitor capsazepine (1 microM). We conclude that functional TRPV4 is expressed in human airway smooth muscle cells and may act as an osmolarity sensor in the airway.

We have previously shown that N/OFQ, the endogenous peptide ligand for the 'opioid-like' NOP receptor, inhibits cough in guinea pigs and cats. In the present study we sought to continue our characterization of the cough-suppressant effects of NOP stimulation by profiling the pulmonary and antitussive effects of a novel non-peptide NOP agonist, Ro-64-6198, in guinea pigs. In receptor-binding assays, we confirmed that Ro-64-6198 selectively binds to NOP receptors over other opioid receptors. The Ki values for Ro-64-6198 at NOP, MOP, KOP and DOP receptors was 0.3, 36, 214 and 3,787 nmol/l, respectively. In GTPgammaS-binding assays, Ro-64-6198 displayed >900-fold functional selectivity at NOP relative to MOP receptors. We evaluated the effects of Ro-64-6198 (3 and 10 micromol/l) in isolated guinea pig nodose ganglia cells on the increases in intracellular Ca2+ concentration evoked by capsaicin stimulation (1 x 10(-8)-1 x 10(-6) mol/l). Similar to previously reported data with N/OFQ, Ro-64-6198 (3 and 10 micromol/l) significantly attenuated Ca2+ responses in nodose ganglia cells produced by exposure to capsaicin. The effect of Ro-64-6198 (3 micromol/l) on capsaicin-induced intracellular Ca2+ responses was blocked by the NOP antagonist, J113397 (3 micromol/l). In guinea pig in vivo studies, aerosolized capsaicin (10-300 micromol/l) produced a dose-dependent increase in cough number. Ro-64-6198 given i.p. significantly inhibited cough due to capsaicin (300 micromol/l) exposure. In a duration study we found that the maximum antitussive effect (42 +/- 8% inhibition) of Ro-64-6198 (3 mg/kg) was observed at 1 h after i.p. administration. Also at 1 h after administration, Ro-64-6198 (0.003-3.0 mg/kg, i.p.) produced a dose-dependent inhibition of cough. The antitussive effect of Ro-64-6198 (3 mg/kg, i.p.) was blocked by J113397 (12 mg/kg, i.p.) but not by the classical opioid antagonist naltrexone (10 mg/kg, i.p.). Although the antitussive action of Ro-64-6198 may be mediated by a central and/or a peripheral site of action, we hypothesize that selective oral NOP agonists that do not penetrate the blood-brain barrier may provide a novel approach for the treatment of cough. Moreover, because these drugs do not interact at MOP receptors, they may be devoid of codeine-like side effects such as respiratory depression, sedation, constipation or proclivities for addictive liabilities., (Copyright 2004 S. Karger AG, Basel)

This study investigated the effects of antigen challenge on the cough reflex in dogs that were neonatally sensitized to ragweed. Tidal volume (V(T)), respiratory rate (f), pulmonary resistance (R(L)), dynamic lung compliance (C(Dyn)) and the number and amplitude (increase in mean peak expiratory pressure) of coughs induced by mechanical stimulation of the intrathoracic trachea were measured in propofol-anesthetized dogs. Aerosolized ragweed challenge had no effect to induce spontaneous cough but increased f and R(L) and reduced V(T) and C(Dyn). Mechanical stimulation of the intrathoracic trachea at this time produced 19+/-5 coughs with an average increase in cough amplitude of 11+/-1 cm H(2)O which differed significantly from the number (9+/-2 coughs) and amplitude (30+/-5.5 cm H(2)O) of mechanically induced coughs after treatment with aerosolized saline. Both the number and amplitude of mechanically induced coughs returned to baseline values by 24-48 h after the ragweed challenge. Similar results were obtained after challenge with aerosolized histamine (0.3-1% histamine) that did not induce spontaneous coughs but increased f, reduced V(T) and decreased C(Dyn) and increased the number but reduced the amplitude of the mechanically induced coughs. In conclusion, both antigen and histamine bronchoprovocation changed the characteristics of the mechanically induced cough in dogs to a response of increased cough number but reduced mean expiratory cough amplitude.

CP-99994 [(+)-(2S,3S)-3-(2-methoxybenzylamino)-2-phenylpiperidine] is a selective tachykinin NK(1) receptor antagonist that inhibits cough in guinea pigs and cats. This study examined the antitussive effects of CP-99994 in dogs produced by mechanical stimulation of the intrathoracic trachea. CP-99994 (10 mg/kg, p.o.) inhibited cough frequency by 52% at 2 h, 31% at 6 h and by 21% at 24 h. Cough amplitude was inhibited by 45% at 6 h but unchanged at 2 and 24 h after CP-99994. Plasma levels of CP-99994 were highest at 2 h (75+/-26 ng/ml) and fell to 22+/-6 ng/ml at 6 h. These results demonstrate antitussive activity of CP-99994 in dogs at a dose proven to antagonize tachykinin NK(1) receptors in this species.

