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.

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.

A Review of Deflazacort for Patients With Duchenne Muscular Dystrophy.

Objective: The objective of this article is to review the pharmacology, pharmacokinetics, efficacy, safety, dosage and administration, and formulary considerations of deflazacort. Data Sources: A search of MEDLINE and EMBASE (1946 to December 31, 2019) was conducted using the terms deflazacort and Duchenne muscular dystrophy (DMD). Results were limited to clinical trials, humans, and English. Additional sources and data were obtained from the references of included articles and prescribing information. Study Selection and Data Extraction: All articles published after July 2014 related to pharmacology, pharmacokinetics, efficacy, or safety of the therapy in human subjects were included. Data Synthesis: Deflazacort 0.9 mg/kg/d is a once-daily oral corticosteroid and is the first drug of its class to be Food and Drug Administration (FDA) approved for DMD. Studies with deflazacort show improved functional outcomes, delayed onset of cardiomyopathy, reduction in scoliosis surgery, and improved survival, but these improvements are supported by relatively weak evidence. Relevance to Patient Care and Clinical Practice: This review presents data from studies published after the most recent DMD 2016 treatment guidelines and offers prescribing considerations, including pharmacology, pharmacokinetics, adverse effects, formulary considerations, and areas of uncertainty. Conclusions: Deflazacort presents an additional, FDA-approved corticosteroid option for patients that offers improved quality of life for DMD patients. However, there is weak evidence to support these benefits; a full risk-benefit analysis considering adverse events, efficacy, cost, and previous trials of steroid therapy is necessary when selecting therapy. Further research will help clarify deflazacort's optimal dose, duration of treatment, and impact on quality of life.

In-vitro metabolism, CYP profiling and metabolite identification of E- and Z- guggulsterone, a potent hypolipidmic agent.

The polyphenol E- and Z-gugggulsterone (GS) is an antagonist ligand for the Farnesoid X Receptor (FXR) and known to possess potent hypolipidemic properties as shown in various preclinical and clinical studies. In the present study, we examined drug-like properties of GS by assessing the isomers plasma protein binding, metabolic stability, CYP profiling, CYP inhibition, and phase I and II metabolite identification of GS using liver microsomes and S9 fractions. GS followed Lipinski and Veber rules and were substrates of CYP3A CYP2C19 and CYP2D6 isoforms. GS was also found to be an inhibitor of CYP2C19 with an IC50 value of 2.1 µM. GS showed high plasma protein binding (<96%), and low to moderate binding with human serum albumin (∼70%). Unbound intrinsic clearances (CLint, in-vitro) was determined to be low at 0.029 ±â€¯0.0009 and 0.027 ±â€¯0.008 mL/min/mg protein for E- and Z-isomer, respectively in human liver microsomes. Nineteen phase I and II metabolites were identified and hydroxylation was found to be major metabolic pathway using human liver microsomes and S9 fractions. The results of in-vitro drug-metabolism studies provide impetus for further structural modification of this pharmacophore in order to improve the stability of drugs with potent hypolipidemic effects.

Deposition and metabolism of inhaled ciclesonide in the human lung.

Ciclesonide is an inhaled corticosteroid, administered as a prodrug via a metered-dose inhaler. Following deposition in the lung, ciclesonide is hydrolysed by esterases to form the pharmacologically active metabolite desisobutyryl-ciclesonide (des-CIC). Formation of des-CIC, as well as reversible esterification of des-CIC with fatty acids, has been demonstrated in vitro. The aim of this study was to investigate the in vivo metabolism of ciclesonide in the human lung. This single-dose, open-label, nonrandomised study was performed in 20 patients undergoing planned lung surgery for treatment of malignant pulmonary lesions. Patients inhaled a single dose of 1,280 µg ciclesonide at various time-points between 2 and 24 h prior to lung tissue resection. The concentration of ciclesonide, des-CIC and fatty acid conjugates of des-CIC in tissue samples was determined. Serum samples for pharmacokinetic analysis were taken at several time-points after inhalation. The pharmacokinetics in serum indicated that the inhalation by the patients was adequate. Metabolites (des-CIC, des-CIC oleate and des-CIC palmitate) were detected in the resected central and peripheral lung tissues. A substantial portion of ciclesonide was already activated to des-CIC at the first time-point of tissue analysis. Activation of ciclesonide and formation of des-CIC fatty acid conjugates was confirmed in vivo in the human lung.

