The effect of food and liquid pH on the integrity of enteric-coated beads from cysteamine bitartrate delayed-release capsules.

BACKGROUND: Cysteamine bitartrate delayed-release (DR) capsule (Procysbi®) is approved for treatment of nephropathic cystinosis in the USA, Canada, and the EU. The capsules contain cysteamine bitartrate beads that are enteric coated with acid-resistant Eudragit L 30 D-55, preventing drug release in the acidic stomach environment while allowing dissolution of the beads in the more alkaline environment of the small intestine. Patients who have difficulty swallowing capsules can open capsules, sprinkle beads onto 4 ounces of a suitable food or liquid, gently mix, and consume the entire content within 30 minutes. Foods found to be suitable for administration, and described in the Procysbi US labeling, include fruit juices (except grapefruit juice), applesauce, and berry jelly; there are minor variations in the foods and liquids recommended by regulatory authorities in other countries. This study aimed to assess the stability of enteric-coated beads exposed to additional foods at different conditions to expand the list of suitable foods for drug administration. METHODS: For each test condition, beads from eight opened 75 mg cysteamine bitartrate DR capsules were gently mixed with test food and maintained at a prespecified temperature and duration; remaining undissolved beads were then recovered from the food. The recovered beads were split into two portions: one assayed for remaining drug content and the other subjected to dissolution testing to assess the effect on the drug-release profile. RESULTS: The results show that bead integrity was maintained when mixed with foods at pH values <5.5 at all time points when refrigerated (2°C-8°C) and at room (20°C-22°C) and lukewarm (37°C-41°C) temperatures. Bead integrity was not maintained when mixed with foods at pH values of ≥5.5 at room temperature. CONCLUSION: The results from this in vitro dissolution study help in identifying additional foods that may be used for the administration of cysteamine bitartrate DR beads from opened capsules using the sprinkle method.

QM-polarized ligand docking accurately predicts the trend in binding affinity of a series of arylmethylene quinuclidine-like derivatives at the α4ß2 and α3ß4 nicotinic acetylcholine receptors (nAChRs).

Compounds containing a quinuclidine scaffold are promising drug candidates for pharmacological management of the central nervous system (CNS) pathologies implicating nAChRs. We have carried out binding affinity and in-silico docking studies of arylmethylene quinuclidine-like derivatives at the α4ß2 receptor using in-vitro receptor binding assay and comparative modeling, respectively. We found that introducing a hydrogen-bond acceptor into the 3-benzylidene quinuclidine derivative resulted in a 266-fold increase in binding affinity and confers agonism properties. By contrast, addition of a phenyl group to 3-benzylidene quinuclidine derivative only results in an 18-fold increase in binding affinity, without conferring agonism. We also found that docking into the orthosteric binding site of the α4ß2 nAChR is consistent with the fact that the basic nitrogen atom donates a hydrogen-bond to the carbonyl group of the highly conserved Trp-149, as initially observed by Dougherty and co-workers.(1) The experimentally-observed trend in binding affinity at both α4ß2 and α3ß4 nAChRs was accurately and independently confirmed by quantum mechanics (QM)-polarized docking. The reduction in binding affinity to the α3ß4 subtype primarily results from a dampening of both coulombic and cation-π interactions.

Novel nicotinic acetylcholine receptor agonists containing carbonyl moiety as a hydrogen bond acceptor.

A novel series of α4ß2 nAChR agonists lacking common pyridine or its bioisosteric heterocycle have been disclosed. Essential pharmacophoric elements of the series are exocyclic carbonyl moiety as a hydrogen bond acceptor and secondary amino group within diaza- or azabicyclic scaffold. Computer modeling studies suggested that molecular shape of the ligand also contributes to promotion of agonism. Proof of concept for improving working memory performance in a novel object recognition task has been demonstrated on a representative of the series, 3-propionyl-3,7-diazabicyclo[3.3.0]octane (34).

Pharmacological properties and predicted binding mode of arylmethylene quinuclidine-like derivatives at the α3ß4 nicotinic acetylcholine receptor (nAChR).

