Hair removal and bioavailability of chemicals: Effect of physicochemical properties of drugs and surfactants on skin permeation ex vivo.

Cosmetic hair removal procedures are everyday routines in our society. However, it is unclear if such routines lead to increased uptake of applied substances such as drugs or formulation compounds, potentially resulting in skin irritation or sensitization. The aim of this study was to elucidate the effect of common depilation and epilation methods on skin penetration of two surfactants and four model drugs of different physicochemical properties using the porcine ear model. It should be elucidated whether the substances' skin penetration behavior would be affected by hair removal procedures and if potential effects would be related to their polarity. Confocal Raman spectroscopy revealed no effect of hair removal on total penetration depths of SDS and sulfathiazole. Significantly higher relative penetrated amounts within 0-6 µm of stratum corneum depth were found for SDS after dry shaving, depilatory cream and waxing and for sulfathiazole after all depilation methods and partly after epilation. ATR-FTIR spectroscopy revealed no effect of hair removal on the penetration depth of lecithin LPC80, but higher relative amounts at the skin surface after wet shaving and electric epilation. Diffusion cell experiments using a lecithin-based microemulsion as carrier system for fluconazole, fludrocortisone acetate and flufenamic acid showed higher cumulative amounts, higher drug fluxes and shorter lag times for the more lipophilic drugs for some of the methods, but only shorter lag times in some cases for fluconazole. In summary, the observed effects appeared to depend on drug polarity and experimental setup.

Fludrocortisone stimulates erythropoietin production in the intercalated cells of the collecting ducts.

Erythropoietin has been thought to be secreted to plasma soon after the production because of the difficulty of Western blot analysis and immunohistochemistry. We established the new methods of Western blot analysis and immunohistochemistry. Using the new methods, we investigated the effects of aldosterone and fludrocortisone, an analogue of aldosterone on erythropoietin mRNA and protein production by the kidneys. Aldosterone stimulated Epo and HIF2α mRNA expressions in tubule suspensions and microdissected medullary thick ascending limbs and outer medullary collecting ducts. Western blot analysis showed a recombinant erythropoietin at 34-45 kDa and kidney erythropoietin at 36-40 and 42 kDa, both of which shifted to 22 kDa by deglycosylation. Erythropoietin protein expression was observed in the nephrons but not in the interstitial cells in control condition. Fludrocortisone stimulated erythropoietin mRNA and protein expressions in the distal nephrons, particularly in the intercalated cells of the collecting ducts. These data show that erythropoietin is produced by the nephrons by the regulation of renin-angiotensin-aldosterone system and not by the renal interstitial cells in control condition.

The urinary steroidome of treated children with classic 21-hydroxylase deficiency.

Monitoring treatment of children with classic congenital adrenal hyperplasia (CAH) is difficult and biochemical targets are not well defined. We retrospectively analysed 576 daily urinary steroid hormone metabolite profiles determined by gas chromatography-mass spectrometry of 150 children aged 3.0-17.9 years with classic 21-hydroxylase deficiency (21-OHD) on hydrocortisone and fludrocortisone treatment. Daily urinary excretion of glucocorticoid-, 17α-hydroxyprogesterone (17-OHP)-, and androgen metabolites as well as growth and weight gain are presented. Children with classic CAH exhibited increased height velocity during prepubertal age, which was then followed by diminished growth velocity during pubertal age until final height was reached. Final height was clearly below the population mean. 11ß-Hydroxyandrosterone was the dominant urinary adrenal-derived androgen metabolite in CAH children. Adrenarche is blunted in children with CAH under hydrocortisone treatment and androgen metabolites except 11ß-hydroxyandrosterone were suppressed. Cortisol metabolite excretion reflected supraphysiological hydrocortisone treatment dosage, which resulted in higher body-mass-indices in children with CAH. Reference values of daily urinary steroid metabolite excretions of treated children with CAH allow the clinician to adequately classify the individual patient regarding the androgen-, 17-OHP-, and glucocorticoid status in the context of the underlying disorder. Additionally, urinary 21-OHD-specific reference ranges will be important for research studies in children with CAH.