Rapid effect of inhaled ciclesonide in asthma: a randomized, placebo-controlled study.

BACKGROUND: Ciclesonide is a novel inhaled corticosteroid for the treatment of asthma, and it is important to measure the onset of effect of this therapy on airway hyperresponsiveness (AHR), exhaled nitric oxide (NO), and levels of eosinophils in induced sputum. METHODS: In a randomized, double-blind, crossover study, 21 patients with mild asthma inhaled ciclesonide 320 microg (ex-actuator) qd, ciclesonide 640 microg (ex-actuator) bid, and placebo for 7 days. Exhaled NO and AHR to adenosine monophosphate (AMP), measured as the provocative concentration of AMP producing a 20% reduction in FEV1 (PC20FEV1), were assessed after inhalation on days 1, 3 and 7. Eosinophil levels in induced sputum were also measured. RESULTS: Ciclesonide 320 microg qd and 640 microg bid produced significantly greater improvements in PC20FEV1 compared with placebo on day 1 (within 2.5 h), and on days 3 and 7 (all p < 0.0001). On day 3, both ciclesonide doses significantly reduced exhaled NO levels by - 17.7 parts per billion (p < 0.0001) and - 15.4 parts per billion (p < 0.003) vs placebo, respectively. Significant reductions were maintained during the study with both ciclesonide doses (p < 0.01). A nonsignificant trend towards a decrease in eosinophil cell numbers was observed after 7 days of ciclesonide treatment, especially in patients receiving the higher dose. CONCLUSIONS: A single dose of ciclesonide decreased AHR to AMP and exhaled NO within 3 h, while FEV, improved at 3 days and 7 days.

The hypolipidemic natural product guggulsterone is a promiscuous steroid receptor ligand.

Guggulsterone (GS) is the active substance in guggulipid, an extract of the guggul tree, Commiphora mukul, used to treat a variety of disorders in humans, including dyslipidemia, obesity, and inflammation. The activity of GS has been suggested to be mediated by antagonism of the receptor for bile acids, the farnesoid X receptor (FXR). Here, we demonstrate that both stereoisomers of the plant sterol, (E)- and (Z)-GS, bind to the steroid receptors at a much higher affinity than to FXR. Both stereoisomers bind to the mineralocorticoid receptor (MR) with a Ki value of approximately 35 nM, which is greater than 100 times more potent than their affinity for FXR. Both (E)- and (Z)-GS also displayed high affinity for other steroid receptors, including the androgen (AR), glucocorticoid (GR), and progesterone receptors (PR) with Ki values ranging from 224 to 315 nM. In cell-based functional cotransfection assays, GSs behaved as antagonists of AR, GR, and MR, but as agonists of PR. Agonist activity was also demonstrated with estrogen receptor (ER) alpha; however, the potency was very low (EC50 > 5000 nM). In addition, GS displayed activity in functional assays in cell lines expressing endogenous AR, GR, ER, and PR. These data suggest that the variety of pharmacological effects exhibited by GS may be mediated by targeting several steroid receptors.

Treatment of active and postactive ileal and colonic Crohn's disease with oral pH-modified-release budesonide. German Budesonide Study Group.

