Sexual dimorphism in the gonad lipidome of blue mussels (Mytilus sp.): New insights from a global lipidomics approach.

Blue mussels (Mytilus sp.) are an economically important species for European aquaculture. Their importance as a food source is expected to increase in the coming net-zero society due to their low environmental footprint; however, their production is affected by anthropogenic stressors and climate change. During reproduction, lipids are key molecules for mussels as they are the main source of energy on which newly hatched embryos depend in the first days of their development. In this work, blue mussels of different origins are analysed, focusing on the differences in lipid composition between the ovary (BMO) and the testis (BMT). The lipidome of blue mussel gonads (BMG) is studied here by combining traditional lipid profiling methods, such as fatty acid and lipid class analysis, with untargeted liquid chromatography-mass spectrometry (LC-MS) lipidomics. The approach used here enabled the identification of 770 lipid molecules from 23 different lipid classes in BMG. BMT, which consists of billions of spermatocytes, had greater amounts of cell membrane and membrane lipid components such as FA18:0, C20 polyunsaturated fatty acids (PUFA), free sterols (ST), ceramide phosphoethanolamines (CerPE), ceramide aminoethylphosphonates (CAEP), cardiolipins (CL), glycerophosphocholines (PC), glycerophosphoethanolamines (PE) and glycerophosphoserines (PS). In BMO, saturated fatty acids (FA14:0 and FA16:0), monounsaturated fatty acids (MUFA) and other storage components such as C18-PUFA accumulated in triradylglycerolipids (TG) and alkyldiacylglycerols (neutral plasmalogens, TG O-), which, together with terpenes, wax esters and cholesterol esters, make up most of oocytes yolk reserves. BMO also had higher levels of ceramides (Cer) and generally alkyl/alkenyl glycerophospholipids (mainly plasmanyl/plasmenyl PC), suggesting a role for these lipids in vitellogenesis. Non-methylene interrupted dienoic fatty acids (NMID FA), typically found in plasmalogens, were the only membrane-forming PUFA predominantly detected in BMO. The results of this study are of great importance for clarifying the lipid composition of BMG and provide an important basis for future studies on the reproductive physiology of these organisms.

Lung-restricted ALK5 inhibition avoids systemic toxicities associated with TGFß pathway inhibition.

Systemic therapies targeting transforming growth factor beta (TGFß) or TGFßR1 kinase (ALK5) have been plagued by toxicities including cardiac valvulopathy and bone physeal dysplasia in animals, posing a significant challenge for clinical development in pulmonary indications. The current work aims to demonstrate that systemic ALK5-associated toxicities can be mitigated through localized lung delivery. Lung-selective (THRX-144644) and systemically bioavailable (galunisertib) ALK5 inhibitors were compared to determine whether lung selectivity is sufficient to maintain local tissue concentrations while mitigating systemic exposure and consequent pathway-related findings. Both molecules demonstrated potent ALK5 activity in rat precision cut lung slices (PCLS; p-SMAD3 half-maximal inhibitory concentration [IC50], 141 nM and 1070 nM for THRX-144644 and galunisertib, respectively). In 14-day repeat-dose studies in rats, dose-related cardiac valvulopathy was recapitulated with oral galunisertib at doses ≥150 mg/kg/day. In contrast, inhaled nebulized THRX-144644 did not cause similar systemic findings up to the maximally tolerated doses in rats or dogs (10 and 1.5 mg/kg/day, respectively). THRX-144644 lung-to-plasma ratios ranged from 100- to 1200-fold in rats and dogs across dose levels. THRX-144644 lung trough (24 h) concentrations in rats and dogs ranged from 3- to 17-fold above the PCLS IC50 across tolerated doses. At a dose level exceeding tolerability (60 mg/kg/day; 76-fold above PCLS IC50) minimal heart and bone changes were observed when systemic drug concentrations reached pharmacologic levels. In conclusion, the current preclinical work demonstrates that localized pulmonary delivery of an ALK5 inhibitor leads to favorable TGFß pathway pharmacodynamic inhibition in lung while minimizing key systemic toxicities.