The peripheral histamine H3 receptor is a presynaptic heterologous receptor located on postganglionic sympathetic nerve fibers innervating sympathetic effector systems such as blood vessels and the heart. An extensive body of evidence shows that activation of the histamine H3 receptor attenuates sympathetic tone by presynaptic inhibition of noradrenaline release. It is proposed that this sympathoinhibitory action, in vivo, leads to reduced vasoconstriction, thereby eliciting a vasodilatory effect. In humans, the peripheral histamine H3 receptor has also been shown to exert a sympathoinhibitory function on specific peripheral autonomic effector systems. For example, human saphenous vein and heart possess functional presynaptic histamine H3 receptors on the sympathetic nerve terminals that upon activation decrease the sympathetic tone to these respective organs. The present studies were conducted to define the role of histamine H3 receptors on neurogenic sympathetic vasoconstrictor responses in human nasal turbinate mucosa. Contractility studies were conducted to evaluate the effect of histamine H3 receptor activation on sympathetic vasoconstriction in surgically isolated human nasal turbinate mucosa. We found that the histamine H3 receptor agonist, (R)-alpha-methylhistamine (30 and 300 nM), inhibited electrical field stimulation-induced (neurogenic) sympathetic vasoconstriction in a concentration-dependent fashion. Pretreatment with the selective histamine H3 receptor antagonist, clobenpropit (100 nM), blocked the sympathoinhibitory effect of (R)-alpha-methylhistamine on the neurogenic sympathetic vasoconstriction. In addition, analysis of Taqman mRNA expression studies showed a specific, high level of distribution of the histamine H3 receptor localized in the human nasal mucosa. Taken together, these studies indicate that histamine H3 receptors modulate vascular contractile responses in human nasal mucosa most likely by inhibiting noradrenaline release from sympathetic nerve terminals in nasal mucosa. It is further suggested that histamine H3 receptors may play a role in the regulation of vascular tone and nasal patency in histamine-dependent allergic nasal congestive disease.

We have previously reported on a model of lipopolysaccharide (LPS)-induced pulmonary inflammation in rats, where LPS-challenged animals develop a significant pulmonary neutrophilia and mucus hypersecretion. In the current studies, we utilized whole body plethysmography and computer assisted data acquisition to examine changes in pulmonary parameters, e.g. frequency (f) tidal volume and Penh as a measure of bronchoconstriction, due to LPS-challenge in conscious rats. Compared to saline challenge, LPS-challenged rats displayed a significant increase in (f) which began within 30 min, peaked by 2 h and remained elevated up to 24 h. Mirroring this increase in (f) was a decrease in the observed tidal volume of LPS-challenged rats. Additionally, compared to saline challenge, LPS-challenge provoked a significant and spontaneous bronchoconstriction, as measured by Penh, 2 h after challenge. In order to further understand these observed LPS-induced pulmonary changes, we utilized two classes of pulmonary obstructive disease standards, namely, bronchodilators and anti-inflammatory agents, and examined their ability to affect the spontaneous bronchoconstriction and the increase in (f) seen at two discrete time points, i.e. 2 and 24 h after LPS-challenge. While ineffective on either the 2 h increase in (f) or the LPS-induced inflammation, animals pretreated with salbutamol (10 mg/kg, p.o.) were protected from the increase in (f) seen at the 24 h time point after LPS-challenge. In contrast, when animals were pretreated with theophylline (10 mg/kg, p.o.) no effect on the LPS-induced pulmonary inflammation or increase in (f) was noted. Meanwhile, in animals pretreated with either betamethasone (3 mg/kg, p.o.) or SB207499 (10 mg/kg, p.o.), a PDE4 inhibitor, doses previously shown to block the LPS-induced neutrophilic inflammation, the persistent increase in (f) seen at 24 h was attenuated, but neither compound was able to attenuate either the increase in (f) or the spontaneous bronchoconstriction seen at 2 h. In summary, the intra-tracheal LPS-challenge of rats results in pulmonary inflammation and dysfunction, which is similar to that seen in COPD patients. We conclude that the early increase in (f) and bronchoconstriction are not dependent upon airway inflammation, but airway inflammation most likely contributes to the persistent increase in (f) seen at 24 h.