BACKGROUND/AIMS: Budesonide is a glucocorticoid with a high topical anti-inflammatory but low systemic activity due to its rapid hepatic inactivation. The aim of this open, multicenter study was to investigate efficacy and safety of oral pH-modified-release budesonide in patients with active Crohn's disease of the ileum and colon and in maintaining budesonide-induced remission in postactive Crohn's disease. MATERIALS AND METHODS: 81 patients (intention-to-treat) received 3 x 3 mg budesonide/day for 6 weeks, followed by 3 x 2 mg budesonide for another 6 weeks in case of response to initial treatment. Clinical and laboratory parameters were assessed at study entry as well as after 2, 4, 6 and 12 weeks of treatment. RESULTS: On an intention-to-treat basis remission was induced in 54.3% of 81 patients with active Crohn's disease, 71.4% of 35 patients stayed in remission after the acute-phase treatment until the end of the trial. Typical steroid-related side effects were observed during the acute-phase treatment in only 18% of the patients. Duration, severity and extent of disease at study entry played no significant role in the outcome of the trial, but there was a tendency towards better results during the acute-phase treatment in patients with moderate disease activity and affection of the terminal ileum and proximal colon. CONCLUSIONS: Budesonide could be an alternative to conventional steroid treatment in patients with active Crohn's disease.

Budesonide. A review of its pharmacological properties and therapeutic efficacy in inflammatory bowel disease.

Budesonide is a glucocorticoid with high topical activity, but low systemic bio-availability which results in reduced systemic effects in comparison with other glucocorticoids. To date, it has been evaluated for use in patients with inflammatory bowel disease when administered either orally as a controlled ileal release formulation or rectally as an enema. In comparative trials, daily treatment with budesonide enema 2 mg/100ml for 4 weeks produced endoscopic remission or improvement in 46 to 84% of patients with active distal ulcerative colitis and/or proctitis and histological remission or improvement in 45 to 68%. In general, this regimen was effective as regimens of hydrocortisone, methylprednisolone, prednisolone or mesalazine (5-amino-salicylic acid, mesalamine) enemas, but caused less suppression of plasma cortisol levels than the other glucocorticoids. Oral treatment with controlled release budesonide 9 mg/day for 8 weeks produces clinical remission in 42 to 67% of patients with active Crohn's disease of the ileum, ileocaecal region and/or ascending colon and significantly reduces Crohn's disease activity index scores compared with baseline and placebo. Results of a quality-of-life questionnaire reflected these clinical improvements. Budesonide has similar efficacy to prednisolone. Response to budesonide is maintained after dosage tapering at 8 weeks. Compared with placebo, maintenance treatment with oral budesonide 3 or 6 mg/day increases the duration of remission in patients with Crohn's disease, but does not appear to affect the 1-year relapse rate. Thus, budesonide, administered rectally to patients with distal ulcerative colitis or proctitis or orally to patients with Crohn's disease of the ileum, ileocaecal region and/or ascending colon, is a favourable option for the treatment of acute exacerbations of inflammatory bowel disease. Because of the low incidence of adverse glucocorticoid-related effects associated with oral budesonide, it may also be a useful agent for longer term maintenance therapy if further clinical trials confirm its efficacy in this indication.

Binding of the anti-inflammatory steroid deflazacort to glucocorticoid receptors in brain and peripheral tissues. In vivo and in vitro studies.

Deflazacort (DFC) is a heterocyclic glucocorticoid with anti-inflammatory activity but with decreased side effects. In this study, we have evaluated the capacity of DFC and other glucocorticoids to reach the central nervous system (CNS) in vivo by measuring changes of [3H]dexamethasone (DEX) binding to glucocorticoid receptors (GR) in vitro. GR occupation was effected by DEX in the cerebral cortex, hippocampus, pituitary, liver and thymus, with DFC showing a similar profile except for the cerebral cortex. In contrast, corticosterone weakly occupied GR in the thymus, pituitary and hippocampus and methyl-prednisolone was active only in peripheral tissues. Furthermore, IC50 for DEX in vitro amounted to 15-17 nM in the hippocampus and liver, whereas IC50 for the active metabolite 21-deacetyl-DFC (21-OH-DFC) was 4 times higher. 21-OH-DFC bound to type II and was absent from type I GR. When tested in equipotent doses based on IC50 analysis, DFC and DEX similarly induced in vivo ornithine decarboxylase activity in hippocampus and liver, although body weight loss after chronic treatment was significantly less for DFC. The results show that DFC distributes on the CNS similarly to DEX, induces ornithine decarboxylase activity but presents less intensive catabolic effects, making it suitable for use as an anti-inflammatory steroid during chronic therapeutic regimes.