Corticosteroid Fludrocortisone Acetate Targets Multiple End Points in Zebrafish (Danio rerio) at Low Concentrations.

Synthetic corticosteroids may pose an environmental risk to fish. Here, we describe multiend point responses of adult zebrafish (8 months old) upon 21-day exposure to a commonly prescribed corticosteroid, fludrocortisone acetate (FLU), at concentrations between 0.006 and 42 µg/L. No remarkable reproductive impacts were observed, while physiological effects, including plasma glucose level and blood leukocyte numbers were significant altered even at 42 ng/L. Ovary parameters and transcriptional analysis of hypothalamic-pituitary-gonadal-liver axis revealed negligible effects. Significant alterations of the circadian rhythm network were observed in the zebrafish brain. Transcripts of several biomarker genes, including per1a and nr1d1, displayed strong transcriptional changes, which occurred at environmental relevant concentrations of 6 and 42 ng/L FLU. Importantly, the development and behavior of F1 embryos were significant changed. Heartbeat, hatching success and swimming behavior of F1 embryos were all increased even at 6 and 42 ng/L. All effects were further confirmed by exposure of eleuthero-embryos. Significant transcriptional changes of biomarker genes involved in gluconeogenesis, immune response and circadian rhythm in eleuthero-embryos confirmed the observations in adult fish. Hatching success, heartbeat, and swimming activity were increased at 81 ng/L and higher, as with F1 embryos. These results provide novel insights into the understanding of potential environmental risks of corticosteroids.

The Role of Fludrocortisone in Cognition and Mood in Patients with Primary Adrenal Insufficiency (Addison's Disease).

BACKGROUND: Primary adrenal insufficiency (AI) requires hormone replacement therapy with fludrocortisone and hydrocortisone stimulating glucocorticoid (GR) and mineralocorticoid receptors (MR). Evidence from animal and human studies shows that MR function is crucial for cognitive function and mood. Regarding patients with AI, very little is known about the role of MR in cognitive function and mood. METHODS: A repeated-measures within-subject design was used to determine whether cognitive function and mood are related to MR occupation in patients with AI. Intraindividually, patients were examined twice, with 1 week between testing days: once with fludrocortisone (high MR occupation) and once without fludrocortisone (low MR occupation). All patients kept their stable regimen of hydrocortisone. The assessment of cognitive function included executive function, attention, and verbal, visuospatial and working memory. Additionally, mood and blood pressure were measured. RESULTS: Verbal memory improved significantly during high MR occupation (after fludrocortisone intake) compared to low MR occupation [without fludrocortisone, t(29) = -2.1, p = 0.046]. There were trend level differences in the Number-Combination test [t(29) = -1.9, p = 0.074] and in the Stroop interference task [t(29) = -1.9, p = 0.068]. No significant differences in visuospatial and working memory were found. Furthermore, the current mood state was better during high MR occupation compared to low MR occupation [t(29) = -2.4, p = 0.023] as was diastolic blood pressure [F(2, 29) = 3.6, p = 0.07]. CONCLUSIONS: Cognitive function and mood in patients with AI depend in part on MR occupation. Because the medium effect size indicates a potential clinical significance, further studies should systematically examine which dosages of fludrocortisone are associated with optimal cognitive function and mood in AI patients.

Auto-amplification of cortisol actions in human carotid atheroma is linked to arterial remodeling and stroke.