Comparing the effectiveness of twine- and binder-seeding in the Laminariales species Alaria esculenta and Saccharina latissima.

The continuing expansion of seaweed cultivation could assist in ensuring future global food security. The Laminariales species Alaria esculenta and Saccharina latissima are each cultivated for food across their European ranges. The predominant method for cultivating European kelps involves growing juveniles on twine within a hatchery which is then deployed at a farm site. The associated hatchery and deployment cost of this approach are relatively high. A new and innovative methodology-called binder-seeding-can reduce these costs, but, has yet to be validated. We compare the biomass yield and morphology of A. esculenta and S. latissima cultured using either the traditional twine-longline method or binder-seeding onto AlgaeRope and AlgaeRibbon, specially designed textiles. In a controlled growth experiment, A. esculenta had a similar biomass yield on all materials, but fronds were shorter (23 ± 7%) and thinner on the AlgaeRibbon (42 ± 4%) due to a 3-4-fold higher density of developing sporophytes compared to the twine-longline. In contrast, S. latissima gave a 4-fold higher biomass yield on the AlgaeRibbon in June (4.0 kg m-1), but frond morphology was not different between materials, despite a 4-fold higher sporophyte density on the AlgaeRibbon. The stipe length of both species also increased at the higher sporophyte density on the AlgaeRibbon. The AlgaeRope gave an intermediate response or was similar to the twine-longline. These results show that binder-seeding onto the AlgaeRibbon significantly increases the achieved biomass yield in S. latissima. These results can assist cultivators to select the most appropriate method of kelp cultivation depending on morphological/yield requirements of the end use. Further study is needed on the optimisation of the binder-seeding density and its impact on thallus morphology.

Lipidomics analysis of juveniles' blue mussels (Mytilus edulis L. 1758), a key economic and ecological species.

Blue mussels (Mytilus edulis L. 1758) are important components of coastal ecosystems and in the economy of rural and coastal areas. The understanding of their physiological processes at key life stages is important both within food production systems and in the management of wild populations. Lipids are crucial molecules for bivalve growth, but their diversity and roles have not been fully characterised. In this study, traditional lipid profiling techniques, such as fatty acid (FA) and lipid class analysis, are combined to untargeted lipidomics to elucidate the lipid metabolism in newly settled spat fed on a range of diets. The evaluated diets included single strains treatments (Cylindrotheca fusiformis CCAP 1017/2 -CYL, Isochrysis galbana CCAP 927/1- ISO, Monodopsis subterranean CCAP 848/1 -MONO, Nannochloropsis oceanica CCAP 849/10- NANNO) and a commercial algae paste (SP). Spat growth was influenced by the diets, which, according to their efficacy were ranked as follows: ISO>NANNO/CYL>SP>MONO. A higher triacylglycerols (TG) content, ranging from 4.23±0.82 µg mgashfree Dry weight (DW)-1 at the beginning of the trial (T0) to 51±15.3 µg mgashfreeDW-1 in ISO, characterised significant growth in the spat, whereas, a reduction of TG (0.3±0.08 µg mgashfreeDW-1 in MONO), mono unsaturated FA-MUFA (from 8.52±1.02 µg mgFAashfreeDW-1 at T0 to 2.81±1.02 µg mgFAashfreeDW-1 in MONO) and polyunsaturated FA-PUFA (from 17.57±2.24 µg mgFAashfreeDW-1 at T0 to 6.19±2.49 µg mgFAashfreeDW-1 in MONO) content characterised poor performing groups. Untargeted lipidomics evidenced how the availability of dietary essential PUFA did not influence only neutral lipids but also the membrane lipids, with changes in lipid molecular species in relation to the essential PUFA provided via the diet. Such changes have the potential to affect spat production cycle and their ability to respond to the surrounding environment. This study evidenced the advantages of coupling different lipid analysis techniques, as each technique disclosed relevant information on nutritional requirements of M. edulis juveniles, expanding the existing knowledge on the physiology of this important species.