Pharmacokinetics of budesonide enema in patients with distal ulcerative colitis or proctitis.

Pharmacokinetic data obtained after one dose of a 2-mg budesonide enema were compared with data obtained after the last dose of four weeks of daily treatment in 24 patients with active distal ulcerative colitis or proctitis. This open multicentre study involved 28 eligible patients. Sigmoidoscopy and biopsy scores improved significantly (P < 0.002) during the four-week treatment period. Maximal plasma concentration (Cmax) of budesonide was 2.1 nmol/L 1.3 h after the first dose and 2.5 nmol/L 1.2 h after the last dose; the difference was not significant. The area under the curve (AUC) of plasma concentration vs. time was after the first dose 9.7 nmol h/L and after the last dose 11.6 nmol h/L (P < 0.03). The small increase in AUC may be attributed to improved absorption. During the last dose interval, minimal plasma concentration was below the limit of quantitation in most subjects. The Cmax and AUC of budesonide increased slightly after four weeks of treatment, but budesonide did not accumulate. Mean morning plasma cortisol values did not change significantly during treatment (P = 0.083), although a small change in cortisol levels between the first visit (pre-treatment) and last visit was positively correlated to the Cmax of budesonide measured at the last visit (P = 0.012).

Fluorine-18-labeled progestin ketals: synthesis and target tissue uptake selectivity of potential imaging agents for receptor-positive breast tumors.

We have studied two new fluorine-substituted progestins as potential imaging agents for progesterone-receptor-positive human breast tumors. The steroids are 16 alpha, 17 alpha-fluoroacetophenone ketals of 16 alpha, 17 alpha-dihydroxyprogesterone and 16 alpha, 17 alpha, 21-trihydroxy-19-norprogesterone. Synthesis of the latter compound in seven steps from 19-norandrost-4-ene-3,17-dione is reported. Both compounds demonstrate high affinity for the progesterone receptor (PgR) (52.5 and 240%, respectively, relative to R5020 = 100). The syntheses were adapted to 18F-labeling with 4'-[18F]-fluoroacetophenone, prepared from 4'-nitroacetophenone by nucleophilic substitution with K18F/Kryptofix. Considerable adjustment of reaction conditions was required to effect ketalization using tracer quantities of the ketone. In tissue distribution studies in estrogen-primed immature female rats, both ketals showed selective uterine uptake, which was blocked by coinjection of a saturating dose of the unlabeled progestin ORG 2058. Additionally, metabolic stability of the radiolabel was indicated by the low radioactivity levels seen in bone. Both compounds showed relatively high uptake in fat, in accord with their relative lipophilicities demonstrated by HPLC-derived octanol-water partition coefficients. The selective uterine uptake and metabolic stability of these compounds suggests that this class of PgR ligands might be promising for the selective imaging of receptor-positive tumors if derivatives of reduced lipophilicity can be prepared.

Target tissue uptake selectivity of three fluorine-substituted progestins: potential imaging agents for receptor-positive breast tumors.