High cortisol and aldosterone levels increase cardiovascular risk, but the respective roles of each hormone within the arterial wall remain controversial. We tested the hypothesis that cortisol production within the arterial wall may contribute to atherosclerotic remodeling and act through illicit activation of the mineralocorticoid receptor (MR). Gene expression studies of the corticoid system components and marker genes of the atherosclerotic process in human carotid atheroma plaque and nearby macroscopically intact tissue (MIT) were considered together with clinical data and compared with pharmacological stimulations of human vascular smooth muscle cells (VSMCs) in contractile or lipid-storing phenotypes. The components of corticoid production and action were present and active within the human carotid wall and VSMCs. Atheroma plaque and lipid-storing VSMCs expressed 11ß-hydroxysteroid deshydrogenase-1 (11ß-HSD1) at two- to tenfold higher levels than MIT or contractile VSMCs. The 11ß-HSD1 expression was stimulated by cortisol and cortisone, especially in lipid-storing VSMCs. MR mRNA level was lower in atheroma and lipid-storing VSMCs and downregulated via MR by fludrocortisone and cortisol. Cortisol upregulated collagen1 and MCP-1 mRNAs via the glucocorticoid receptor (GRα), in both VSMC phenotypes, whereas fludrocortisone stimulated the collagen1 expression only in lipid-storing VSMCs. The GRα mRNA level in MIT was higher in patients with previous stroke and correlated positively with the collagen1 mRNA but negatively with diastolic blood pressure. Local cortisol production by 11ß-HSD1, and its action via high parietal GRα could be relevant from the first step of atherosclerotic remodeling and auto-amplify with transdifferentiation of VSMCs during atheroma progression.

Influence of drug content, type of semi-solid vehicle and rheological properties on the skin penetration of the model drug fludrocortisone acetate.

Throughout Europe, topical creams containing corticosteroids are diluted with various neutral cream bases to meet the specific needs of patients. Even though this practice has been common for years, its effect has not been thoroughly investigated and so the effectiveness of the diluted topical steroidal creams is difficult to predict. In the present study, the model drug fludrocortisone acetate was incorporated into three cream bases of different hydrophilicity that are commonly used in Austria. Different final drug concentrations were chosen for comparative studies. Additionally, a semi-solid preparation developed by our group was investigated for comparison. These formulations were tested in diffusion and tape stripping experiments. Diffusion cell studies showed that changes in drug concentration do not necessarily change the skin permeation behaviour in vitro. The tape stripping protocol was successfully optimised for investigation of semi-solid preparations to provide reproducible and accurate results despite the challenges of investigating semi-solid formulations. The results showed that tape stripping experiments are more suitable to elucidate subtle differences between formulations. The composition of the cream bases exhibited stronger effects on the skin penetration of the steroidal drug irrespective of its concentration than the rheological properties. No correlation between formulation viscosity and skin penetration was found.

Effect of γ-cyclodextrin on the in vitro skin permeation of a steroidal drug from nanoemulsions: impact of experimental setup.

Numerous reports on the enhancement effect of cyclodextrins (CDs) on the skin permeation of dermally applied drugs exist, the majority of which is based on in vitro diffusion cell studies. The specific experimental setup of such studies may skew the obtained results, which is rarely discussed in the context of CD studies. Thus, the aim of this work was to conduct a systematic in vitro investigation of the permeation enhancement potential of γ-CD on a steroidal drug from a nanoemulsion. The role of critical diffusion cell parameters such as the dose of application, occlusive conditions, the nature of the receptor medium and the skin thickness were investigated. The results showed that significantly enhanced skin permeation rates of fludrocortisone acetate were indeed caused by 1% (w/w) of γ-CD at both finite and infinite dose conditions. At 0.5% (w/w) of γ-CD, significant enhancement was only achieved at infinite dose application. Additional in vitro tape stripping experiments confirmed these tendencies, but the observed effects did not reach statistical significance. It may be concluded that the full permeation enhancement potential of the CD as observed in the Franz-cell setup can only be realised at infinite dose conditions while preserving the formulation structure.

A bulking agent may lead to adrenal insufficiency crisis: a case report.

Adrenal insufficiency is a life-threatening disorder which must be treated with glucocorticoid replacement and needs permanent dose adjustment during patient's different somatic situations. Insufficient glucocorticoid doses result in adrenal crisis and must be treated with intravenous hydrocortisone. The patient was known with Adrenal insufficiency and was treated optimally with fludrocortisone and prednisolone since seven years with no history of adrenal crisis. The patient was admitted with abdominal pain, weakness, fatigue and nausea developed 3-4 days after taking psyllium, a bulking agent, prescribed by a surgeon to diagnose anal fissure. Detailed medical history, physical examinations, laboratory and imaging examinations did not approve any other cause of adrenal crisis. Psyllium may interfere with gastrointestinal absorption of prednisolone and/or fludrocortisone and trigger acute adrenal crisis in patients with adrenal insufficiency.

