Biological evaluation and synthesis of calcitroic acid.

We describe the synthesis and broad profiling of calcitroic acid (CTA) as vitamin D receptor (VDR) ligand. The x-ray co-crystal structure of the Danio Rerio VDR ligand binding domain in complex with CTA and peptide MED1 confirmed an agonistic conformation of the receptor. CTA adopted a similar conformation as 1,25(OH)2D3 in the binding pocket. A hydrogen bond with His333 and a water molecule were observed in the binding pocket, which was accommodated due to the shorter CTA side chain. In contrast, 1,25(OH)2D3 interacted with His423 and His333 due to its longer side chain. In vitro, the EC50 values of CTA and CTA-ME for VDR-mediated transcription were 2.89 µM and 0.66 µM, respectively, confirming both compounds as VDR agonists. CTA was further evaluated for interaction with fourteen nuclear receptors demonstrating selective activation of VDR. VDR mediated gene regulation by CTA in intestinal cells was observed for the VDR target gene CYP24A1. CTA at 10 µM upregulated CYP24A1 with similar efficacy as 1,25(OH)2D3 at 20 nM and 100-fold stronger compared to lithocholic acid at 10 µM. CTA reduced the transcription of iNOS and IL-1ß in interferon γ and lipopolysaccharide stimulated mouse macrophages resulting in a reduction of nitric oxide production and secretion of IL-1ß. These observed anti-inflammatory properties of 20 µM CTA were similar to 20 nM 1,25(OH)2D3.

Synthesis and biological evaluation of calcioic acid.

Herein, we describe the synthesis of calcioic acid following a recently developed synthetic strategy for calcitroic acid. Several improvements to reaction conditions were made, which resulted in higher yields. The improved workup and isolation procedures are described. Furthermore, we investigated the interaction between the vitamin D receptor (VDR) and calcioic acid. Calcioic acid was able to bind VDR with a binding constant of 71 µM. In cells, calcioic acid reduced the transcription of VDR target gene CYP24A1 in the presence 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) but did not induce the transcription of CYP24A1. Therefore, calcioic acid is a very weak VDR antagonist. With the generation of gram quantities, further studies are expected to reveal if calcioic acid is solely a water-soluble metabolite of vitamin D or if it mediates other biological functions through biomolecules other than VDR.

Alternative binding sites at the vitamin D receptor and their ligands.

In recent decades, the majority of ligands developed for the vitamin D receptor (VDR) bind at its deeply buried genomic ligand binding pocket. Theses ligands can be categorized into agonists and partial agonists/antagonists. A limited number of ligands, most of them peptides, bind the VDR‒coactivator binding site that is formed in the presence of an agonist and inhibit coactivator recruitment, and therefore transcription. Another solvent exposed VDR‒ligand binding pocket was identified for lithocholic acid, improving the overall stability of the VDR complex. Additional proposed interactions with VDR are discussed herein that include the alternative VDR‒ligand binding pocket that may mediate both non-genomic cellular responses and binding function 3 that was identified for the androgen receptor. Many VDR ligands increase blood calcium levels at therapeutic concentrations in vivo, thus the identification of alternative VDR‒ligand binding pockets might be crucial to develop non-calcemic and potent ligands for VDR to treat cancer and inflammatory disease.

A Novel Orally Available Asthma Drug Candidate That Reduces Smooth Muscle Constriction and Inflammation by Targeting GABAA Receptors in the Lung.