We have studied three new fluorine-substituted progestins (1-3) as potential imaging agents for progesterone receptor (PgR)-positive human breast tumors. Two of these are fluorine-substituted analogs of the potent progestin R5020 (promegestone), derived from (21S)-hydroxy R 5020 (RU 27987) and (21R)-hydroxy R 5020 (RU 27988), known metabolites of R 5020, which have affinities for PgR that are 116 and 4%, respectively (relative to R 5020 = 100%). These precursors were protected as their 3,3-dioxolane derivatives and converted to the 21-trifluoromethanesulfonate derivatives. Fluoride ion displacement, followed by acid-catalyzed deprotection, furnished in good yield the epimeric fluoroanalogs, (21S)- and (21R)-fluro R 5020 (1 and 2, affinities for PgR, 11 and 45%, respectively). These compounds were also prepared in 18F labeled form by the same route, in 14-32% overall radiochemical yield (decay corrected; synthesis time 90 min; sp. act. 370-1060 Ci/mmol). In tissue distribution studies in estrogen-primed immature rats, uterus-to-muscle ratios were 4.3 at 1 h for the 21S-epimer and 1.1 for the 21R-epimer, paralleling their relative binding affinities. Considerable metabolic defluorination was observed. The third fluorine-substituted progestin, DU 41165, has a novel retroprogesterone (9 beta, 10 alpha) structure, substituted with fluorine at C-6; its binding affinity is 145% relative to R 5020, and it was prepared in tritium-labeled form by acetylation of DU 41231, the 17 alpha-hydroxy precursor, with [3H]acetic anhydride. In estrogen-primed immature rats, this compound shows uterus-to-muscle ratios of 15 at 1 h, and 18-71 between 2 and 6 h, suggesting that compounds in this retroprogesterone series may be very promising candidates for selective imaging of PgR-positive tissues and tumors.

Progestin inhibition of progesterone receptor gene expression in human breast cancer cells.

The present study was designed to investigate whether inhibition of progesterone receptor (PR) gene transcription and/or regulation of PR mRNA half-life were involved in the progestin-mediated decrease of PR in T-47D human breast cancer cells. Cells were treated with the progestin ORG 2058 and PR mRNA measured by Northern blot analysis of total RNA. A major PR mRNA around 13.5 kilobases and minor species around the 28S ribosomal RNA subunit were decreased upon ORG 2058 treatment. The decrease was not detectable until 2-3 h after treatment and was the same at all ORG 2058 concentrations (1-100 nM) tested. The decrease in PR mRNA was unaffected by actinomycin D in the first 3 h but was inhibited thereafter. There was a partial recovery of PR mRNA levels 24 h after ORG 2058 exposure. Immunoblot analysis showed that immunoreactive PR decreased in parallel with PR mRNA. The rate of protein loss in the first 12 h after progestin treatment was related to the ORG 2058 concentration used. Nuclear run-on experiments showed that ORG 2058 caused a decrease of up to 70% in the transcription rate of the PR gene. The half-life of PR mRNA was shown to be 2-2.5 h by [3H]uridine incorporation, which was much shorter than estimates obtained using actinomycin D, and was unaffected by ORG 2058. In summary, these data have shown that the mechanism by which progestins decrease the concentration of PR includes inhibition of transcription of the PR gene.

Uptake of three [3H]progestins by target tissues in vivo: implications for the design of diagnostic imaging agents.

We have investigated the tissue distribution of radioactivity for 0.5-4 h following the i.v. injection of three tritium-labeled progestins in estrogen-primed, immature rats. Whereas [3H]progesterone shows minimal uterine uptake (less than 0.7% injected dose per gram; %ID/g), the two higher affinity, synthetic progestins [3H]R 5020 (promegestrone) and [3H]ORG 2058 show highly selective uptake that reaches 4-5% ID/g by 1-3 h. The uterus to non-target tissue activity ratio at 2-4 h is approximately 12-20 for R 5020 and ORG 2058, but less than 2 for progesterone; the uterus to blood activity ratio for R 5020 is also high (approximately 15), but is lower for ORG 2058, possibly due to the accumulation of radiolabeled metabolites in the blood. The uterine uptake is selectively blocked by simultaneous injection of a large dose of unlabeled steroid, indicating that the uptake is mediated by a high affinity, low capacity binding system, presumably the progesterone receptor. Pronounced uptake is also observed by the liver and into fat, but is not receptor-mediated. The highly selective target tissue uptake by the two synthetic steroids, but not by progesterone, indicates that one must have ligands with sufficiently high affinity for the target tissue receptor, as well as low affinity for certain non-receptor binding proteins, in order to obtain adequate contrast between target and non-target tissues in dynamic uptake studies. These guidelines will be important in the development of suitable in vivo imaging agents based on the progesterone receptor.