Protective effects of guggulsterone against colitis are associated with the suppression of TREM-1 and modulation of macrophages.

Triggering receptor expressed on myeloid cells 1 (TREM-1)-expressing intestinal macrophages are significantly increased in the colons of patients with inflammatory bowel disease (IBD). We focused here on the effects of guggulsterone on macrophage modulation in colitis as a potential therapeutic molecule in human IBD and explore the underlying mechanisms. Gene expression in macrophages was examined and wound-healing assay using HT-29 cells was performed. Colitis in wild-type and IL-10-, Toll-like receptor 4 (TLR4)-, and myeloid differentiation primary response 88 (MyD88)-deficient mice was induced via the administration of 2,4,6-trinitrobenzene sulfonic acid (TNBS) into the colon. In both in vitro and in vivo experiments, guggulsterone suppressed intestinal inflammation amplified by TREM-1 stimulation, in which the suppression of NF-κB, activating protein-1, and proteasome pathways was involved. In the TNBS-induced colitis model, guggulsterone reduced disease activity index scores and TREM-1 expression, stimulated IL-10 production, and improved survival in wild-type mice. These effects were not observed in IL-10-, TLR4-, and MyD88-deficient mice. Guggulsterone also suppressed M1 polarization, yet induced the M2 phenotype in macrophages from IBD patients as well as from mice. These findings indicate that guggulsterone blocks the hyperactivation of macrophages via TREM-1 suppression and induces M2 polarization via IL-10 mediated by the TLR4 signaling pathway. Furthermore, this study provides a new rationale for the therapeutic potential of guggulsterone in the treatment of IBD. NEW & NOTEWORTHY We found that guggulsterone attenuates triggering receptor expressed on myeloid cells 1 (TREM-1)-mediated hyperactivation of macrophages and polarizes macrophages toward the M2 phenotype. This was mediated by IL-10 and partly Toll-like receptor 4 signaling pathways. Overall, these data support that guggulsterone as a natural plant sterol modulates macrophage phenotypes in colitis, which may be of novel therapeutic importance in inflammatory bowel disease treatment.

In vitro metabolism of dexamethasone cipecilate, a novel synthetic corticosteroid, in human liver and nasal mucosa.

Dexamethasone cipecilate (DX-CP, 9-fluoro-11ß,17,21-trihydroxy-16α-methylpregna-1,4-diene-3,20-dione 21-cyclohexanecarboxylate 17-cyclopropanecarboxylate) is a novel synthetic corticosteroid used to treat allergic rhinitis. The pharmacological effect of DX-CP is considered to be mainly due to its active de-esterified metabolite (DX-17-CPC). To investigate the in vitro metabolism of DX-CP in human liver, DX-CP was incubated with human liver microsomes and S9. In addition, a metabolism study of DX-CP with human nasal mucosa was carried out in order to elucidate whether DX-17-CPC is formed in nasal mucosa, the site of action of DX-CP. DX-17-CPC was the major metabolite in both liver microsomes and S9. Two new epoxide metabolites, UK1 and UK2, were detected in liver S9, while only UK1 was detected in liver microsomes. This suggests that cytosol enzymes are responsible for the formation of UK2. In human nasal mucosa, DX-CP was mainly transformed into DX-17-CPC. By using recombinant human carboxylesterases (CESs), the reaction was shown to be catalyzed by CES2. These results provide the evidence that the active metabolite DX-17-CPC is the main contributor to the pharmacological action after the intranasal administration of DX-CP to humans.

Visualizing the spatial localization of ciclesonide and its metabolites in rat lungs after inhalation of 1-µm aerosol of ciclesonide by desorption electrospray ionization-time of flight mass spectrometry imaging.

