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Fecal microbiota transplantation (FMT) is a highly effective therapy for recurrent Clostridium difficile infection (R-CDI), but its mechanisms remain poorly understood. Emerging evidence suggests that gut bile acids have significant influence on the physiology of C. difficile, and therefore on patient susceptibility to recurrent infection. We analyzed spore germination of 10 clinical C. difficile isolates exposed to combinations of bile acids present in patient feces before and after FMT. Bile acids at concentrations found in patients' feces prior to FMT induced germination of C. difficile, although with variable potency across different strains. However, bile acids at concentrations found in patients after FMT did not induce germination and inhibited vegetative growth of all C. difficile strains. Sequencing of the newly identified germinant receptor in C. difficile, CspC, revealed a possible correspondence of variation in germination responses across isolates with mutations in this receptor. This may be related to interstrain variability in spore germination and vegetative growth in response to bile acids seen in this and other studies. These results support the idea that intra-colonic bile acids play a key mechanistic role in the success of FMT, and suggests that novel therapeutic alternatives for treatment of R-CDI may be developed by targeted manipulation of bile acid composition in the colon.

Schistosomiasis is a debilitating tropical disease caused by infection with parasitic blood flukes. Approximately 260 million people are infected worldwide, underscoring the clinical and socioeconomic impact of this chronic infection. Schistosomiasis is treated with the drug praziquantel (PZQ), which has proved the therapeutic mainstay for over three decades of clinical use. However, the molecular target(s) of PZQ remain undefined. Here we identify a molecular target for the antischistosomal eutomer —  (R) -PZQ — which functions as a partial agonist of the human serotoninergic 5HT 2B receptor. (R) -PZQ modulation of serotoninergic signaling occurs over a concentration range sufficient to regulate vascular tone of the mesenteric blood vessels where the adult parasites reside within their host. These data establish (R) -PZQ as a G-protein-coupled receptor ligand and suggest that the efficacy of this clinically important anthelmintic is supported by a broad, cross species polypharmacology with PZQ modulating signaling events in both host and parasite. Schistosomiasis is caused by infection with the flatworm Schistosoma , and praziquantel is the drug of choice for its treatment. Here, Chan and colleagues identify praziquantel as a ligand for the human serotoninergic 5-HT 2B G-protein-coupled receptor, and reveal a function for praziquantel as a regulator of vascular tone in treated hosts.

There is demand for non-dimethyl sulfoxide (DMSO) cryoprotective agents that maintain cell viability without causing poor postthaw function or systemic toxicity. The focus of this investigation involves expanding our understanding of multicomponent osmolyte solutions and their ability to preserve cell viability during freezing. Controlled cooling rate freezing, Raman microscopy, and differential scanning calorimetry (DSC) were utilized to evaluate the differences in recovery and ice crystal formation behavior for solutions containing multiple cryoprotectants, including sugars, sugar alcohols, and small molecule additives. Postthaw recovery of mesenchymal stem cells (MSCs) in solutions containing multiple osmolytes have been shown to be comparable or better than that of MSCs frozen in 10% DMSO at 1°C/min when the solution composition is optimized. Maximum postthaw recovery was observed in these multiple osmolyte solutions with incubation times of up to 2 h before freezing. Raman images demonstrate large ice crystal formation in cryopreserved cells incubated for shorter periods of time (∼30 min), suggesting that longer permeation times are needed for these solutions. Recovery was dependent upon the concentration of each component in solution, and was not strongly correlated with osmolarity. It is noteworthy that the postthaw recovery varied significantly with the composition of solutions containing the same three components and this variation exhibited an inverted U-shape behavior, indicating that there may be a “sweet spot” for different combinations of osmolytes. Raman images of freezing behavior in different solution compositions were consistent with the observed postthaw recovery. Phase change behavior (solidification patterns and glass-forming tendency) did not differ for solutions with similar osmolarity, but differences in postthaw recovery suggest that biological, not physical, methods of protection are at play. Lastly, molecular substitution of glucose (a monosaccharide) for sucrose (a disaccharide) resulted in a significant drop in recovery. Taken together, the information from these studies increases our understanding of non-DMSO multicomponent cryoprotective solutions and the manner by which they enhance postthaw recovery.

