Microfluidic cell cultures enable investigation of complex physiological tissue properties and functionalities. For convenience, they are often implemented with immortalized cell lines, but primary cells more closely approximate the in vivo biology. Our aim was to develop a biomimetic microfluidic model of the human airway using all primary cells. The model is comprised of airway epithelial cells cultured at an air-liquid interface, lung fibroblasts and polarized microvascular endothelial cells, respectively positioned in three vertically stacked, individually accessible compartments separated by nanoporous membranes. We report device fabrication, a gravity fed microfluidic system, and culture medium able to support functional co-cultures of all three primary human cell types. As characterized by imaging and permeability measurements, airway epithelial cells in microfluidic devices displayed mucociliary differentiation and barrier function. Subjacent fibroblasts and microvascular endothelial cells were added under conditions enabling co-culture for at least 5 days. Microfluidic airway models based on primary human cells in a relevant biomimetic configuration will improve physiological relevance and will enable novel disease modeling and drug development studies.

GPR88 is an orphan G-protein-coupled receptor (GPCR) enriched in the striatum. Genetic deletion and gene expression studies have suggested that GPR88 plays an important role in the regulation of striatal functions and is implicated in psychiatric disorders. The signal transduction pathway and receptor functions of GPR88, however, are still largely unknown due to the lack of endogenous and synthetic ligands. In this paper, we report the synthesis of a GPR88 agonist 2-PCCA and its pure diastereomers, which were functionally characterized in both transiently and stably expressing GPR88 HEK293 cells. 2-PCCA inhibited isoproterenol-stimulated cAMP accumulation in a concentration-dependent manner in cells expressing GPR88 but not in the control cells, suggesting that the observed cAMP inhibition is mediated through GPR88 and that GPR88 is coupled to Gαi. 2-PCCA did not induce calcium mobilization in GPR88 cells, indicating no Gαq-mediated response. A structure-activity relationship (SAR) study of 2-PCCA was also conducted to explore the key structural features for GPR88 agonist activity.

The recent discovery of allosteric modulators of the CB1 receptor including PSNCBAM-1 (4) has generated significant interest in CB1 receptor allosteric modulation. Here in the first SAR study on 4, we have designed and synthesized a series of analogs focusing on modifications at two positions. Pharmacological evaluation in calcium mobilization and binding assays revealed the importance of alkyl substitution at the 2-aminopyridine moiety and electron deficient aromatic groups at the 4-chlorophenyl position for activity at the CB1 receptor, resulting in several analogs with comparable potency to 4. These compounds increased the specific binding of [3H]CP55,940, in agreement with previous reports. Importantly, 4 and two analogs dose-dependently reduced the Emax of the agonist curve in the CB1 calcium mobilization assays, confirming their negative allosteric modulator characteristics. Given the side effects associated with CB1 receptor orthosteric antagonists, negative allosteric modulators provide an alternative approach to modulate the pharmacologically important CB1 receptor.

Increasing evidence implicates the orexin 1 (OX1) receptor in reward processes, suggesting OX1 antagonism could be therapeutic in drug addiction. In a program to develop an OX1 selective antagonist, we designed and synthesized a series of substituted tetrahydroisoquinolines and determined their potency in OX1 and OX2 calcium mobilization assays. Structure-activity relationship (SAR) studies revealed limited steric tolerance and preference for electron deficiency at the 7-position. Pyridylmethyl groups were shown to be optimal for activity at the acetamide position. Computational studies resulted in a pharmacophore model and confirmed the SAR results. Compound 72 significantly attenuated the development of place preference for cocaine in rats.

Salvinorin A (1) is a hallucinogenic neoclerodane diterpene isolated from the widely available psychoactive plant Salvia divinorum and is the first example of a non-nitrogenous opioid receptor ligand. At present, there is little information available as to why this compound is selective for κ opioid receptors. One approach to better understanding the mode of binding of 1 at κ receptors is to systematically alter the structure of 1 and examine the effects on opioid receptor affinity and activity. Currently, there is a paucity of methods described for the preparation of analogues derived from 1. Here, we report the investigation of several chemical transformations of 1 isolated from S. divinorum. In particular, this work provides a semisynthesis of salvinicins A (2) and B (3) and has identified 10a as the first neoclerodane diterpene with δ opioid antagonist activity. Keywords salvinorin A; salvinicin A; salvinicin B; neoclerodane diterpenes; and Salvia divinorumExtracts of the opium poppy, Papaver somniferum, have been used for centuries to relieve pain and to induce sleep. 1 Among the most important constituents in opium are the alkaloids morphine and codeine. Many of the agonists and antagonists derived from these alkaloids are essential for the practice of modern medicine. While many potent agonists are effective analgesics, they have undesirable side effects, such as tolerance, dependence, and respiratory depression. 2Recently, opioid receptors have been implicated in the actions of salvinorin A (1), the major active ingredient of Salvia divinorum Epling & Játiva (Lamiaceae), a hallucinogenic plant that has been used historically in the traditional practices of the Mazatecs in Oaxaca, This finding is unique because 1 represents the only known lipid-like small molecule that † This work was presented in part at the American Society of Pharmacognosy 46 th Annual Meeting, July 23 -July 27, 2005.*To whom correspondence should be addressed, S327 PHAR, MNPC, 115 S. Grand Ave. Iowa City, Iowa 52242-1112, Telephone: (319) Fax: (319) selectively and potently activates a peptidergic G-protein coupled receptor (GPCR). 7, 8 Diterpene 1 was found to be a high efficacy agonist for κ opioid receptors (κOR). 6, 9 Thus, 1 provides a truly unique template for the development of novel agents to attenuate pain with a potential for reduced abuse liability.Recently, we 10, 11 and others 12-15 have described the isolation and synthesis of several novel neoclerodane diterpenes with opioid receptor activity. Among these compounds were salvinicin A (2) and salvinicin B (3). 11 Diterpene 2 was identified as a partial κOR agonist, whereas, 3 was found to be the first neoclerodane diterpene with μ antagonist activity. There is a growing body of information as to why 1 and related analogues have activity at opioid receptors. 10, 11, 13-17 However, there are few synthetic methods described for related analogues of 1. Based on previous reports, 10, 11, 13,14 the C-2 position and furan ring appear to be important sites for the op...