Inhaled ciclesonide (CIC), a corticosteroid used to treat asthma that is also being investigated for the treatment of corona virus disease 2019, hydrolyzes to desisobutyryl-ciclesonide (des-CIC) followed by reversible esterification when exposed to fatty acids in lungs. While previous studies have described the distribution and metabolism of the compounds after inhalation, spatial localization in the lungs remains unclear. We visualized two-dimensional spatial localization of CIC and its metabolites in rat lungs after administration of a single dose of a CIC aerosol (with the mass median aerodynamic diameter of 0.918-1.168 µm) using desorption electrospray ionization-time of flight mass spectrometry imaging (DESI-MSI). In the analysis, CIC, des-CIC, and des-CIC-oleate were imaged in frozen lung sections at high spatial and mass resolutions in negative-ion mode. MSI revealed the coexistence of CIC, des-CIC, and des-CIC-oleate on the airway epithelium, and the distribution of des-CIC and des-CIC-oleate in peripheral lung regions. In addition, a part of CIC independently localized on the airway epithelium. These results suggest that distribution of CIC and its metabolites in lungs is related to both the intended delivery of aerosols to pulmonary alveoli and peripheral regions, and the potential deposition of CIC particles on the airway epithelium.

CYP3A4 mediated pharmacokinetics drug interaction potential of Maha-Yogaraj Gugglu and E, Z guggulsterone.

Maha yogaraja guggulu (MYG) is a classical herbomineral polyherbal formulation being widely used since centuries. The aim of this study was to investigate the effect of MYG formulation and its major constituents E & Z guggulsterone on CYP3A4 mediated metabolism. In vitro inhibition of MYG and Guggulsterone isomers on CYP3A4 was evaluated by high throughput fluorometric assay. Eighteen Adult male Sprague-Dawley rats (200 ± 25 g body weight) were randomly divided into three groups. Group A, Group B and Group C were treated with placebo, MYG and Standard E & Z guggulsterone for 14 days respectively by oral route. On 15th day, midazolam (5 mg/kg) was administered orally to all rats in each group. Blood samples (0.3 mL) were collected from the retro orbital vein at 0.25, 0.5, 0.75, 1, 2, 4, 6, 12 and 24 h of each rat were collected. The findings from the in vitro & in vivo study proposed that the MYG tablets and its guggulsterone isomers have drug interaction potential when consumed along with conventional drugs which are CYP3A4 substrates. In vivo pharmacokinetic drug interaction study of midazolam pointed out that the MYG tablets and guggulsterone isomers showed an inhibitory activity towards CYP3A4 which may have leads to clinically significant interactions.

In vitro evaluation of reactive nature of E- and Z-guggulsterones and their metabolites in human liver microsomes using UHPLC-Orbitrap mass spectrometer.

Guggulipid is known to be useful for hypercholesterolemia, arthritis, acne, and obesity. These activities are attributed to its two principal isomeric active constituents, viz., E- and Z-guggulsterones. There are several side effects reported for guggulipid, which include widespread erythematous papules in a morbilliform pattern and macules localized to the arms; swelling and erythema of the face with burning sensation; pruritis; and bullous lesions on the lower legs with associated headaches, myalgia and itching. We hypothesized that one probable reason for these toxic reactions could be the formation of electrophilic reactive metabolites (RMs) of guggulsterones and their subsequent reaction with cellular proteins. Unfortunately, no report exists in the literature highlighting detection of RMs of guggulsterone isomers. Accordingly, the present study was undertaken to investigate the potential of E- and Z-guggulsterones to form RMs in human liver microsomes (HLM) using glutathione (GSH) and N-acetylcysteine (NAC) as trapping agents. The generated samples were analysed using ultra-high performance liquid chromatography (UHPLC) coupled to an Orbitrap mass spectrometer. The analysis of incubations with trapping agents highlighted that hydroxylated metabolites of guggulsterone isomers showed adduction with GSH and NAC. Even direct adducts of guggulsterone isomers were observed with both the trapping agents. The in silico toxicity potential of E- and Z-guggulsterones and their RMs was predicted using ADMET Predictor™ software and comparison was made against reported toxicities of guggulipid.

Determination of hormones inducing oocyte maturation in Chalcalburnus tarichi (Pallas, 1811).