We describe lead compound MIDD0301 for the oral treatment of asthma based on previously developed positive allosteric α5ß3γ2 selective GABAA receptor (GABAAR) ligands. MIDD0301 relaxed airway smooth muscle at single micromolar concentrations as demonstrated with ex vivo guinea pig tracheal rings. MIDD0301 also attenuated airway hyperresponsiveness (AHR) in an ovalbumin murine model of asthma by oral administration. Reduced numbers of eosinophils and macrophages were observed in mouse bronchoalveolar lavage fluid without changing mucous metaplasia. Importantly, lung cytokine expression of IL-17A, IL-4, and TNF-α were reduced for MIDD0301-treated mice without changing antiinflammatory cytokine IL-10 levels. Automated patch clamp confirmed amplification of GABA induced current mediated by α1-3,5ß3γ2 GABAARs in the presence of MIDD0301. Pharmacodynamically, transmembrane currents of ex vivo CD4+ T cells from asthmatic mice were potentiated by MIDD0301 in the presence of GABA. The number of CD4+ T cells observed in the lung of MIDD0301-treated mice were reduced by an oral treatment of 20 mg/kg b.i.d. for 5 days. A half-life of almost 14 h was demonstrated by pharmacokinetic studies (PK) with no adverse CNS effects when treated mice were subjected to sensorimotor studies using the rotarod. PK studies also confirmed very low brain distribution. In conclusion, MIDD0301 represents a safe and improved oral asthma drug candidate that relaxes airway smooth muscle and attenuates inflammation in the lung leading to a reduction of AHR at a dosage lower than earlier reported GABAAR ligands.

Alleviation of Multiple Asthmatic Pathologic Features with Orally Available and Subtype Selective GABAA Receptor Modulators.

We describe pharmacokinetic and pharmacodynamic properties of two novel oral drug candidates for asthma. Phenolic α4ß3γ2 GABAAR selective compound 1 and acidic α5ß3γ2 selective GABAAR positive allosteric modulator compound 2 relaxed airway smooth muscle ex vivo and attenuated airway hyperresponsiveness (AHR) in a murine model of asthma. Importantly, compound 2 relaxed acetylcholine contracted human tracheal airway smooth muscle strips. Oral treatment of compounds 1 and 2 decreased eosinophils in bronchoalveolar lavage fluid in ovalbumin sensitized and challenged mice, thus exhibiting anti-inflammatory properties. Additionally, compound 1 reduced the number of lung CD4+ T lymphocytes and directly modulated their transmembrane currents by acting on GABAARs. Excellent pharmacokinetic properties were observed, including long plasma half-life (up to 15 h), oral availability, and extremely low brain distribution. In conclusion, we report the selective targeting of GABAARs expressed outside the brain and demonstrate reduction of AHR and airway inflammation with two novel orally available GABAAR ligands.

Optimization of substituted imidazobenzodiazepines as novel asthma treatments.

We describe the synthesis of analogs of XHE-III-74, a selective α4ß3γ2 GABAAR ligand, shown to relax airway smooth muscle ex vivo and reduce airway hyperresponsiveness in a murine asthma model. To improve properties of this compound as an asthma therapeutic, a series of analogs with a deuterated methoxy group in place of methoxy group at C-8 position was evaluated for isotope effects in preclinical assays; including microsomal stability, cytotoxicity, and sensorimotor impairment. The deuterated compounds were equally or more metabolically stable than the corresponding non-deuterated analogs and increased sensorimotor impairment was observed for some deuterated compounds. Thioesters were more cytotoxic in comparison to other carboxylic acid derivatives of this compound series. The most promising compound 16 identified from the in vitro screens also strongly inhibited smooth muscle constriction in ex vivo guinea pig tracheal rings. Smooth muscle relaxation, determined by reduction of airway hyperresponsiveness with a murine ovalbumin sensitized and challenged model, showed that 16 was efficacious at low methacholine concentrations. However, this effect was limited due to suboptimal pharmacokinetics of 16. Based on these findings, further analogs of XHE-III-74 will be investigated to improve in vivo metabolic stability while retaining the efficacy at lung tissues involved in asthma pathology.

Calcitroic Acid-A Review.