Elevated intraocular pressure is the only treatable risk factor for glaucoma, an eye disease that is the leading cause of irreversible blindness worldwide. We have identified cromakalim prodrug 1 (CKLP1), a novel water-soluble ATP-sensitive potassium channel opener, as a new ocular hypotensive agent. To evaluate the pharmacokinetic and safety profile of CKLP1 and its parent compound levcromakalim, Dutch-belted pigmented rabbits were treated intravenously (0.25 mg/kg) or topically (10 mM; 4.1 mg/ml) with CKLP1. Body fluids (blood, aqueous and vitreous humor) were collected at multiple time points and evaluated for the presence of CKLP1 and levcromakalim using a liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) based assay. Histology of tissues isolated from Dutch-belted pigmented rabbits treated once daily for 90 days was evaluated in a masked manner by a certified veterinary pathologist. The estimated plasma parameters following intravenous administration of 0.25 mg/kg of CKLP1 showed CKLP1 had a terminal half-life of 61.8 ± 55.2 min, Tmax of 19.8 ± 23.0 min and Cmax of 1968.5 ± 831.0 ng/ml. Levcromakalim had a plasma terminal half-life of 85.0 ± 37.0 min, Tmax of 61.0 ± 32.0 min and Cmax of 10.6 ± 1.2 ng/ml. Topical CKLP1 treatment in the eye showed low levels (<0.3 ng/mL) of levcromakalim in aqueous and vitreous humor, and trace amounts of CKLP1 and levcromakalim in the plasma. No observable histological changes were noted in selected tissues that were examined following topical application of CKLP1 for 90 consecutive days. These results suggest that CKPL1 is converted to levcromakalim in the eye and likely to some extent in the systemic circulation.

The anthelmintic praziquantel (±PZQ) serves as a highly effective antischistosomal therapy. ±PZQ causes a rapid paralysis of adult schistosome worms and deleterious effects on the worm tegument. In addition to these activities against the parasite, ±PZQ also modulates host vascular tone in blood vessels where the adult worms reside. In resting mesenteric arteries ±PZQ causes a constriction of basal tone, an effect mediated by (R)-PZQ activation of endogenous serotoninergic G protein coupled receptors (GPCRs). Here, we demonstrate a novel vasodilatory action of ±PZQ in mesenteric vessels that are precontracted by high potassium-evoked depolarization, an effect previously reported to be associated with agonists of the transient receptor potential melastatin 8 channel (TRPM8). Pharmacological profiling a panel of 17 human TRPs demonstrated ±PZQ activity against a subset of human TRP channels. Several host TRP channels (hTRPA1, hTRPC3, hTRPC7) were activated by both (R)-PZQ and (S)-PZQ over a micromolar range whereas hTRPM8 showed stereoselective activation by (S)-PZQ. The relaxant effect of ±PZQ in mesenteric arteries was caused by (S)-PZQ, and mimicked by TRPM8 agonists. However, persistence of both (S)-PZQ and TRPM8 agonist evoked vessel relaxation in TRPM8 knockout tissue suggested that canonical TRPM8 does not mediate this (S)-PZQ effect. We conclude that (S)-PZQ is vasoactive over the micromolar range in mesenteric arteries although the molecular mediators of this effect remain to be identified. These data expand our knowledge of the polypharmacology and host vascular efficacy of this clinically important anthelmintic.

Elevated intraocular pressure (IOP) is the most prevalent and only treatable risk factor for glaucoma, a leading cause of irreversible blindness worldwide. Unfortunately, all current therapeutics used to treat elevated IOP and glaucoma have significant and sometimes irreversible side effects necessitating the development of novel compounds. We evaluated the IOP lowering ability of the broad spectrum KATP channel opener cromakalim. Cultured human anterior segments when treated with 2 μM cromakalim showed a decrease in pressure (19.33 ± 2.78 mmHg at 0 hours to 13.22 ± 2.64 mmHg at 24 hours; p<0.001) when compared to vehicle treated controls (15.89 ± 5.33 mmHg at 0 h to 15.56 ± 4.88 mmHg at 24 hours; p = 0.89). In wild-type C57BL/6 mice, cromakalim reduced IOP by 18.75 ± 2.22% compared to vehicle treated contralateral eyes (17.01 ± 0.32 mmHg at 0 hours to 13.82 ± 0.37 mmHg at 24 hours; n = 10, p = 0.002). Cromakalim demonstrated an additive effect when used in conjunction with latanoprost free acid, a common ocular hypotensive drug prescribed to patients with elevated IOP. To examine KATP channel subunit specificity, Kir6.2(-/-) mice were treated with cromakalim, but unlike wild-type animals, no change in IOP was noted. Histologic analysis of treated and control eyes in cultured human anterior segments and in mice showed similar cell numbers and extracellular matrix integrity within the trabecular meshwork, with no disruptions in the inner and outer walls of Schlemm's canal. Together, these studies suggest that cromakalim is a potent ocular hypotensive agent that lowers IOP via activation of Kir6.2 containing KATP channels, its effect is additive when used in combination with the commonly used glaucoma drug latanoprost, and is not toxic to cells and tissues of the aqueous humor outflow pathway, making it a candidate for future therapeutic development.

