Hedgehog regulates cerebellar progenitor cell and medulloblastoma apoptosis.

The external granule layer (EGL) is a proliferative region that produces over 90% of the neurons in the cerebellum but can also malignantly transform into a cerebellar tumor called the medulloblastoma (the most common malignant brain tumor in children). Current dogma considers Hedgehog stimulation a potent proliferative signal for EGL neural progenitor cells (NPCs) and medulloblastomas. However, the Hedgehog pathway also acts as a survival signal in the neural tube where it regulates dorsoventral patterning by controlling NPC apoptosis. Here we show that Hedgehog stimulation is also a potent survival signal in the EGL and medulloblastomas that produces a massive apoptotic response within hours of signal loss in mice. This toxicity can be produced by numerous Hedgehog antagonists (vismodegib, cyclopamine, and jervine) and is Bax/Bak dependent but p53 independent. Finally, since glucocorticoids can also induce EGL and medulloblastoma apoptosis, we show that Hedgehog's effects on apoptosis can occur independent of glucocorticoid stimulation. This effect may play a major role in cerebellar development by directing where EGL proliferation occurs thereby morphologically sculpting growth. It may also be a previously unknown major therapeutic effect of Hedgehog antagonists during medulloblastoma therapy. Results are discussed in terms of their implications for both cerebellar development and medulloblastoma treatment.

Metabolic pathways of inhaled glucocorticoids by the CYP3A enzymes.

Asthma is one of the most prevalent diseases in the world, for which the mainstay treatment has been inhaled glucocorticoids (GCs). Despite the widespread use of these drugs, approximately 30% of asthma sufferers exhibit some degree of steroid insensitivity or are refractory to inhaled GCs. One hypothesis to explain this phenomenon is interpatient variability in the clearance of these compounds. The objective of this research is to determine how metabolism of GCs by the CYP3A family of enzymes could affect their effectiveness in asthmatic patients. In this work, the metabolism of four frequently prescribed inhaled GCs, triamcinolone acetonide, flunisolide, budesonide, and fluticasone propionate, by the CYP3A family of enzymes was studied to identify differences in their rates of clearance and to identify their metabolites. Both interenzyme and interdrug variability in rates of metabolism and metabolic fate were observed. CYP3A4 was the most efficient metabolic catalyst for all the compounds, and CYP3A7 had the slowest rates. CYP3A5, which is particularly relevant to GC metabolism in the lungs, was also shown to efficiently metabolize triamcinolone acetonide, budesonide, and fluticasone propionate. In contrast, flunisolide was only metabolized via CYP3A4, with no significant turnover by CYP3A5 or CYP3A7. Common metabolites included 6ß-hydroxylation and Δ(6)-dehydrogenation for triamcinolone acetonide, budesonide, and flunisolide. The structure of Δ(6)-flunisolide was unambiguously established by NMR analysis. Metabolism also occurred on the D-ring substituents, including the 21-carboxy metabolites for triamcinolone acetonide and flunisolide. The novel metabolite 21-nortriamcinolone acetonide was also identified by liquid chromatography-mass spectrometry and NMR analysis.

Endogenous tenascin-C enhances glioblastoma invasion with reactive change of surrounding brain tissue.

Tenascin-C is an extracellular matrix glycoprotein implicated in embryogenesis, wound healing and tumor progression. We previously revealed that tenascin-C expression is correlated with the prognosis of patients with glioblastoma. However, the exact role of endogenous tenascin-C in regulation of glioblastoma proliferation and invasion remains to be established. We show here that endogenous tenascin-C facilitates glioblastoma invasion, followed by reactive change of the surrounding brain tissue. Although shRNA-mediated knockdown of endogenous tenascin-C does not affect proliferation of glioblastoma cells, it abolishes cell migration on a two-dimensional substrate and tumor invasion with brain tissue changes in a xenograft model. The tyrosine phosphorylation of focal adhesion kinase, a cytoplasmic tyrosine kinase that associates with integrins, was decreased in tenascin-C-knockdown cells. In the analysis of clinical samples, tenascin-C expression correlates with the volume of peritumoral reactive change detected by magnetic resonance imaging. Interestingly, glioblastoma cells with high tenascin-C expression infiltrate brain tissue in an autocrine manner. Our results suggest that endogenous tenascin-C contributes the invasive nature of glioblastoma and the compositional change of brain tissue, which renders tenascin-C as a prime candidate for anti-invasion therapy for glioblastoma.

Flunisolide metabolism and dynamics of a metabolite.