Early development and substrate twine selection for the cultivation of Sargassum muticum (Yendo) Fensholt under laboratory conditions.

The phaeophyte macroalgae Sargassum muticum is under investigation as a cultivation crop within its native range in SE Asia, alongside other members of the Sargassum genus. During the critical hatchery phase, germlings are grown to ≥ several millimeters ready for outplanting. By optimising the growth medium and twine substrate used for the germling attachment, hatcheries can become more efficient and cost-effective. An 8-week replicated laboratory experiment investigated these factors. It found that adding 0.125 mL L-1 of saturated germanium dioxide during the first week increased mean germling size by 23% (p < 0.005), whereas additional nutrients in the form of F/2 medium made no difference (p > 0.05). Six twine substrates were also tested: jute, cotton, polyamide/cotton, polyester, polyvinyl alcohol and polypropylene. Sargassum muticum grew similarly well on all, although attachment success during the first week was highest on the rougher natural fibres, particularly jute. A negative density-dependent effect of germling density on growth was seen across all materials, with the highest growth seen on the materials with the lowest germling density. Jute is recommended as a highly suitable substrate for hatchery cultivation in this species, although the initial density should be carefully controlled to prevent intraspecific competition.

Decontamination treatments to eliminate problem biota from macroalgal tank cultures of Osmundea pinnatifida, Palmaria palmata and Ulva lactuca.

The effect of a range of chemical disinfectants at different concentration and exposure times was investigated on five macroalgal species and the marine gastropod Littorina spp. Palmaria palmata, Osmundea pinnatifida and Ulva lactuca are commercially valuable and are often cultivated in tanks for food or feed. Ectocarpus siliculosus and Ulva intestinalis are common epiphytes of P. palmata and O. pinnatifida cultures, whilst Littorina spp. are common herbivorous epibionts within U. lactuca culture tanks. These contaminants reduce the productivity and quality of the culture as a food. Differential tolerance to the treatments was seen between the algal species using pulse-amplitude modulation (PAM) chlorophyll a fluorescence, a few hours and a week following treatment. We identified treatments that selectively damaged the epiphyte but not the basiphyte species. Ectocarpus siliculosus had a significantly lower tolerance to 1 % sodium hypochlorite than P. palmata, and to 25 % methanol than O. pinnatifida, with a 1-5 min exposure appearing most suitable. Ulva intestinalis had a significantly lower tolerance than P. palmata and O. pinnatifida to many disinfectants: 0.1-1 % sodium hypochlorite for 10 min, 0.5 % potassium iodide for up to 10 min, and 0.25 % Kick-start (a commercial aquaculture disinfectant solution) for 1-5 min. No treatment was able to kill the gastropod snails without also damaging U. lactuca, although agitation in freshwater for an hr may cause them to detach from the basiphyte, with little to no photophysiological impact seen to U. lactuca. This experiment forms the basis for more extended commercial trials.

Discovery of (R)-1-(3-((2-chloro-4-(((2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethyl)amino)methyl)-5-methoxyphenyl)amino)-3-oxopropyl)piperidin-4-yl [1,1'-biphenyl]-2-ylcarbamate (TD-5959, GSK961081, batefenterol): first-in-class dual pharmacology multivalent muscarinic antagonist and ß2 agonist (MABA) for the treatment of chronic obstructive pulmonary disease (COPD).