Chalcalburnus tarichi is an endemic cyprinid species living in the Lake Van basin, in eastern Anatolia, Turkey. The present study was undertaken to determine which hormones induce oocyte maturation in C. tarichi. The levels of 17alpha,20beta,21-trihydroxyprogesterone (20beta-S), progesterone (P), 17alpha-hydroxyprogesterone (17alpha-HOP), 11-deoxycortisol (11-DOC), and 17alpha-hydroxy-20beta-dihydroprogesterone (17,20beta-P) were measured in fish caught from Lake Van and the Karasu River, and injected with human chorionic hormone (hCG) (1,000 and 1,500 IU/kg). Oocytes of fish caught from the lake were also incubated in vitro with different doses (50, 200, and 1,000 ng/ml) of 20beta-S, 17alpha-HOP, 11-DOC, and 17,20beta-P. 11-DOC was found to be the most effective hormone among those measured for inducing oocyte maturation in vivo and in vitro. 17,20beta-P could not be determined in the plasma of any fish in vivo (P < 0.05). 1,000 IU/kg dose of hCG given by injection caused a statistically significant increase in all plasma hormone levels (P < 0.05). It was found that there was a significant decrease in the P level only at 1,500 IU/kg dose of hCG injected (P < 0.05), while the level of other hormones increased at this dose (P < 0.05). It was also determined that all the hormones were effective in germinal vesicle breakdown (GVBD) in in vitro oocyte culture (P < 0.05). However, 11-DOC was found to be the most effective hormone in GVBD at a dose of 200 ng/ml (70% GVBD). In conclusion, 11-DOC synthesized during final oocyte maturation in C. tarichi was found to be a potent inducer of GVBD, which shows that 11-DOC may be described as an oocyte maturation steroid in this species.

Binding studies of guggulsterone-E to calf thymus DNA by multi-spectroscopic, calorimetric and molecular docking studies.

Guggulsterone, a sterol found in plants is used as an ayurvedic medicine for many diseases such as obesity, internal tumors, ulcers etc. E and Z are two isoforms of guggulsterone, wherein guggulsterone-E (GUGE) has also been shown to have anticancer potential. Most of the anticancer drugs target nucleic acids. Therefore, we studied the mode of interaction between ctDNA and GUGE using UV-Vis, fluorescence and CD spectroscopy, isothermal calorimetry along with molecular docking studies. Hoechst 3325, ethidium bromide and rhodamine-B displacement experiments confirms that GUGE binds in the minor groove of DNA. ITC results further suggest these interactions to be feasible and spontaneous with hydrogen bond formation and van der waals interactions. Lastly, molecular docking also suggests GUGE to be a minor groove binder interacting through a single hydrogen bond formation between OH group of GUGE and nitrogen (N3) of adenosine (A6).

Modulation of human lung fibroblast functions by ciclesonide: evidence for its conversion into the active metabolite desisobutyryl-ciclesonide.