Flunisolide (6 alpha-fluoro-11 beta,16 alpha,17 alpha,21-tetrahydroxypregna-1,4-diene-3,20-dione 16,17-acetonide) is a potent corticoid used clinically in topical formulations. Three men were given single 2-mg intravenous and oral doses of 14C-labeled flunisolide and plasma and urine concentrations of flunisolide and a major metabolite, 6 beta,11 beta,16 alpha,17 alpha,21-penta-hydroxypregna-1,4-diene-3,20-dione 16,17-acetonide (6 beta-OH metabolite) were determined. Oral flunisolide was metabolized rapidly and extensively to the 6 beta-OH metabolite and to conjugates; comparison in the intravenous dose kinetics suggested significant first-pass metabolism. In a separate study in 12 normal subjects, flunisolide in plasma was quantitated by radioimmunoassay (RIA); average systemic availability was 20%. The apparent volume of distribution (Vd beta) of flunisolide was large and systemic clearance and apparent oral clearance values were high. The 6 beta-OH metabolite had corticoid activities no more than 3 times that of hydrocortisone in rats as measured by thymolytic, anti-inflammatory, and adrenal-suppressive assays, whereas flunisolide had 180 to 550 times the activity of hydrocortisone. These data offer a metabolic explanation for the clinical observation that flunisolide can be administered intranasally and by inhalation in therapeutically effective doses without causing significant reduction in adrenal function.

Morning plasma cortisol levels in infants treated with topical fluorinated glucocorticosteroids.

The plasma cortisol levels of 17 infants of mean age 12.1 months taken at 8 AM were measured before and after application of a topical fluorinated glucocorticosteroid. There was a statistically significant depression of plasma cortisol value in 11 infants at days 3, 7, and 14 after therapy. All of the plasma cortisol levels returned to normal. For the entire group of 17 infants, however, there was no significant depression of plasma cortisol levels. Even in the usual office use of topical glucocorticosteroids, one must be aware of the possiblity of percutaneous absorption.

Evidence of P-glycoprotein mediated apical to basolateral transport of flunisolide in human broncho-tracheal epithelial cells (Calu-3).

1. Transepithelial transport of flunisolide was studied in reconstituted cell monolayers of Calu-3, LLC-PK1 and the MDR1-P-glycoprotein transfected LLC-MDR1 cells. 2. Flunisolide transport was polarized in the apical (ap) to basolateral (bl) direction in Calu-3 cells and was demonstrated to be ATP-dependent. In LLC-MDR1 cells, flunisolide was transported in the bl to ap direction and showed no polarization in LLC-PK1 cells. 3. Non-specific inhibition of cellular metabolism at low temperature (4 degrees C) or by 2-deoxy-D-glucose (2-d-glu) and sodium azide (NaN(3)) abolished the polarized transport. Polarized flunisolide transport was also inhibited by the specific Pgp inhibitors verapamil, SDZ PSC 833 and LY335979. 4. Under all experimental conditions and in the presence of all used inhibitors, no decrease in the TransEpithelial Electrical Resistance (TEER) values was detected. From all inhibitors used, only the general metabolism inhibitors 2-deoxy-D-glucose and NaN(3), decreased the survival of Calu-3 cells. 5. Western blotting analysis and confocal laser scanning microscopy demonstrated the presence of MDR1-Pgp at mainly the basolateral side of the plasma membrane in Calu-3 cells and at the apical side in LLC-MDR1 cells. Mass spectroscopy studies demonstrated that flunisolide is transported unmetabolized across Calu-3 cells. 6. In conclusion, these results show that the active ap to bl transport of flunisolide across Calu-3 cells is facilitated by MDR1-Pgp located in the basolateral plasma membrane.

Flunisolide: a review of its pharmacological properties and therapeutic efficacy in rhinitis.

Flunisolide, a derivative of fluocinolone acetonide, is advocated for intranasal inhalation for the treatment of perennial and seasonal allergic rhinitis. It is rapidly absorbed by all routes of administration, but it quickly undergoes extensive first-pass metabolism to a 6 beta-hydroxylated metabolite, which possesses only weak corticosteroid effects. Intranasal flunisolide relieves nasal symptoms (but not eye symptoms) in both perennial and seasonal allergic rhinitis, being most effective in patients who have an allergic component to their rhinitis; and like other corticosteroids it may reduce the need for systemic antihistamines in such patients, expecially during peak pollen periods. A few well designed comparative studies have shown flunisolide to be as effective as intranasal beclomethasone, and (in a single study) more effective than intranasal sodium cromoglycate solution. Only transient side effects have occurred, including nasal stinging and throat irritation. No Candida infections have been clinically apparent in short or longer term trials. Resting morning plasma cortisol levels have not been suppressed by usual therapeutic doses of intranasal flunisolide, but the drug's effects on hypothalamo-pituitary-adrenal (HPA) axis integrity during conditions of stress have not been evaluated.

Corticosteroid aerosols in the treatment of asthma.