Through application of our multivalent approach to drug discovery we previously reported the first discovery of dual pharmacology MABA bronchodilators, exemplified by 1. Herein we describe the subsequent lead optimization of both muscarinic antagonist and ß2 agonist activities, through modification of the linker motif, to achieve 24 h duration of action in a guinea pig bronchoprotection model. Concomitantly we targeted high lung selectivities, low systemic exposures and identified crystalline forms suitable for inhalation devices. This article culminates with the discovery of our first clinical candidate 12f (TD-5959, GSK961081, batefenterol). In a phase 2b trial, batefenterol produced statistical and clinically significant differences compared to placebo and numerically greater improvements in the primary end point of trough FEV1 compared to salmeterol after 4 weeks of dosing in patients with moderate to severe chronic obstructive pulmonary disease (COPD).

Pharmacologic characterization of GSK-961081 (TD-5959), a first-in-class inhaled bifunctional bronchodilator possessing muscarinic receptor antagonist and ß2-adrenoceptor agonist properties.

The objective of the present studies was to characterize the pharmacologic properties of GSK-961081 [TD-5959; (R)-1-(3-((2-chloro-4-(((2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethyl)amino)methyl)-5-methoxyphenyl)amino)-3-oxopropyl) piperidin-4-yl [1,1'-biphenyl]-2-ylcarbamate], a novel first-in-class inhaled bifunctional compound possessing both muscarinic antagonist (MA) and ß2-adrenoceptor agonist (BA) properties (MABA). In competition radioligand binding studies at human recombinant receptors, GSK-961081 displayed high affinity for hM2 (Ki = 1.4 nM), hM3 muscarinic receptors (Ki = 1.3 nM) and hß2-adrenoceptors (Ki = 3.7 nM). GSK-961081 behaved as a potent hß2-adrenoceptor agonist (EC50 = 0.29 nM for stimulation of cAMP levels) with 440- and 320-fold functional selectivity over hß1- and hß3-adrenoceptors, respectively. In guinea pig isolated tracheal tissues, GSK-961081 produced smooth muscle relaxation through MA (EC50 = 50.2 nM), BA (EC50=24.6 nM), and MABA (EC50 = 11 nM) mechanisms. In the guinea pig bronchoprotection assay, inhaled GSK-961081 produced potent, dose-dependent inhibition of bronchoconstrictor responses via MA (ED50 = 33.9 µg/ml), BA (ED50 = 14.1 µg/ml), and MABA (ED50 = 6.4 µg/ml) mechanisms. Significant bronchoprotective effects of GSK-961081 were evident in guinea pigs via MA, BA, and MABA mechanisms for up to 7 days after dosing. The lung selectivity index of GSK-961081 in guinea pigs was 55- to 110-fold greater than that of tiotropium with respect to systemic antimuscarinic antisialagogue effects and was 10-fold greater than that of salmeterol with respect to systemic ß2-adrenoceptor hypotensive effects. These preclinical findings studies suggest that GSK-961081 has the potential to be a promising next-generation inhaled lung-selective bronchodilator for the treatment of airway diseases, including chronic obstructive pulmonary disease.

Fucosylated chondroitin sulfates from the body wall of the sea cucumber Holothuria forskali: conformation, selectin binding, and biological activity.

Fucosylated chondroitin sulfate (fCS) extracted from the sea cucumber Holothuria forskali is composed of the following repeating trisaccharide unit: → 3)GalNAcß4,6S(1 → 4) [FucαX(1 → 3)]GlcAß(1 →, where X stands for different sulfation patterns of fucose (X = 3,4S (46%), 2,4S (39%), and 4S (15%)). As revealed by NMR and molecular dynamics simulations, the fCS repeating unit adopts a conformation similar to that of the Le(x) blood group determinant, bringing several sulfate groups into close proximity and creating large negative patches distributed along the helical skeleton of the CS backbone. This may explain the high affinity of fCS oligosaccharides for L- and P-selectins as determined by microarray binding of fCS oligosaccharides prepared by Cu(2+)-catalyzed Fenton-type and photochemical depolymerization. No binding to E-selectin was observed. fCS poly- and oligosaccharides display low cytotoxicity in vitro, inhibit human neutrophil elastase activity, and inhibit the migration of neutrophils through an endothelial cell layer in vitro. Although the polysaccharide showed some anti-coagulant activity, small oligosaccharide fCS fragments had much reduced anticoagulant properties, with activity mainly via heparin cofactor II. The fCS polysaccharides showed prekallikrein activation comparable with dextran sulfate, whereas the fCS oligosaccharides caused almost no effect. The H. forskali fCS oligosaccharides were also tested in a mouse peritoneal inflammation model, where they caused a reduction in neutrophil infiltration. Overall, the data presented support the action of fCS as an inhibitor of selectin interactions, which play vital roles in inflammation and metastasis progression. Future studies of fCS-selectin interaction using fCS fragments or their mimetics may open new avenues for therapeutic intervention.