BACKGROUND: Ciclesonide, an inhaled corticosteroid administered as inactive compound with almost no binding affinity for the glucocorticoid receptor, is clinically effective in asthma being converted by airway epithelial cells into its active metabolite desisobutyryl-(des)-ciclesonide. AIM: To evaluate whether ciclesonide could directly modulate in vitro bronchial fibroblast functions being converted into des-ciclesonide by these pluripotent cells involved in the regulation of airway inflammation and remodelling. METHODS: Ciclesonide (0.09-9.0 microM) was added to a human adult lung fibroblast cell line (CCL-202), seeded in medium in the presence of the following cytokines and growth factors: (a) basic fibroblast growth factor (bFGF) for cell proliferation, measured by tritiated thymidine ([3H]TdR) incorporation; (b) tumour necrosis factor (TNF)-alpha, to stimulate intercellular adhesion molecule (ICAM)-1 expression and monocyte chemoattractant protein-1 (MCP-1) and eotaxin release, evaluated by flow cytometry and ELISA, respectively; (c) transforming growth factor (TGF)-beta1, for induction of alpha smooth muscle actin (alpha-SMA) protein expression and modification of the organization of alpha-SMA stress fibres, evaluated by Western blot analysis and fluorescence microscopy. RESULTS: The presence of ciclesonide in cell cultures induced a significant downregulation of: (a) bFGF-induced fibroblast proliferation and TNF-alpha-induced ICAM-1 expression, at the 0.3-9.0 microM concentrations (p<0.05); (b) TNF-alpha-induced MCP-1 release, at all the concentrations tested (p<0.05); (c) TNF-alpha-induced eotaxin release, at the three highest concentrations (0.9-9.0 microM) (p<0.05); (d) TGF-beta1-induced of alpha-SMA protein expression at the 0.3-3.0 microM concentrations, associated with a reduction in the organization of alpha-SMA stress fibres. CONCLUSIONS: These data show at cellular level an effective anti-inflammatory activity of ciclesonide on human lung fibroblasts and support the hypothesis that also these cells, in addition to airway epithelial cells, may be involved in converting the parental compound into its active metabolite in the airways.

The role of esterases in the metabolism of ciclesonide to desisobutyryl-ciclesonide in human tissue.

Ciclesonide (CIC) is an inhaled glucocorticosteroid. This study aimed to identify esterases involved in the metabolism of CIC to the active metabolite desisobutyryl-ciclesonide (des-CIC), and to measure hydrolysis rates in human liver, lung and plasma and normal human bronchial epithelial (NHBE) cells in vitro. Ciclesonide (5 microM and 500 microM) was incubated with microsomal or cytosolic fractions from liver, lung and plasma (n=4 for each) and des-CIC formation was determined by reverse-phase high-performance liquid chromatography with U.V. detection. The roles of carboxylesterase, cholinesterase and A-esterase in CIC hydrolysis were determined using a range of inhibitors. Inhibitor concentrations for liver and NHBE cells were 100 microM and 5 microM, respectively. Liver tissue had a higher activity for 500 microM CIC hydrolysis (microsomes: 25.4; cytosol: 62.9 nmol/g tissue/min) than peripheral lung (microsomes: 0.089; cytosol: 0.915 nmol/g tissue/min) or plasma (0.001 nmol/mL plasma/min), corresponding with high levels of carboxylesterase and cholinesterase in the liver compared with the lung. CIC (5 microM) was rapidly hydrolyzed by NHBE cells (approximately 30% conversion at 4h), with almost complete conversion by 24h. In liver and NHBE cells, major involvement of cytosolic carboxylesterases, with some contribution by cholinesterases, was indicated. The highest level of conversion was found in the liver, the site of inactivation of des-CIC through rapid oxidation by cytochrome P450. Carboxylesterases in bronchial epithelial cells probably contribute significantly to the conversion to des-CIC in the target organ, whereas low systemic levels of des-CIC are a result of the high metabolic clearance by the liver following CIC inhalation.

In vitro metabolism of beclomethasone dipropionate, budesonide, ciclesonide, and fluticasone propionate in human lung precision-cut tissue slices.