We have carried out a pharmacological evaluation of arylmethylene quinuclidine derivatives interactions with human α3ß4 nAChRs subtype, using cell-based receptor binding, calcium-influx, electrophysiological patch-clamp assays and molecular modeling techniques. We have found that the compounds bind competitively to the α3ß4 receptor with micromolar affinities and some of the compounds behave as non-competitive antagonists (compounds 1, 2 and 3), displaying submicromolar IC(50) values. These evidences suggest a mixed mode of action for these compounds, having interactions at the orthosteric site and more pronounced interactions at an allosteric site to block agonist effects. One of the compounds, 1-benzyl-3-(diphenylmethylene)-1-azoniabicyclo[2.2.2]octane chloride (compound 3), exhibited poorly reversible use-dependent block of α3ß4 channels. We also found that removal of a phenyl group from compound 1 confers a partial agonism to the derived analog (compound 6). Introducing a hydrogen-bond acceptor into the 3-benzylidene quinuclidine derivative (compound 7) increases agonism potency at the α3ß4 receptor subtype. Docking into the orthosteric binding site of a α3ß4 protein structure derived by comparative modeling accurately predicted the experimentally-observed trend in binding affinity. Results supported the notion that binding requires a hydrogen bond formation between the ligand basic nitrogen and the backbone carbonyl oxygen atom of the conserved Trp-149.

3D molecular descriptors important for clinical success.

The pharmacokinetic and safety profiles of clinical drug candidates are greatly influenced by their requisite physicochemical properties. In particular, it has been shown that 2D molecular descriptors such as fraction of Sp3 carbon atoms (Fsp3) and number of stereo centers correlate with clinical success. Using the proteomic off-target hit rate of nicotinic ligands, we found that shape-based 3D descriptors such as the radius of gyration and shadow indices discriminate off-target promiscuity better than do Fsp3 and the number of stereo centers. We have deduced the relevant descriptor values required for a ligand to be nonpromiscuous. Investigating the MDL Drug Data Report (MDDR) database as compounds move from the preclinical stage toward the market, we have found that these shape-based 3D descriptors predict clinical success of compounds at preclinical and phase1 stages vs compounds withdrawn from the market better than do Fsp3 and LogD. Further, these computed 3D molecular descriptors correlate well with experimentally observed solubility, which is among well-known physicochemical properties that drive clinical success. We also found that about 84% of launched drugs satisfy either Shadow index or Fsp3 criteria, whereas withdrawn and discontinued compounds fail to meet the same criteria. Our studies suggest that spherical compounds (rather than their elongated counterparts) with a minimal number of aromatic rings may exhibit a high propensity to advance from clinical trials to market.

Discovery of (2S,3R)-N-[2-(pyridin-3-ylmethyl)-1-azabicyclo[2.2.2]oct-3-yl]benzo[b]furan-2-carboxamide (TC-5619), a selective α7 nicotinic acetylcholine receptor agonist, for the treatment of cognitive disorders.

(2S,3R)-N-[2-(Pyridin-3-ylmethyl)-1-azabicyclo[2.2.2]oct-3-yl]benzo[b]furan-2-carboxamide (7a, TC-5619), a novel selective agonist of the α7 neuronal nicotinic acetylcholine receptor, has been identified as a promising drug candidate for the treatment of cognitive impairment associated with neurological disorders. 7a demonstrated more than a thousand-fold separation between the affinities for the α7 and α4ß2 receptor subtypes and had no detectable effects on muscle or ganglionic nicotinic receptor subtypes, indicating a marked selectivity for the central nervous system over the peripheral nervous system. Results obtained from homology modeling and docking explain the observed selectivity. 7a had positive effects across cognitive, positive, and negative symptoms of schizophrenia in animal models and was additive or synergistic with the antipsychotic clozapine. Compound 7a, as an augmentation therapy to the standard treatment with antipsychotics, demonstrated encouraging results on measures of negative symptoms and cognitive dysfunction in schizophrenia and was well tolerated in a phase II clinical proof of concept trial in patients with schizophrenia.

Analgesic effects mediated by neuronal nicotinic acetylcholine receptor agonists: correlation with desensitization of α4ß2* receptors.