Heterotrophic compensation: a possible mechanism for resilience of coral reefs to global warming or a sign of prolonged stress?

Thermally induced bleaching has caused a global decline in corals and the frequency of such bleaching events will increase. Thermal bleaching severely disrupts the trophic behaviour of the coral holobiont, reducing the photosynthetically derived energy available to the coral host. In the short term this reduction in energy transfer from endosymbiotic algae results in an energy deficit for the coral host. If the bleaching event is short-lived then the coral may survive this energy deficit by depleting its lipid reserves, or by increasing heterotrophic energy acquisition. We show for the first time that the coral animal is capable of increasing the amount of heterotrophic carbon incorporated into its tissues for almost a year following bleaching. This prolonged heterotrophic compensation could be a sign of resilience or prolonged stress. If the heterotrophic compensation is in fact an acclimatization response, then this physiological response could act as a buffer from future bleaching by providing sufficient heterotrophic energy to compensate for photoautotrophic energy losses during bleaching, and potentially minimizing the effect of subsequent elevated temperature stresses. However, if the elevated incorporation of zooplankton is a sign that the effects of bleaching continue to be stressful on the holobiont, even after 11 months of recovery, then this physiological response would indicate that complete coral recovery requires more than 11 months to achieve. If coral bleaching becomes an annual global phenomenon by mid-century, then present temporal refugia will not be sufficient to allow coral colonies to recover between bleaching events and coral reefs will become increasingly less resilient to future climate change. If, however, increasing their sequestration of zooplankton-derived nutrition into their tissues over prolonged periods of time is a compensating mechanism, the impacts of annual bleaching may be reduced. Thus, some coral species may be better equipped to face repeated bleaching stress than previously thought.

Fatty acid profiles during gametogenesis in sea urchin (Paracentrotus lividus): effects of dietary inputs on gonad, egg and embryo profiles.

The effects of dietary fatty acids on the composition of Paracentrotus lividus gonads were investigated to determine whether dietary inputs affect their relative abundance during gametogenesis. Egg and embryo FA compositions were compared with that of mature gonads to understand how maternal FA is transferred to the offspring. Urchins were fed an experimental Pellet diet in comparison to brown Kelp (Laminaria digitata). FA profiles of diets, gonads, eggs and embryos revealed the presence in gonads of FA that was absent in the diets and/or higher in contents of some long-chain polyunsaturated fatty acid (LC-PUFA). Moreover, some unusual FA, such as non-methylene interrupted (NMI), was found in gonads, eggs and embryos, but not in the diets, suggesting that P. lividus may be capable of synthesizing this FA and accumulating them in the eggs. A description of gonad FA profiles during gametogenesis is reported for the first time and data suggest that eicosapentaenoic and docosahexaenoic acids are accumulated during gametogenesis, while arachidonic acid is highly regulated and is the only LC-PUFA clearly accumulated into the eggs along with NMI. Further studies are required to determine if maternal provisioning of FA has the potential to influence sea urchin production outputs and to increase hatchery profitability.

Biogas from Macroalgae: is it time to revisit the idea?