BACKGROUND: The therapeutic effect of inhaled corticosteroids (ICS) may be affected by the metabolism of the drug in the target organ. We investigated the in vitro metabolism of beclomethasone dipropionate (BDP), budesonide (BUD), ciclesonide (CIC), and fluticasone propionate (FP) in human lung precision-cut tissue slices. CIC, a new generation ICS, is hydrolyzed by esterases in the upper and lower airways to its pharmacologically active metabolite desisobutyryl-ciclesonide (des-CIC). METHODS: Lung tissue slices were incubated with BDP, BUD, CIC, and FP (initial target concentration of 25 microM) for 2, 6, and 24 h. Cellular viability was assessed using adenosine 5'-triphosphate content and protein synthesis in lung slices. Metabolites and remaining parent compounds in the tissue samples were analyzed by HPLC with UV detection. RESULTS: BDP was hydrolyzed to the pharmacologically active metabolite beclomethasone-17-monopropionate (BMP) and, predominantly, to inactive beclomethasone (BOH). CIC was hydrolyzed initially to des-CIC with a slower rate compared to BDP. A distinctly smaller amount (approximately 10-fold less) of fatty acid esters were formed by BMP (and/or BOH) than by BUD or des-CIC. The highest relative amounts of fatty acid esters were detected for BUD. For FP, no metabolites were detected at any time point. The amount of drug-related material in lung tissue (based on initial concentrations) at 24 h was highest for CIC, followed by BUD and FP; the smallest amount was detected for BDP. CONCLUSION: The in vitro metabolic pathways of the tested ICS in human lung tissue were differing. While FP was metabolically stable, the majority of BDP was converted to inactive polar metabolites. The formation of fatty acid conjugates was confirmed for BMP (and/or BOH), BUD, and des-CIC.

Optical probes to identify the glucocorticoid receptor ligands in living cells.

Glucocorticoids act through glucocorticoid receptor (GR) and are used for the treatment of several diseases. Ligand-induced recruitment of coregulator protein(s), coactivator/corepressor, to GR is an initial step in transcriptional activation/inhibition of GR. We describe herein genetically encoded fluorescent probes for screening of glucocorticoids, natural and synthetic, in single living cells. The GR ligand binding domain was connected to the GR interacting peptide sequence from coactivator or corepressor protein via a flexible linker sequence. This fusion protein was sandwiched between cyan and yellow fluorescent proteins (CFP and YFP, respectively) to complete the construct of the probe. This construct functions as an optical probe for imaging ligand-induced interaction between the glucocorticoid receptor and the coregulator protein (GLUCOCOR) in live cells. The interaction between GR LBD and coregulator peptide within GLUCOCOR brings CFP in close proximity of YFP to induce fluorescence resonance energy transfer from CFP to YFP. The GLUCOCORs can identify functionally active GR ligands, rapidly and conveniently, in a high-throughput screen; and are capable of distinguishing GR agonists, antagonists, and selective GR modulators in intact living cells. Therefore, the present method may play a significant role in developing new glucocorticoids for clinical use.

Ciclesonide uptake and metabolism in human alveolar type II epithelial cells (A549).

BACKGROUND: Ciclesonide is a novel inhaled corticosteroid for the treatment of airway inflammation. In this study we investigated uptake and in vitro metabolism of ciclesonide in human alveolar type II epithelial cells (A549). Ciclesonide uptake was compared with fluticasone propionate, an inhaled corticosteroid that is not metabolized in lung tissue. A549 cells were incubated with 2 x 10(-8) M ciclesonide or fluticasone propionate for 3 to 30 min to determine uptake; or with 2 x 10(-8) M ciclesonide for 1 h, followed by incubation with drug-free buffer for 3, 6, and 24 h to analyze in vitro metabolism. High performance liquid chromatography with tandem mass spectrometry was used to measure the concentrations of both corticosteroids and metabolites. RESULTS: At all time points the mean intracellular concentration was higher for ciclesonide when compared with fluticasone propionate. Activation of ciclesonide to desisobutyryl-ciclesonide (des-CIC) was confirmed and conjugates of des-CIC with fatty acids were detected. The intracellular concentration of ciclesonide decreased over time, whereas the concentration of des-CIC remained relatively stable: 2.27 to 3.19 pmol/dish between 3 and 24 h. The concentration of des-CIC fatty acid conjugates increased over time, with des-CIC-oleate being the main metabolite. CONCLUSION: Uptake of ciclesonide into A549 cells was more efficient than that of the less lipophilic fluticasone propionate. Intracellular concentrations of the pharmacologically active metabolite des-CIC were maintained for up to 24 h. The local anti-inflammatory activity of ciclesonide in the lung may be prolonged by the slow release of active drug from the depot of fatty acid esters.