Development of sucrose stearate-based nanoemulsions and optimisation through γ-cyclodextrin.

Nanoemulsions aimed at dermal drug delivery are usually stabilised by natural lecithins. However, lecithin has a high tendency towards self-aggregation and is prone to chemical degradation. Therefore, the aim of this study was to develop nanoemulsions with improved structure and long-term stability by employing a natural sucrose ester mixture as sole surfactant. A thorough comparison between the novel sucrose stearate-based nanoemulsions and corresponding lecithin-based nanoemulsions revealed that the sucrose ester is superior in terms of emulsifying efficiency, droplet formation as well as physical and chemical stability. The novel formulations exhibited a remarkably homogeneous structure in cryo TEM investigations, as opposed to the variable structure observed for lecithin-based systems. The in vitro skin permeation rates of lipophilic drugs from sucrose stearate nanoemulsions were comparable to those obtained with their lecithin-based counterparts. Furthermore, it was observed that addition of γ-cyclodextrin led to enhanced skin permeation of the steroidal drug fludrocortisone acetate from 9.99±0.46 to 55.10±3.67 µg cm(-2) after 24 h in the case of sucrose stearate-based systems and from 9.98±0.64 to 98.62±24.89 µg cm(-2) after 24 h in the case of lecithin-based systems. This enhancement effect was significantly stronger in formulations based on lecithin (P<0.05), which indicates that synergistic mechanisms between the surfactant and the cyclodextrin are involved. Cryo TEM images suggest that the cyclodextrin is incorporated into the interfacial film, which might alter drug release rates and improve the droplet microstructure.

Impaired renal 11 beta-oxidation of 9 alpha-fluorocortisol: an explanation for its mineralocorticoid potency.

9 alpha-Fluorocortisol (9 alpha FF) is about 200 times more potent as a mineralocorticoid than cortisol (F) in man, although it binds with the same affinity as F and aldosterone to the human mineralocorticoid receptor. The low mineralocorticoid activity of F has been shown to be due to its rapid conversion by the kidney to cortisone (E), which does not bind to the receptor. Therefore, we compared the conversion of F to E with that of 9 alpha FF to 9 alpha-fluorocortisone (9 alpha FE) by 11-hydroxysteroid dehydrogenases in man in vivo and in vitro. Single oral doses of 9 alpha FF, 9 alpha FE, and F were given to normal males, and the excretion of free 9 alpha FF, 9 alpha FE, F, and E was measured in urine. Human kidney and liver slices were incubated with unlabeled steroids, and the free 11-hydroxy- and 11-oxosteroids were quantitated after high performance liquid chromatography separation by UV absorption. Oral F (5 mg) is excreted 70% as free E and 30% as free F (percentage of free steroids only). Oral 9 alpha FF (5 mg) is excreted 90% as free 9 alpha FF and 10% as free 9 alpha FE. Free 9 alpha FF excretion is 14 times greater than that of F after ingesting an identical dose. Oral 9 alpha FE (4 mg) is also excreted 90% as 9 alpha FF and 10% as 9 alpha FE. Kidney slices convert F much faster to E than 9 alpha FF to 9 alpha FE. The conversion of 9 alpha FE to 9 alpha FF is, on the contrary, much faster than that of E to F. Thus, the equilibrium of the reaction is on the 11-oxo side for F/E and on the 11-hydroxy side for 9 alpha FF/9 alpha FE. The interconversion of both pairs of steroids is inhibited by glycyrrhetinic acid in a dose-dependent manner. Liver slices do not measurably convert 9 alpha FF to 9 alpha FE, but do rapidly convert 9 alpha FE into 9 alpha FF. Reflecting this negligible conversion of 9 alpha FF to 9 alpha FE and the low plasma-protein binding of 9 alpha FF, free urinary 9 alpha FF excretion is much higher than that of F after the same oral dose.(ABSTRACT TRUNCATED AT 400 WORDS)

Non-classical receptors for aldosterone in plasma membranes from pig kidneys.