Since the 1950s, corticosteroid aerosols have proved useful in the treatment of asthma. Although their precise mechanism of action is not known, these topical agents have beneficial antiinflammatory and decongestive effects on the bronchial tree in both the allergic and nonallergic forms of this disease. Four of the newer aerosolized steroids--beclomethasone dipropionate, triamcinolone acetonide, flunisolide and budesonide--have been evaluated in clinical trials. The last drug is still investigational. Their side effects are minimal, the major ones being oral candidiasis and dysphonia. They are most effective when used prophylactically and should not be administered during acute asthmatic attacks, as insufficient amounts of drug are inhaled when the airways are obstructed. Patients must be instructed in the correct techniques of administering steroid aerosols to ensure optimal therapy.

Preliminary studies into percorneal penetration and elemental content of the stratum corneum using X-ray microanalysis.

A technique is described which measures the penetration of substances through the stratum corneum (SC) and the distribution of elements in this structure employing scanning electron microscopy and energy dispersive analysis. Preliminary observations show that the normal SC shows a concentration gradient of potassium, high in the surface layers and lowest deeper down whereas the reverse is true for phosphorus. It has been shown that sulphur rapidly tranverses the SC and seems to penetrate through all parts of the horny layer whereas lead and zinc do not easily enter this structure.

Deep tissue penetration of bases and steroids after dermal application in rat.

The local deep tissue penetration of bases such as diazepam, antipyrine, iodoantipyrine, haloperidol and steroids such as hydrocortisone, fluocinolone acetonide, testosterone and progesterone after dermal application as aqueous solutions was studied in a rat model. The extent of local, as distinct from systemic delivery, for each solute was assessed by comparing the tissue concentrations obtained below a treated site with those in contralateral tissues. Local direct penetration was evident for all solutes below the applied site, although depth of penetration varied between individual solutes. A physiological pharmacokinetic model was employed to estimate local tissue concentrations of various compounds after dermal application.

[Microbiological dehydrogenation of intermediate of fluocinanide acetate].

Both compound I and II are intermediates in fluocinanide acetate synthesis. I could be dehydrogenated to II in 62% approximately 63% yields by Arthrobactor simplex No. A-1, which was selected in our laboratory. When concentration of I was 0.1%, it was transformed so fast that II could not be accumulated. When concentration was increased to 0.2%, four intermediates IV, V, VI and VII were formed in addition to a little amount of product II. When concentration of substrate I was increased to 0.5% and B-CS buffer solution and 4% alcohol (95%, V/V) were added, compound II in cuboidal microcrystalline form was obtained. Under this condition, the yield was steady and melting point was above 250 degrees C.

[Pharmacologic study of the glucocorticoid activity of flunisolide compared with other steroids in the rat]. / Etude pharmacologique de l'activité glucocorticoïde du flunisolide comparativement à d'autres stéroïdes chez le rat.

Flunisolide (FLU), beclomethasone dipropionate (BDP) and its pulmonary metabolites beclomethasone monopropionate (BMP) and beclomethasone (B) were studied in rat for: their relative binding affinity (RBA) for the 5 classes of steroid receptors, their in vitro glucocorticoid activity on rat thymocytes, their in vivo glucocorticoid activity by oral route. These compounds displayed a strong RBA for rat lung, thymus and liver glucocorticoid receptors (FLU > or = BMP > BDP > or = B). They were also shown to have a moderate RBA for both mineralocorticoid and progestin receptors, while being devoid of any binding to androgen and oestrogen receptors. On rat thymocytes FLU exhibited the highest glucocorticoid activity (FLU > B > or = BMP > BDP). In rat oral FLU displayed a strong glucocorticoid activity with a slight first-pass metabolism as opposed to what has been reported in human.

Photoactivation of corticosteroids in UVB-exposed skin.

The photodegradation of flumethasone (FM) and fluocinolone acetonide (FC) was studied in solution and in the pig skin. Both glucocorticosteroids applied to the pig skin were unstable under UVB light. The photoproducts formed in the skin were the lumi-, photolumi- and andro-derivatives for FM, the same found in vitro. Instead, FC hydroperoxide formed in solution was not found in the skin: the reactivity and oxidative ability of this photoproduct towards biological substrates (lipids, proteins) seems the reason of the lack of its detection in the ex vivo model. In fact, it demonstrated to quickly oxidize amino acids and peptides, and to react with BSA both in the dark and under irradiation. Moreover, the presence in the irradiated pig skin of the FC andro-derivative, which usually forms in H-donating environment, seems consistent with the mechanism of Norrish I fragmentation followed by H-abstraction, likely from the surrounding biological substrates. These findings indicate that photoreactivity of these compounds may take place in the skin of patients exposing themselves to sunlight and is a warning about possible skin damage as a result of that. Furthermore, photolability of these drugs in the skin might cause loss of their therapeutic activity.