Uptake and metabolism of ciclesonide and retention of desisobutyryl-ciclesonide for up to 24 hours in rabbit nasal mucosa.

BACKGROUND: The nasal tissue uptake and metabolism of ciclesonide, a new-generation corticosteroid under investigation for treatment of allergic rhinitis, to its active metabolite, desisobutyryl-ciclesonide (des-CIC), was evaluated when administered to rabbits in a hypotonic versus an isotonic ciclesonide suspension. Nasal mucosa extracts from normal Japanese white rabbits were evaluated by high-performance liquid chromatography with tandem mass spectrometry detection after a single 143-mug dose of ciclesonide. Retention and formation of fatty acid conjugates of des-CIC were also measured in nasal mucosa extracts postadministration of a hypotonic ciclesonide suspension (143-mug single dose). RESULTS: Versus an isotonic suspension, the hypotonic suspension achieved higher concentrations of des-CIC (5.6-fold, 11.4-fold, and 13.4-fold; p < 0.05 for all) and ciclesonide (25.3-fold, 34.2-fold [p = not significant], and 16-fold [p < 0.05]) at 30, 120, and 240 min postadministration. Additionally, when administered via a hypotonic suspension, des-CIC was retained up to 24 h postadministration (45.46 pmol/g tissue). Highest concentration of major fatty acid ester conjugate, des-CIC-oleate, was detected in nasal mucosa at 8 h postadministration. CONCLUSION: These data suggest that a hypotonic ciclesonide suspension provides higher intracellular concentrations of des-CIC up to 24 h, thereby providing a rationale for investigation of ciclesonide as a convenient once-daily nasal spray for treatment of allergic rhinitis.

Tibolone and metabolites induce prolactin production in human endometrial stromal cells in vitro: evidence for cell-specific metabolism.

In this study, we assessed the effects of tibolone and its metabolites on the production of a progesterone sensitive parameter, prolactin, in human endometrium stroma cells in vitro. In addition, the metabolism of the compounds by isolated stromal and epithelial cells was evaluated. The reference compounds, progesterone, Org 2058, and DHT all induced prolactin production. Oestradiol also slightly induced prolactin production and enhanced the response to Org 2058. Tibolone and Delta4-tibolone were similar with regard to potency to induce prolactin levels in the culture supernatant. Their potency was lower than that of Org 2058, similar to that of progesterone and higher than that of DHT. The efficacies of tibolone, Delta4-tibolone and Org 2058 were similar (approximately 200-fold induction). The estrogenic tibolone metabolites 3alpha- and 3beta-OH tibolone also significantly stimulated prolactin production. Their potency, however, was low since significance was reached only at the highest concentrations tested. The PR antagonist Org 31710 inhibited both tibolone- and Delta4-tibolone-induced prolactin production. The responses of tibolone and Delta4-tibolone were not affected by co-incubation with the androgen receptor antagonist OH-flutamide. The effect of tibolone, but not Delta4-tibolone, was antagonized approximately 50% in combination with the highest dose (1 microM) estrogen receptor antagonist, ICI 164384. The induction of prolactin by 3alpha- and 3beta-OH tibolone was antagonized most potently by Org 31710, but also by ICI 164384 and OH-flutamide. Tibolone is metabolized differently in epithelial and stromal cells of the human endometrium. The epithelial cells mostly produce the progestagenic/androgenic Delta4-tibolone. The stromal cells produce predominantly the 3beta-OH tibolone, and some Delta4-tibolone, but the net effect observed with regard to prolactin production is progestagenic. When the metabolites 3alpha-OH, 3beta-OH, and Delta4-tibolone were added to the cultures no conversions were observed. The HPLC analyses showed no evidence for the production of sulfated metabolites. In conclusion, the net effects on endometrial stromal cells are predominantly progestagenic. Tibolone is converted by epithelial cells into Delta4-tibolone which displays progestagenic and androgenic activities, whereas in stromal cells also the estrogenic metabolites 3alpha- and 3beta-OH tibolone are formed.