Calcitroic acid was isolated and characterized almost four decades ago, but little is known about this important vitamin D metabolite. Four reported synthetic strategies to generate calcitroic acid are presented that highlight the scientific progress in the field of chemistry directed to vitamin D analog synthesis. The most recent synthesis described the generation of calcitroic acid with an overall yield of 12.8% in 13 steps. The endogenous formation of calcitroic acid has been demonstrated in perfused rat kidney using 24,25,26,27-tetranor-1,23(OH)2D3. Although, the majority of vitamin D metabolism is mediated by 24-hydoxylase (CYP24A1), it is not clear why the formation of calcitroic acid was not observed in the presence of recombinant CYP24A1 enzyme. Furthermore, it is not known if enzyme 1α-hydroxylase (CYP27B1) can convert calcioic acid into calcitroic acid. In addition to the lack of research investigating the endogenous formation of calcitroic acid, the physiological role of calcitroic acid remains unknown. Only a few reports mentioned the biological activity of calcitroic acid in connection with the vitamin D receptor (VDR). When administered subcutaneously, calcitroic acid has anthracitic properties and elevates calcium blood levels when administered intravenously. In vitro, calcitroic acid at higher concentrations has been shown to bind VDR and induce gene transcription. However, these studies were not carried out in cells derived from target organs of calcitroic acid such as kidney, liver, and intestine. We can conclude that our current knowledge of calcitroic acid is limited, and more studies are needed to identify its physiological role.

Development of GABAA Receptor Subtype-Selective Imidazobenzodiazepines as Novel Asthma Treatments.

Recent studies have demonstrated that subtype-selective GABAA receptor modulators are able to relax precontracted human airway smooth muscle ex vivo and reduce airway hyper-responsiveness in mice upon aerosol administration. Our goal in this study was to investigate systemic administration of subtype-selective GABAA receptor modulators to alleviate bronchoconstriction in a mouse model of asthma. Expression of GABAA receptor subunits was identified in mouse lungs, and the effects of α4-subunit-selective GABAAR modulators, XHE-III-74EE and its metabolite XHE-III-74A, were investigated in a murine model of asthma (ovalbumin sensitized and challenged BALB/c mice). We observed that chronic treatment with XHE-III-74EE significantly reduced airway hyper-responsiveness. In addition, acute treatment with XHE-III-74A but not XHE-III-74EE decreased airway eosinophilia. Immune suppressive activity was also shown in activated human T-cells with a reduction in IL-2 expression and intracellular calcium concentrations [Ca(2+)]i in the presence of GABA or XHE-III-74A, whereas XHE-III-74EE showed only partial reduction of [Ca(2+)]i and no inhibition of IL-2 secretion. However, both compounds significantly relaxed precontracted tracheal rings ex vivo. Overall, we conclude that the systemic delivery of a α4-subunit-selective GABAAR modulator shows good potential for a novel asthma therapy; however, the pharmacokinetic properties of this class of drug candidates have to be improved to enable better beneficial systemic pharmacodynamic effects.

Synthesis and evaluation of vitamin D receptor-mediated activities of cholesterol and vitamin D metabolites.

A systematic study with phase 1 and phase 2 metabolites of cholesterol and vitamin D was conducted to determine whether their biological activity is mediated by the vitamin D receptor (VDR). The investigation necessitated the development of novel synthetic routes for lithocholic acid (LCA) glucuronides (Gluc). Biochemical and cell-based assays were used to demonstrate that hydroxylated LCA analogs were not able to bind VDR. This excludes VDR from mediating their biological and pharmacological activities. Among the synthesized LCA conjugates a novel VDR agonist was identified. LCA Gluc II increased the expression of CYP24A1 in DU145 cancer cells especially in the presence of the endogenous VDR ligand 1,25(OH)2D3. Furthermore, the methyl ester of LCA was identified as novel VDR antagonist. For the first time, we showed that calcitroic acid, the assumed inactive final metabolite of vitamin D, was able to activate VDR-mediated transcription to a higher magnitude than bile acid LCA. Due to a higher metabolic stability in comparison to vitamin D, a very low toxicity, and high concentration in bile and intestine, calcitroic acid is likely to be an important mediator of the protective vitamin D properties against colon cancer.