The economic and environmental viability of dedicated terrestrial energy crops is in doubt. The production of large scale biomass (macroalgae) for biofuels in the marine environment was first tested in the late 1960's. The culture attempts failed due to the engineering challenges of farming offshore. However the energy conversion via anaerobic digestion was successful as the biochemical composition of macroalgae makes it an ideal feedstock. The technology for the mass production of macroalgae has developed principally in China and Asia over the last 50 years to such a degree that it is now the single largest product of aquaculture. There has also been significant technology transfer and macroalgal cultivation is now well tried and tested in Europe and America. The inherent advantage of production of biofuel feedstock in the marine environment is that it does not compete with food production for land or fresh water. Here we revisit the idea of the large scale cultivation of macroalgae at sea for subsequent anaerobic digestion to produce biogas as a source of renewable energy, using a European case study as an example.

Echinoderms display morphological and behavioural phenotypic plasticity in response to their trophic environment.

The trophic interactions of sea urchins are known to be the agents of phase shifts in benthic marine habitats such as tropical and temperate reefs. In temperate reefs, the grazing activity of sea urchins has been responsible for the destruction of kelp forests and the formation of 'urchin barrens', a rocky habitat dominated by crustose algae and encrusting invertebrates. Once formed, these urchin barrens can persist for decades. Trophic plasticity in the sea urchin may contribute to the stability and resilience of this alternate stable state by increasing diet breadth in sea urchins. This plasticity promotes ecological connectivity and weakens species interactions and so increases ecosystem stability. We test the hypothesis that sea urchins exhibit trophic plasticity using an approach that controls for other typically confounding environmental and genetic factors. To do this, we exposed a genetically homogenous population of sea urchins to two very different trophic environments over a period of two years. The sea urchins exhibited a wide degree of phenotypic trophic plasticity when exposed to contrasting trophic environments. The two populations developed differences in their gross morphology and the test microstructure. In addition, when challenged with unfamiliar prey, the response of each group was different. We show that sea urchins exhibit significant morphological and behavioural phenotypic plasticity independent of their environment or their nutritional status.

Dual-pharmacology muscarinic antagonist and ß2 agonist molecules for the treatment of chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD) is one of the leading causes of death in the world today. Bronchodilators, particularly muscarinic antagonists and ß(2) agonists, are recommended for patients with moderate to severe COPD. Dual-pharmacology muscarinic antagonist- ß(2) agonist (MABA) molecules present an exciting new approach to the treatment of COPD by combining muscarinic antagonism and ß(2) agonism in a single entity. They have the potential to demonstrate additive or synergistic bronchodilation over either pharmacology alone. Due to this enticing prospect, several companies have now reported MABA discovery efforts through a conjugated/linked strategy with one candidate (GSK-961081) demonstrating clinical proof of concept. Several MABA crystal forms have been identified, satisfying the requirements for inhaled dosing devices. There are significant challenges in designing MABAs, but the potential to achieve enhanced bronchoprotection in patients and facilitate 'triple therapy' makes this an extremely important and exciting area of pharmaceutical research.

Discovery of muscarinic acetylcholine receptor antagonist and beta 2 adrenoceptor agonist (MABA) dual pharmacology molecules.

We sought to design dual pharmacology bronchodilators targeting both the M(3) muscarinic acetylcholine and beta-2 adrenergic (ß(2)) receptors by applying our multivalent approach to drug discovery. Herein, we describe our initial discovery and the SAR of the first such compounds with matched potencies at both receptors.

THRX-198321 is a bifunctional muscarinic receptor antagonist and beta2-adrenoceptor agonist (MABA) that binds in a bimodal and multivalent manner.