Nicotinic α4ß2* agonists are known to be effective in a variety of preclinical pain models, but the underlying mechanisms of analgesic action are not well-understood. In the present study, we characterized activation and desensitization properties for a set of seventeen novel α4ß2*-selective agonists that display druggable physical and pharmacokinetic attributes, and correlated the in vitro pharmacology results to efficacies observed in a mouse formalin model of analgesia. ABT-894 and Sazetidine-A, two compounds known to be effective in the formalin assay, were included for comparison. The set of compounds displayed a range of activities at human (α4ß2)(2)ß2 (HS-α4ß2), (α4ß2)(2)α5 (α4ß2α5) and (α4ß2)(2)α4 (LS-α4ß2) receptors. We report the novel finding that desensitization of α4ß2* receptors may drive part of the antinociceptive outcome. Our molecular modeling approaches revealed that when receptor desensitization rather than activation activitiesat α4ß2* receptors are considered, there is a better correlation between analgesia scores and combined in vitro properties. Our results suggest that although all three α4ß2 subtypes assessed are involved, it is desensitization of α4ß2α5 receptors that plays a more prominent role in the antinociceptive action of nicotinic compounds. For modulation of Phase I responses, correlations are significantly improved from an r(2) value of 0.53 to 0.67 and 0.66 when HS- and LS-α4ß2 DC(50) values are considered, respectively. More profoundly, considering the DC(50) at α4ß2α5 takes the r(2) from 0.53 to 0.70. For Phase II analgesia scores, adding HS- or LS-α4ß2 desensitization potencies did not improve the correlations significantly. Considering the α4ß2α5 DC(50) value significantly increased the r(2) from 0.70 to 0.79 for Phase II, and strongly suggested a more prominent role for α4ß2α5 nAChRs in the modulation of pain in the formalin assay. The present studies demonstrate that compounds which are more potent at desensitization of α4ß2* receptors display better analgesia scores in the formalin test. Consideration of desensitization propertiesat α4ß2* receptors, especially at α4ß2α5, in multiple linear regression analyses significantly improves correlations with efficacies of analgesia. Thus, α4ß2* nicotinic acetylcholine receptor desensitization may contribute to efficacy in the mediation of pain, and represent a mechanism for analgesic effects mediated by nicotinic agonists.

Discovery of 3-(5-chloro-2-furoyl)-3,7-diazabicyclo[3.3.0]octane (TC-6683, AZD1446), a novel highly selective α4ß2 nicotinic acetylcholine receptor agonist for the treatment of cognitive disorders.

Diversification of essential nicotinic cholinergic pharmacophoric elements, i.e., cationic center and hydrogen bond acceptor, resulted in the discovery of novel potent α4ß2 nAChR selective agonists comprising a series of N-acyldiazabicycles. Core characteristics of the series are an exocyclic carbonyl moiety as a hydrogen bond acceptor and endocyclic secondary amino group. These features are positioned at optimal distance and with optimal relative spatial orientation to provide near optimal interactions with the receptor. A novel potent and highly selective α4ß2 nAChR agonist 3-(5-chloro-2-furoyl)-3,7-diazabicyclo[3.3.0]octane (56, TC-6683, AZD1446) with favorable pharmaceutical properties and in vivo efficacy in animal models has been identified as a potential treatment for cognitive deficits associated with psychiatric or neurological conditions and is currently being progressed to phase 2 clinical trials as a treatment for Alzheimer's disease.

Comparison of acetylcholine receptor interactions of the marine toxins, 13-desmethylspirolide C and gymnodimine.