Relationship of cytosolic estrogen and progesterone receptor content and the growth fraction in human mammary carcinomas.

In this study of breast cancer specimens, the relationship between cytosolic estrogen (ER) and progesterone receptor (PR) content to the size of the respective growth fraction (GF) (expressed as percentage of proliferating tumor cells) was investigated. We applied the recently developed ligand-binding assay for extracts of frozen sections and Ki-67 immunocytochemistry for the assessment of the GF to adjoining serial sections of a single tissue block. If the receptor content is plotted against the percentage of Ki-67 labeled cells, an inverse relationship between receptor content and proliferation becomes obvious, meaning that a high receptor content is associated with a small GF and vice versa. If tumor specimens are grouped according to the evaluated receptor status, the mean percentage of Ki-67-positive cells is 12% for ER-positive/PR-positive (ER+/PR+), 26% for ER-positive/PR-negative (ER+/PR-), 55% for ER-negative/PR-positive (ER-/PR+), and 57% for ER-negative/PR-negative (ER-/PR-) specimens. A significant population of tumors exists, however, which exhibit a high receptor content and a high GF. The percentages of ER+/PR+ samples with a high proliferation index are 16 and 26% if the total ER+ population is considered.

Assessment of in vitro metabolic stability, plasma protein binding, and pharmacokinetics of E- and Z-guggulsterone in rat.

Guggulsterone is a racemic mixture of two stereoisomers (E- and Z-), obtained from the gum resin of Commiphora mukul and it is marketed as an antihyperlipidemic drug. The aim of our study was to assess the in vitro and in vivo absorption, distribution, metabolism, and excretion (ADME) properties namely solubility, in vitro metabolism, plasma protein binding and oral pharmacokinetic studies of E- and Z-guggulsterone. In vitro metabolism experiments were performed by using rat liver and intestinal microsomes. In vitro intrinsic clearance (CLint ) was found to be 33.34 ± 0.51 and 39.23 ± 8.12 µL/min/mg protein in rat liver microsomes for E- and Z-isomers, respectively. Plasma protein binding was determined by equilibrium dialysis method and in vivo pharmacokinetic studies were performed in male Sprague Dawley (SD) rats. Both isomers were highly bound to rat plasma proteins (>95% bound). Plasma concentration of E- and Z-isomers decreased rapidly following oral administration and were eliminated from systemic circulation with a terminal half-life of 0.63 ± 0.25 and 0.74 ± 0.35 h, respectively. The clearance (CL) for E-isomer was 2.79 ± 0.73 compared to 3.01 ± 0.61 L/h/kg for Z-isomer, indicating no significant difference (student t test; p <0.05) in their elimination.The pharmacokinetics of both isomers was characterized by extensive hepatic metabolism as seen with rat liver microsomes with high clearance and low systemic availability in rats. In brief, first-pass metabolism seems to be responsible factor for low bioavailability of guggulsterone. Copyright © 2015 John Wiley & Sons, Ltd.

Ciclesonide: A Pro-Soft Drug Approach for Mitigation of Side Effects of Inhaled Corticosteroids.

Inhaled corticosteroids are used as one of the first-line drug therapy in patients with asthma. However, their long-term use is associated with various oropharyngeal and systemic side and adverse effects. Design of pro-soft drug is one of the strategies, which was adopted in the design of ciclesonide for mitigation of side effects usually observed with the use of inhaled corticosteroids. Ciclesonide, a pro-soft drug, is converted to an active metabolite desisobutyryl-ciclesonide in the lungs. The anti-inflammatory effect of desisobutyryl-ciclesonide is much higher than ciclesonide, and therefore, the local effect of the metabolite is higher with lower systemic side effects. Ciclesonide has favorable pharmacokinetic and pharmacodynamic properties as inhaled corticosteroid including low oral bioavailability, high plasma protein binding and rapid systemic clearance, high pulmonary deposition and distribution and long pulmonary residence duration. These advantageous properties make ciclesonide a very effective treatment option with low side effects. Various clinical studies support safety and efficacy of ciclesonide use in mild, moderate, and severe asthma patients.