Activities of aldo-keto reductase 1 enzymes on two inhaled corticosteroids: implications for the pharmacological effects of inhaled corticosteroids.

Inhaled corticosteroids (ICS) are a mainstay anti-inflammatory therapy for the management of asthma. ICS are synthetic glucocorticoids that are structurally similar to the natural active human glucocorticoid cortisol. Steroid transforming enzymes of the aldo-keto reductase (AKR) family, namely AKR1D1 (5ß-steroid reductase) and AKR1C1-4 (ketosteroid reductases) are implicated in the systemic metabolism of cortisol in liver. In this study, the activities of these AKR1 enzymes on cortisol and two ICS compounds budesonide (BUD) and flunisolide (FLU) were investigated. It was found that the catalytic efficiency of AKR1D1 for the reduction of the double bond in cortisol was 4- and 10-fold higher than the catalytic efficiencies of AKR1D1 with FLU and BUD, respectively. This suggests that compared to cortisol, for which the 5ß-reduction is a major metabolic pathway, a lower degree of systemic (hepatic) metabolism of BUD and FLU via AKR1D1 takes place. In addition, BUD potently inhibited AKR1D1 and AKR1C4, the key steroid metabolizing enzymes in liver, which may disrupt endogenous steroid hormone metabolism and thus contribute to BUD-induced systemic effects. Activities of AKR1C1-3 on cortisol and the two ICS compounds (targeting the 20-keto group) suggest these enzymes may be involved in the local (lung) metabolism of these glucocorticoids.

Trabecular meshwork and lens partitioning of corticosteroids: implications for elevated intraocular pressure and cataracts.

OBJECTIVE: To determine whether adverse effects such as elevated intraocular pressure and cataracts, which are lower with dexamethasone when compared with fluocinolone acetonide or triamcinolone acetonide, may be explained in part by the differences in drug lipophilicity and partitioning of these drugs into the trabecular meshwork and lens. METHODS: The n-octanol/phosphate-buffered saline (pH 7.4) partition coefficient (log distribution coefficient [D]) and bovine/human ocular tissue partition coefficients were determined for triamcinolone, prednisolone, dexamethasone, fluocinolone acetonide, triamcinolone acetonide, and budesonide at 37°C. RESULTS: The log D of the corticosteroids ranged from 0.712 to 2.970. The ranges of tissue:PBS partition coefficients following drug incubation at 0.4, 2.0, and 10.0 µg/mL were 0.35 to 1.56, 0.30 to 2.12, and 0.30 to 1.95, respectively, for the bovine lens, 0.87 to 4.18, 0.71 to 4.40, and 0.69 to 5.86, respectively, for the human lens, and 2.98 to 9.48, 2.41 to 9.16, and 1.71 to 9.96, respectively, for the bovine trabecular meshwork. In general, tissue partitioning showed a positive correlation with log D. Dexamethasone, with lipophilicity less than triamcinolone acetonide and fluocinolone acetonide, exhibited the least amount of partitioning in the trabecular meshwork and lens among these 3 corticosteroids commonly used for treating diseases at the back of the eye. CONCLUSION: Binding of corticosteroids to the trabecular meshwork and lens increases as drug lipophilicity increases. CLINICAL RELEVANCE: Less lipophilic corticosteroids with limited partitioning to the trabecular meshwork and lens may result in reduced incidence of elevated intraocular pressure and cataracts.

Temperature and pH dependence of fluocinolone acetonide degradation in a topical cream formulation.

We investigated the degradation of fluocinolone acetonide (FA) incorporated into an oil-in-water cream base. The study examined the influence of temperature (23 to 80 degrees C) and cream pH (pH 2.3 to 6) on FA degradation rates. FA degradation followed pseudo-first-order kinetics and adhered to the Arrhenius expression over the entire temperature range investigated. At all temperatures, the pH strongly influenced the observed degradation rate constant (kobs) values, with rate minima observed near pH 4. The FA log(degradation rate)-pH profiles were consistent with a reaction mechanism requiring drug hydrolysis catalyzed by hydroxide and hydrogen ions. Taking into account both the temperature and the pH dependence of FA degradation permits calculating kobs values from the following equation: kobs = exp[22.5 - (17,200/RT)] + exp[38.7 - (22,200/RT)] x [H+] + exp[49.5 - (21,100/RT)] x [OH-] where the three bracketed terms represent Arrhenius expressions for neutral, acid-catalyzed, and base-catalyzed hydrolysis reactions. FA degradation in the cream base parallels the degradation of a related steroid (triamcinolone acetonide) in an aqueous alcohol solution. The equivalence between FA and triamcinolone acetonide kinetics in the different reaction media suggests that in the cream base, FA degradation is limited to an aqueous phase largely unperturbed by the presence of nonaqueous constituents that comprise the cream formulation.