The interaction of 13-desmethylspirolide C (SPX-desMe-C) and gymnodimine with several nicotinic and muscarinic acetylcholine receptors was investigated. Interaction at the muscarinic receptors was minimal. At nicotinic receptors, both SPX-desMe-C and gymnodimine displayed greatest affinity for the α7 receptor. The rank order for binding affinity (Ki) for SPX-desMe-C was α7 > α6ß3ß4α5 >> rat α3ß4, α1ßγδ > α4ß4, human α3ß4 > human α4ß2 > rat α4ß2 and for gymnodimine was α7, α6ß3ß4α5 > rat α3ß4 > human α3ß4, α4ß4 > rat α4ß2, human α4ß2 > α1ßγδ. Both molecules antagonized agonist-induced nicotinic responses. The antagonism rank order of potency (IC(50)) for SPX-desMe-C was α7 > low sensitivity (LS) α4ß2 > human α3ß4 > high sensitivity (HS) α4ß2, α1ßγδ > α4ß4 > rat α3ß4 and for gymnodimine was LS α4ß2 > human α3ß4 > α7 > HS α4ß2 > α4ß4 > rat α3ß4 > α1ßγδ. Neither gymnodimine nor SPX-desMe-C antagonism could be surmounted by increasing concentrations of nicotine. To elucidate the nature of this insurmountable blockade, we carried out homology modelling and molecular docking studies of both ligands with α7 nAChR. Their very high binding affinity results from very tight hydrophobic enclosures, in addition to previously reported hydrogen-bond and cation-π interactions. Also, the higher the hydrophilic surface area of the binding site of nAChRs, the weaker the binding affinity of both ligands. Together these results show the targets of action are nicotinic and define these marine toxins as additional tools to advance our understanding regarding interactions between antagonists and the nAChR ligand binding domain.

Discovery of novel α7 nicotinic acetylcholine receptor ligands via pharmacophoric and docking studies of benzylidene anabaseine analogs.

Based on pharmacophore elucidation and docking studies on interactions of benzylidene anabaseine analogs with AChBPs and α7 nAChR, novel spirodiazepine and spiroimidazoline quinuclidine series have been designed. Binding studies revealed that some of hydrogen-bond donor containing compounds exhibit improved affinity and selectivity for the α7 nAChR subtype in comparison with most potent metabolite of GTS-21, 3-(4-hydroxy-2-methoxybenzylidene)-anabaseine. Hydrophobicity and rigidity of the ligand also contribute into its binding affinity. We also describe alternative pharmacophoric features equidistant from the carbonyl oxygen atom of the conserved Trp-148 of the principal face, which may be exploited to further design diverse focused libraries targeting the α7 nAChR.

Docking studies of benzylidene anabaseine interactions with α7 nicotinic acetylcholine receptor (nAChR) and acetylcholine binding proteins (AChBPs): application to the design of related α7 selective ligands.

AChBPs isolated from Lymnaea stagnalis (Ls), Aplysia californica (Ac) and Bulinus truncatus (Bt) have been extensively used as structural prototypes to understand the molecular mechanisms that underlie ligand-interactions with nAChRs [1]. Here, we describe docking studies on interactions of benzylidene anabaseine analogs with AChBPs and α7 nAChR. Results reveal that docking of these compounds using Glide software accurately reproduces experimentally-observed binding modes of DMXBA and of its active metabolite, in the binding pocket of Ac. In addition to the well-known nicotinic pharmacophore (positive charge, hydrogen-bond acceptor, and hydrophobic aromatic groups), a hydrogen-bond donor feature contributes to binding of these compounds to Ac, Bt, and the α7 nAChR. This is consistent with benzylidene anabaseine analogs with OH and NH(2) functional groups showing the highest binding affinity of these congeners, and the position of the ligand shown in previous X-ray crystallographic studies of ligand-Ac complexes. In the predicted ligand-Ls complex, by contrast, the ligand OH group acts as hydrogen-bond acceptor. We have applied our structural findings to optimizing the design of novel spirodiazepine and spiroimidazoline quinuclidine series. Binding and functional studies revealed that these hydrogen-bond donor containing compounds exhibit improved affinity and selectivity for the α7 nAChR subtype and demonstrate partial agonism. The gain in affinity is also due to conformational restriction, tighter hydrophobic enclosures, and stronger cation-π interactions. The use of AChBPs structure as a surrogate to predict binding affinity to α7 nAChR has also been investigated. On the whole, we found that molecular docking into Ls binding site generally scores better than when a α7 homology model, Bt or Ac crystal structure is used.

An alpha7 nicotinic acetylcholine receptor-selective agonist reduces weight gain and metabolic changes in a mouse model of diabetes.