New Anti-Inflammatory Metabolites by Microbial Transformation of Medrysone.

Microbial transformation of the anti-inflammatory steroid medrysone (1) was carried out for the first time with the filamentous fungi Cunninghamella blakesleeana (ATCC 8688a), Neurospora crassa (ATCC 18419), and Rhizopus stolonifer (TSY 0471). The objective was to evaluate the anti-inflammatory potential of the substrate (1) and its metabolites. This yielded seven new metabolites, 14α-hydroxy-6α-methylpregn-4-ene-3,11,20-trione (2), 6ß-hydroxy-6α-methylpregn-4-ene-3,11,20-trione (3), 15ß-hydroxy-6α-methylpregn-4-ene-3,11,20-trione (4), 6ß,17α-dihydroxy-6α-methylpregn-4-ene-3,11,20-trione (5), 6ß,20S-dihydroxy-6α-methylpregn-4-ene-3,11-dione (6), 11ß,16ß-dihydroxy-6α-methylpregn-4-ene-3,11-dione (7), and 15ß,20R-dihydroxy-6α-methylpregn-4-ene-3,11-dione (8). Single-crystal X-ray diffraction technique unambiguously established the structures of the metabolites 2, 4, 6, and 8. Fungal transformation of 1 yielded oxidation at the C-6ß, -11ß, -14α, -15ß, -16ß positions. Various cellular anti-inflammatory assays, including inhibition of phagocyte oxidative burst, T-cell proliferation, and cytokine were performed. Among all the tested compounds, metabolite 6 (IC50 = 30.3 µg/mL) moderately inhibited the reactive oxygen species (ROS) produced from zymosan-induced human whole blood cells. Compounds 1, 4, 5, 7, and 8 strongly inhibited the proliferation of T-cells with IC50 values between <0.2-10.4 µg/mL. Compound 7 was found to be the most potent inhibitor (IC50 < 0.2 µg/mL), whereas compounds 2, 3, and 6 showed moderate levels of inhibition (IC50 = 14.6-20.0 µg/mL). Compounds 1, and 7 also inhibited the production of pro-inflammatory cytokine TNF-α. All these compounds were found to be non-toxic to 3T3 cells (mouse fibroblast), and also showed no activity when tested against HeLa (human epithelial carcinoma), or against PC3 (prostate cancer) cancer cell lines.

Regulation of P-glycoprotein efflux activity by Z-guggulsterone of Commiphora mukul at the blood-brain barrier.

The present study was to investigate whether Z-guggulsterone had the regulatory effect on the activity and expression of P-glycoprotein in rat brain microvessel endothelial cells (rBMECs) and in rat brain. Inorganic phosphate liberation assay, high performance liquid chromatography, and western blot analysis were performed to assess the P-glycoprotein ATPase activity, the accumulation of NaF and rhodamine 123, and P-glycoprotein and MRP1 expression. The results showed that Z-guggulsterone (0-100 µM) significantly enhanced basal P-glycoprotein ATPase activity in a concentration-dependent manner. Tetrandrine (0.1, 0.3, 1 µM) or cyclosporine A (0.1, 0.3, 1 µM) had non-competitively inhibitory manner on Z-guggulsterone-stimulated P-glycoprotein ATPase activity, suggesting that Z-guggulsterone might have unique binding site or regulating site on P-glycoprotein. However, Z-guggulsterone (30, 100 µM) had almost no influence on MRP1 expression in rBMECs. Further results revealed that Z-guggulsterone (50mg/kg) significantly increased the accumulation of rhodamine 123 by down-regulating P-glycoprotein expression in rat brain, as compared with control (P<0.05). Our studies suggested that Z-guggulsterone potentially inhibited the activity and expression of P-glycoprotein in rBMECs and in rat brain.