Biphenyl-2-yl-carbamic acid 1-{9-[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydro-quinolin-5-yl)-ethylamino]-nonyl}-piperidin-4-yl ester (THRX-198321) is a single molecule composed of a muscarinic acetylcholine receptor (mAChR) antagonist moiety, represented by the fragment MA, linked by a C9 polymethylene chain to a ß(2)-adrenoceptor (ß(2)AR) agonist moiety, represented by the fragment 8-hydroxy-5-((R)-1-hydroxy-2-methylamino-ethyl)-1H-quinolin-2-one (BA). THRX-198321 exhibited high affinity for mAChR (M(2) pK(I,App) = 10.57 ± 0.09; M(3) pK(I,App) = 10.07 ± 0.11) and ß(2)AR (pK(I,App) = 9.54 ± 0.15), with potent mAChR antagonist (M(2) pK(I,Fn) = 9.69 ± 0.23; M(3) pK(I,Fn) = 10.05 ± 0.17) and ß(2)AR agonist (pEC(50) = 9.25 ± 0.02) activities. Consistent with multivalent interactions, THRX-198321 binding affinity was >300-fold higher at mAChR and 29-fold higher at ß(2)AR relative to its monovalent fragments biphenyl carbamic acid piperidinyl ester (MA) and BA, respectively. THRX-198321 was a competitive antagonist at mAChR (M(2) pK(B) = 9.98 ± 0.13; M(3) pK(B) = 10.31 ± 0.89), whereas THRX-198321 agonist activity at ß(2)AR was competitively inhibited by propranolol. Interactions of THRX-198321 with an allosteric site on mAChR and a novel extracellular allosteric site on ß(2)AR, respectively, were detected by measuring THRX-198321-evoked changes in the dissociation rates for the orthosteric radioligands, [N-methyl-(3)H]scopolamine methyl chloride (M(2) pEC(50,diss) = 6.73 ± 0.10; M(3) pEC(50,diss) = 5.02 ± 0.14) and [4,6-propyl-(3)H]dihydroalprenolol (ß(2)AR pEC(50,diss) = 3.82 ± 0.25). The carbostyril-linker fragment (BA-L) binds to the allosteric site of mAChR (M(2) pEC(50,diss) = 5.06 ± 0.03; M(3) pEC(50,diss) = 4.15 ± 0.25), whereas the MA fragment binds to the allosteric site of ß(2)AR (pEC(50,diss) = 3.60 ± 0.18). Collectively, these observations suggest that THRX-198321 exhibits a multivalent bimodal orientation in the orthosteric and allosteric binding pockets of mAChR and ß(2)AR, a phenomenon that may be unique to this class of molecule.

Pharmacological properties of TD-6301, a novel bladder selective muscarinic receptor antagonist.

Existing antimuscarinic drugs for overactive bladder have high affinity for M(3)/M(1) muscarinic receptors and consequently produce M(3)/M(1)-mediated adverse effects including dry mouth, constipation, mydriasis and somnolence. TD-6301 is a M(2/4) muscarinic receptor-selective antagonist developed for the treatment of overactive bladder. The present studies characterize the in vitro and in vivo pharmacological properties of this molecule in comparison to other marketed antimuscarinics agents. In radioligand binding studies, TD-6301 was found to possess high affinity for human M(2) muscarinic receptor (K(i)=0.36 nM) and was 31, 36, 2 and 128-fold selective for the human M(2) muscarinic receptor compared to the M(1), M(3), M(4) and M(5) muscarinic receptors, respectively. The in vivo bladder selectivity of TD-6301 in rats was determined to be 26, 28, >100, 16 and 0.4-fold, respectively, assessed by comparing its potency for inhibition of volume-induced bladder contractions to that for inhibition of oxotremorine-induced salivation, inhibition of small-intestinal transit, decreases in locomotor activity, increases in pupil diameter and increases in heart rate. TD-6301 was more potent in inhibiting volume-induced bladder contractions (ID(50)=0.075 mg/kg) compared to oxotremorine-induced salivation (ID(50)=1.0 mg/kg) resulting in a bladder/salivary gland selectivity ratio greater than that observed for tolterodine, oxybutynin, darifenacin and solifenacin. The preclinical properties of TD-6301 suggest that this molecule is likely to be efficacious in overactive bladder patients with a lower propensity to cause M(3) muscarinic receptor mediated adverse effects.