Rapid, nongenomic in vitro effects of aldosterone on intracellular electrolytes, cell volume and the sodium-proton antiporter have been found in human mononuclear leukocytes (HML), as have related membrane receptors. In the present study, binding of 125I-labeled aldosterone to plasma membrane preparations from pig kidneys was studied, since nongenomic in vitro effects of aldosterone have also been described in cultured kidney cells. In this preparation, binding of aldosterone shares important features with both functional and binding data in HML. These include a very low apparent Ki of approximately 0.1 nM for aldosterone, a high turnover rate and binding selectivity for aldosterone and fludrocortisone. Desoxycorticosterone acetate and corticosterone show intermediate affinity, with apparent Ki values of approximately 1 and 100 nM, with hydrocortisone even less active. Thus binding of aldosterone to kidney plasma membranes is compatible with the major features of its nongenomic renal effects.

Cortisol metabolism by human liver in vitro--IV. Metabolism of 9 alpha-fluorocortisol by human liver microsomes and cytosol.

The oxidative and reductive biotransformations of 9 alpha-fluorocortisol (fluorocortisol) by human liver microsomes and cytosol have been characterized. 9 alpha-Fluorination greatly simplified cortisol metabolism in microsomes: dehydrogenation of the 11 beta-hydroxyl group and A-ring (4-ene-5 beta and 3 alpha-keto) reduction, the principle pathways, were completely blocked. Fluorocortisol was essentially metabolized by the remaining pathways, 20 beta-reduction and 6 beta-hydroxylation. In cytosol, 20 beta-reduction replaced the A-ring reduction of cortisol as the sole biotransformation. The major structure-metabolism relationships of fluorocortisol in man, i.e. complete and extensive inhibition of 11 beta-dehydrogenation and 4-ene-5 beta-reduction, respectively, were attributed to hepatic enzyme systems. Their mechanistic basis is discussed with reference to the electronic and conformational changes induced by 9 alpha-fluorination.

Control of hyperkalemia with fludrocortisone in a patient with systemic lupus erythematosus.

We describe the use of fludrocortisone in a patient with systemic lupus erythematosus, who initially presented with moderate renal insufficiency, high serum potassium, metabolic acidosis, low urine pH, and a high urinary anion gap indicating tubular dysfunction. Renal histology revealed membranous glomerulonephritis and interstitial inflammation. During the course of the disease the patient developed persistent severe hyperkalemia. Dietary potassium restriction and therapy with diuretics combined with cation exchange resins failed to decrease potassium levels sufficiently. Drug therapy including cyclosporin A, enalapril, atenolol, phenytoin, necessary to control active disease and severe hypertension contributed to elevated potassium levels. Effective correction of hyperkalemia was achieved by fludrocortisone treatment. We conclude that fludrocortisone is a potent therapeutic approach for selected patients suffering from life threatening hyperkalemia, in whom potassium elevating drugs cannot be withdrawn.

Adverse effect of phenytoin on mineralocorticoid replacement with fludrocortisone in adrenal insufficiency.

Two patients with longstanding adrenal insufficiency developed severe mineralocorticoid deficiency during concomitant phenytoin treatment. A 64-year-old man with primary adrenal insufficiency of 41 years duration was treated with phenytoin for an acute seizure disorder. He subsequently developed mineralocorticoid insufficiency despite taking his customary dosages of cortisone acetate and fludrocortisone. This responded to volume repletion and increased fludrocortisone requirement from 0.05 mg to 0.4 mg daily, which decreased to the former amount following discontinuation of phenytoin. A 42-year-old woman with primary adrenal insufficiency of 3 years duration and a lifelong seizure disorder treated with phenytoin incurred multiple, life-threatening episodes of mineralocorticoid insufficiency. Her fludrocortisone requirement was ultimately established as 2.0 mg daily with a normal hydrocortisone requirement and clearance rate. Fludrocortisone thus appears to be another synthetic steroid whose metabolism is sensitive to drugs that increase hepatic 6-beta-hydroxylation, such as phenytoin. Treatment with these inducing drugs may markedly alter mineralocorticoid requirements in patients with primary adrenal insufficiency.