Type 2 diabetes has become a pervasive public health problem. The etiology of the disease has not been fully defined but appears to involve abnormalities in peripheral and central nervous system pathways, as well as prominent inflammatory components. Because nicotinic acetylcholine receptors (nAChRs) are known to interact with anti-inflammatory pathways and have been implicated in control of appetite and body weight, as well as lipid and energy metabolism, we examined their role in modulating biological parameters associated with the disease. In a model of type 2 diabetes, the homozygous leptin-resistant db/db obese mouse, we measured the effects of a novel alpha7 nAChR-selective agonist [5-methyl-N-[2-(pyridin-3-ylmethyl)-1-azabicyclo[2.2.2]oct-3-yl]thiophene-2-carboxamide (TC-7020)] on body mass, glucose and lipid metabolism, and proinflammatory cytokines. Oral administration of TC-7020 reduced weight gain and food intake, reduced elevated glucose and glycated hemoglobin levels, and lowered elevated plasma levels of triglycerides and the proinflammatory cytokine tumor necrosis factor-alpha. These changes were reversed by the alpha7-selective antagonist methyllycaconitine, confirming the involvement of alpha7 nAChRs. Prevention of weight gain, decreased food intake, and normalization of glucose levels were also blocked by the Janus kinase 2 (JAK2) inhibitor alpha-cyano-(3,4-dihydroxy)-N-benzylcinnamide (AG-490), suggesting that these effects involve linkage of alpha7 nAChRs to the JAK2-signal transducer and activator of transcription 3 signaling pathway. The results show that alpha7 nAChRs play a central role in regulating biological parameters associated with diabetes and support the potential of targeting these receptors as a new therapeutic strategy for treatment.

First total synthesis of (+/-)-3-hydroxy-11-norcytisine: structure confirmation and biological characterization.

The first total synthesis of the natural product 3-hydroxy-11-norcytisine (1), structurally related to cytisine (2), a benchmark ligand at neuronal nicotinic acetylcholine receptors (NNRs), has been achieved. The synthesis permits the unambiguous confirmation of the structure originally proposed for 1 and has enabled initial biological characterization of 1 and its related compounds against NNRs.

Therapeutic potential of novel selective drugs targeting nicotinic acetylcholine receptors.

The potential therapeutic benefit of nicotinic ligands in a variety of neurodegenerative pathologies involving the CNS has energized research efforts to develop nicotinic acetylcholine receptor (nAChR) subtype-selective ligands (Bencherif and Schmitt, 2005). In particular, there has been a concerted effort to develop nicotinic compounds with selectivity for CNS nAChRs as potential pharmaceutical tools in the management of these disorders. Clinical and experimental data demonstrate a central role for alpha7 and alpha4beta2 nAChRs in cognitive function, sensory processing, mood, and neuroprotection (Bencherif and Schmitt, 2005; Buccafusco et al., 2005). The development of safe alpha7-selective ligands has been hampered by their lack of discrimination with hERG channels and 5-HT3 receptors. We have developed a number of compounds that display nanomolar affinity to the alpha7 and/or the alpha4beta2 receptor. Investigation of alpha7 functional activity showed a full range of activities from antagonists to full agonists without any significant activity at the human 5-HT3 receptor, P450 isozymes, hERG channels, or in the AMES test. Our findings demonstrate that potent and highly selective nAChR ligands can be designed.

2-(Arylmethyl)-3-substituted quinuclidines as selective alpha 7 nicotinic receptor ligands.

A series of 2-(arylmethyl)-3-substituted quinuclidines was developed as alpha7 neuronal nicotinic acetylcholine receptor (nAChR) agonists based on a putative pharmacophore model. The series is highly selective for the alpha7 over other nAChRs (e.g., the alpha4beta2 of the CNS, and the muscle and ganglionic subtypes) and is functionally tunable at alpha7. One member of the series, (+)-N-(1-azabicyclo[2.2.2]oct-3-yl)benzo[b]furan-2-carboxamide (+)-8l), has potent agonistic activity for the alpha7 nAChR (EC(50)=33nM, I(max)=1.0), at concentrations below those that result in desensitization.