Praziquantel (PZQ) is a key therapy for treatment of parasitic flatworm infections of humans and livestock, but the mechanism of action of this drug is unresolved. Resolving PZQ-engaged targets and effectors is important for identifying new druggable pathways that may yield novel antiparasitic agents. Here we use functional, genetic and pharmacological approaches to reveal that serotonergic signals antagonize PZQ action in vivo. Exogenous 5-hydroxytryptamine (5-HT) rescued PZQ-evoked polarity and mobility defects in free-living planarian flatworms. In contrast, knockdown of a prevalently expressed planarian 5-HT receptor potentiated or phenocopied PZQ action in different functional assays. Subsequent screening of serotonergic ligands revealed that several ergot alkaloids possessed broad efficacy at modulating regenerative outcomes and the mobility of both free living and parasitic flatworms. Ergot alkaloids that phenocopied PZQ in regenerative assays to cause bipolar regeneration exhibited structural modifications consistent with serotonergic blockade. These data suggest that serotonergic activation blocks PZQ action in vivo, while serotonergic antagonists phenocopy PZQ action. Importantly these studies identify the ergot alkaloid scaffold as a promising structural framework for designing potent agents targeting parasitic bioaminergic G protein coupled receptors.

RationaleCabergoline (CAB) is an ergot derivative typically prescribed for the treatment of hyperprolactinemia. It suppresses the release of prolactin through agonist actions on dopamine (DA) D2 receptors; however, it possesses binding affinity for other DA and 5-HT receptors. Side effects that exacerbate valvular heart disease can occur with high doses.ObjectiveThe present study examined the acute, subchronic, and chronic dose–response effects of CAB and a derivative dimethylcabergoline (DMC) which acts as an antagonist instead of agonist at 5-HT 2B receptors, on appetitive and consummatory sexual behaviors of male rats.MethodsCAB (0, 0.03, 0.15, or 0.3 mg/kg/ml) was administered daily to sexually experienced male rats (N = 10/dose) by oral gavage for a total of 68 days. Sexual behavior was tested every 4 days during this period for a total of 16 trials. On the 17th trial, rats were administered their dose of CAB, and 4 h after were overdosed with sodium pentobarbital, perfused intracardially, and their brains processed for Fos immunohistochemistry. DMC (0, 0.03, 0.15, 0.3 mg/kg/ml) was administered daily to sexually experienced male rats (N = 10/dose) by oral gavage for a total of 36 days. Sexual behavior was tested every 4 days for a total of 9 trials.ResultsCAB increased anticipatory level changes, intromissions, and ejaculations significantly across all timepoints, with the medium and high doses being most potent. The medium and high doses also increased Fos protein significantly within the medial preoptic area, whereas in the nucleus accumbens shell, the low and medium doses decreased Fos protein but the high dose increased it significantly from control. Similar to CAB, the medium and high doses of DMC increased the number of ejaculations significantly. Rats in all drug dose groups appeared healthy for the duration of the experiments.ConclusionsBoth CAB and DMC facilitate ejaculations, and CAB further facilitates measures of anticipatory sexual motivation and intromissions. These data suggest that both could be used as treatments for sexual arousal disorders and ejaculation/orgasm disorders with little or no untoward side effects at low doses.

ABSTRACT IMPACT: Pharmacological activation of KATP channels may provide analgesia and attenuate opioid tolerance and withdrawal OBJECTIVES/GOALS: Our long term goal is to develop therapeutics for the treatment of the overuse of opioids. The objective of this application is to test novel KATP channel-targeting prodrugs in rodent models of neuropathic and inflammatory pain in addition to opioid tolerance after chronic morphine administration. METHODS/STUDY POPULATION: In one study, two different measures for chronic pain were implemented in mice. Male and female mice (n=10) were subjected to spinal nerve ligation (SNL) or intraplantar injection of Complete Freund’s Adjuvant (CFA) to induce neuropathic and inflammatory pain, respectively. Administration of KATP channel prodrugs (60ug, it) attenuated mechanical hypersensitivity after SNL or CFA compared to vehicle (saline). In a separate study, changes in mechanical hypersensitivity were tested while mice undergo chronic morphine treatment (15mg/kg, 2x, 5 days) with administration of the prodrugs. Tolerance was measured as the loss of antinociception, and withdrawal is measured ˜24 hours after the final morphine injection. RESULTS/ANTICIPATED RESULTS: Intrathecal administration of either KATP channel prodrugs significantly attenuated mechanical hypersensitivity after SNL and significantly attenuated mechanical hypersensitivity after CFA in mice. We predict that intrathecal administration of these prodrugs will also attenuate morphine tolerance and withdrawal in mice. This hypothesis is based off our previous data indicating non-water soluble KATP channel agonists produce analgesia and attenuate morphine tolerance in mice. DISCUSSION/SIGNIFICANCE OF FINDINGS: Pharmaceutical strategies to utilize KATP channels for therapeutics have been hindered due to the low solubility and low ability to cross the neurovascular unit. Newly developed, water-soluble KATP channel openers could be useful pharmaceutical strategy to reduce chronic pain, opioid tolerance, and withdrawal in human populations.