RU-26988--a new tool for the study of the mineralocorticoid receptor.

Aldosterone binds to two renal cytosol receptors, one with high affinity and low capacity (Type I or mineralocorticoid) and another with lower affinity but greater capacity (Type II or glucocorticoid receptor). One of the ways to study Type I receptors isolated from Type II receptors is to block the latter with a steroid which shows high affinity for the glucocorticoid receptor and low affinity for the mineralocorticoid receptor. Dexamethasone has been used widely for this purpose but previous investigations and this study have found that dexamethasone competes with [3H]aldosterone for Type I receptor binding with a relative activity of 26% that of aldosterone and therefore is less than ideal as a glucocorticoid receptor blocker. RU-26988 (11 beta, 17 beta-dihydroxy-17 alpha-pregnane-1,4,6-trien-20-yn-21-methyl-3-one) is a recently synthesized glucocorticoid that shows very low affinity for the mineralocorticoid receptor. RU-26988 competes with [3H]aldosterone and [3H]2 alpha-methyl-9 alpha-fluorocortisol, a powerful mineralocorticoid, for the cytosol receptor from renal slices of adrenalectomized rats with a relative potency of less than 0.5% in comparison to unlabeled aldosterone and 2 alpha-methyl-9 alpha-fluorocortisol. RU-26988 has over twice the ability of unlabeled dexamethasone to compete with [3H]dexamethasone for binding to the renal cytosol glucocorticoid receptor. Scatchard analysis of [3H]aldosterone binding to rat renal cytosol showed two receptors, one with a calculated dissociation constant (Kd) of 2.81 X 10(-9) M and a second with a calculated Kd of 3.33 X 10(-8) M. The addition of a 100-fold concentration of RU-26988 produced a single line with a Kd of 5.02 X 10(-10) M indicating that the specific blocking of the glucocorticoid receptors allows an accurate determination of the kinetic parameters of the mineralocorticoid receptor by itself. Scatchard analysis of [3H]2 alpha-methyl-9 alpha-fluorocortisol binding produced a straight line with a Kd of 3.7 X 10(-9) M, such as would be produced if it were binding to only one single class of receptors. However, when an excess of RU-26988 was added to block the glucocorticoid receptor, a different straight line was produced by Scatchard's analysis with a Kd of 3.78 X 10(-10) M. Whereas the explanation for this is not apparent, it may be that the much larger concentration of glucocorticoid receptors, for which 2 alpha-methyl-9 alpha-fluorocortisol also has a very great affinity, masks the binding to the mineralocorticoid receptors.

Tritiated 9alpha-fluorocortisol metabolism and binding in rat kidney.

Metabolism of 9alpha-fluorocortisol (9alpha-F) has been studied in rat kidney slices, homogenate, and in the isolated perfused kidney. These studies show that the rate of 9alpha-F metabolism varies depending upon the experimental conditions. The major metabolite formed, identified by mass spectrometry, is 20(xi)-dihydro-9alpha-fluorocortisol. The kidney slice experiments show that only 3H-9alpha-F and none of the metabolites bind to cytosolic receptors. In competition experiments performed with tritiated and unlabeled 9alpha-fluorocortisol, aldosterone (A), dexamethasone (DM) and triamcinolone acetonide (TA), 9alpha-F was found to bind to mineralocorticoid receptors with a lower affinity than A but also to glucocorticoid receptors with a higher affinity than A. The Scatchard plot analysis indicated that 3H-9alpha-F is characterized by KD : 8.6 X 10(-9)M and N : 1.9 x 10(-13)moles/mg of protein. In conclusion it is felt that 9alpha-F would not be a better "marker" than aldosterone for the renal mineralocorticoid receptors.