Cystic Fibrosis (CF) is one of the most common autosomal genetic disorders in humans. This disease is caused by mutations within a single gene, coding for the cystic fibrosis transmembrane conductance regulator (CFTR) protein. The phenotypic hallmark of CF is chronic lung infection and associated inflammation from opportunistic microbes such as Pseudomonas aeruginosa (PA), Haemophilus influenzae , and Staphylococcus aureus . This eventually leads to deterioration of lung function and death in most CF patients. Unfortunately, there is no approved therapy for correcting the genetic defect causal to the disease. Hence, controlling inflammation and infection in CF patients are critical to disease management. Accordingly, anti-inflammatory agents and antibiotics are used to manage chronic inflammation and infection in CF patients. However, most of the anti-inflammatory agents in CF have severe limitations due to adverse side effects, and resistance to antibiotics is becoming an even more prominent problem. Thus, new agents that can be used to control chronic inflammation in CF are needed in the absence of a cure for the disease. Activation of the transcription factor NFκB through Toll-like receptors (TLR) following bacterial infection is principally involved in regulating lung inflammation in CF. NFκB regulates the transcription of several genes that are involved in inflammation, anti-apoptosis and anti-microbial activity, and hyper-activation of this transcription factor leads to a potent inflammatory response. Thus, NFκB is a potential anti-inflammatory drug target in CF. Screening of several compounds from natural sources in an in vitro model of CF-related inflammation wherein NFκB is activated by filtrates of a clinically isolated strain of PA (PAF) led us to Withaferin A (WFA), a steroidal lactone from the plant Withania Somnifera L. Dunal . Our data demonstrate that WFA blocks PAF-induced activation of NFκB as determined using reporter assays, IL-8 measurements and high-content fluorescent imaging of NFκB subunit p65 translocation. Since the airways of CF patients can be specifically targeted for delivery of therapeutics, we propose that WFA should be further studied as an anti-inflammatory agent in models of CF related inflammation mediated by NFκB.

A series of 7-substituted 1,1-diphenyl-hexahydro-oxazolo[3,4-a]pyrazin-3-ones were synthesized and tested for Neuropeptide S (NPS) antagonist activity. A concise synthetic route was developed, which features a DMAP catalyzed carbamate formation. 4-Fluorobenzyl urea (1c) and benzyl urea (1d) were identified as the most potent antagonists among the compounds examined. Structure-activity relationships (SARs) demonstrate that a 7-position urea functionality is required for potent antagonist activity and alkylation of the urea nitrogen (1e) or replacement with carbon or oxygen (5a) results in reduced potency. In addition, compounds with alpha-methyl substitution (1b) or elongated alkyl chains (1h and 1j) had reduced potency, indicating a limited tolerance for 7-position substituents.

Procedures were developed for the synthesis of 3-methyl-5-phenylethynyl[1,2,4]triazine (4), 6-methyl-3-phenylethynyl[1,2,4]triazine (5), and 5-methyl-3-phenylethynyl[1,2,4]triazine (6a) as analogues of 2-methyl-6-(phenylethynyl)pyridine (2). The compounds were evaluated for antagonism of glutamate-mediated mobilization of internal calcium in an mGluR5 in vitro efficacy assay. The most potent of the three analogues was 6a. Twenty additional analogues of 6a were synthesized and evaluated for mGluR5 antagonist efficacy. The most potent compounds were 3-(3-methylphenylethynyl)-5-methyl[1,2,4]triazine (6b), 5-(3-chlorophenylethynyl)-5-methyl[1,2,4]triazine (6c), and 3-(3-bromophenylethynyl)-5-methyl[1,2,4]triazine (6d).