Thiobenzothiazole-modified Hydrocortisones Display Anti-inflammatory Activity with Reduced Impact on Islet ß-Cell Function.

Glucocorticoids signal through the glucocorticoid receptor (GR) and are administered clinically for a variety of situations, including inflammatory disorders, specific cancers, rheumatoid arthritis, and organ/tissue transplantation. However, glucocorticoid therapy is also associated with additional complications, including steroid-induced diabetes. We hypothesized that modification of the steroid backbone is one strategy to enhance the therapeutic potential of GR activation. Toward this goal, two commercially unavailable, thiobenzothiazole-containing derivatives of hydrocortisone (termed MS4 and MS6) were examined using 832/13 rat insulinoma cells as well as rodent and human islets. We found that MS4 had transrepression properties but lacked transactivation ability, whereas MS6 retained both transactivation and transrepression activities. In addition, MS4 and MS6 both displayed anti-inflammatory activity. Furthermore, MS4 displayed reduced impact on islet ß-cell function in both rodent and human islets. Similar to dexamethasone, MS6 promoted adipocyte development in vitro, whereas MS4 did not. Moreover, neither MS4 nor MS6 activated the Pck1 (Pepck) gene in primary rat hepatocytes. We conclude that modification of the functional groups attached to the D-ring of the hydrocortisone steroid molecule produces compounds with altered structure-function GR agonist activity with decreased impact on insulin secretion and reduced adipogenic potential but with preservation of anti-inflammatory activity.

Design, synthesis, and local anti-inflammatory activity of 17ß-carboxamide derivatives of glucocorticoids.

Molecular docking studies were performed on 18 17ß-carboxamide steroids in order to select compounds with potential local anti-inflammatory activity. These derivatives are amides of cortienic acids (obtained from hydrocortisone, prednisolone, and methylprednisolone) with methyl or ethyl esters of six amino acids. Interactions with the glucocorticoid receptor (GR), binding energies and ligand efficiency values of these compounds were compared with dexamethasone and cortienic acid obtained from prednisolone (inactive metabolite). On the basis of molecular docking studies, seven compounds were selected and their binding affinities for the GR were predicted by use of the exponential model created in this study. Subsequently, selected compounds were synthesized in good yields by use of modified N,N'-dicyclohexylcarbodiimide (DCC)/1-hydroxybenzotriazole (HOBt) coupling procedure. Finally, the local anti-inflammatory activity of the synthesized compounds was examined by use of the croton oil-induced ear edema test. In vivo evaluation of systemic side effects as well as in silico prediction of metabolism were performed on the derivative with the best local anti-inflammatory activity. The combination of molecular docking studies and the exponential model for the GR binding affinity prediction could be used as an in silico tool for the rational design of novel 17ß-carboxamide steroids with potentially better biological profile than dexamethasone.

Hydrocortisone-loaded poly(ε-caprolactone) nanoparticles for atopic dermatitis treatment.

Atopic dermatitis (AD) is a chronic inflammatory skin condition that affects mostly young infants. The purpose of this research was to achieve a prolonged drug release and the reduction of side effects with hydrocortisone-loaded nanoparticles (NPs), for AD treatment. Poly(ε-caprolactone) (PCL) NPs were prepared by modified solvent displacement method and were characterized in terms of size, potential zeta, morphology, entrapment efficiency (EE), Fourier transform infrared (FT-IR) spectrometry and in vitro permeation studies using Franz cells. Toxicology of this nanosystem was also assessed. The obtained NPs EE showed an increased size and a more homogenous size distribution after loading and were negatively charged. EF was around 62%. In vitro release studies demonstrated a controlled release of drug from the NPs over time. FT-IR analysis showed the system stability for one week. Permeation studies revealed significant differences in the permeation of encapsulated and free hydrocortisone. In vitro toxicity studies showed no effect of drug toxicity after encapsulation. The study seems to indicate that encapsulation of hydrocortisone in PCL NPs could enable a faster control of the disease and a decrease in the side effects associated to the long-term application of corticosteroids.

Designing an anti-inflammatory and tissue-adhesive colloidal dressing for wound treatment.

Wound dressing materials are widely used to protect wounds from the external environment and to promote wound healing. However, conventional wound dressings lack tissue adhesive properties and anti-inflammatory functions, which lead to fibrosis and stricture, in cases such as gastrointestinal wounds after endoscopic surgery. In the current study, we report tissue-adhesive and anti-inflammatory properties of a wound dressing composed of corticosteroid-modified gelatin particles. Hydrocortisone (HC), which is a class of anti-inflammatory corticosteroid, was used to modify Alaska-pollock gelatin (ApGltn) to synthesize HC-modified ApGltn (HC-ApGltn). Microparticles (MPs) of HC-ApGltn were fabricated by adding ethanol in HC-ApGltn aqueous solution and performing thermal crosslinking (TC) without the use of toxic surfactants and crosslinking reagents. Modification of ApGltn with hydrophobic HC containing cholesterol backbone structure improved its adhesion strength to gastric submucosal tissues under wet conditions owing to hydrophobic interactions. This retention of adhesive property under wet conditions allows for stable protection of wounds from the external environment. We found that HC-ApGltn MPs were taken up by macrophages and they effectively suppressed morphological changes of LPS-activated macrophages and the expression level of the inflammatory cytokine. Robust tissue adhesive and anti-inflammatory MPs may serve as an advanced wound dressing that can protect wounds and suppress inflammatory responses for promoting wound healing.

Spectroscopic evaluation of synthesized 5ß-dihydrocortisol and 5ß-dihydrocortisol acetate binding mechanism with human serum albumin and their role in anticancer activity.

Our study focus on the biological importance of synthesized 5ß-dihydrocortisol (Dhc) and 5ß-dihydrocortisol acetate (DhcA) molecules, the cytotoxic study was performed on breast cancer cell line (MCF-7) normal human embryonic kidney cell line (HEK293), the IC50 values for MCF-7 cells were 28 and 25 µM, respectively, whereas no toxicity in terms of cell viability was observed with HEK293 cell line. Further experiment proved that Dhc and DhcA induced 35.6 and 37.7% early apoptotic cells and 2.5, 2.9% late apoptotic cells, respectively, morphological observation of cell death through TUNEL assay revealed that Dhc and DhcA induced apoptosis in MCF-7 cells. The complexes of HSA-Dhc and HSA-DhcA were observed as static quenching, and the binding constants (K) was 4.7 ± .03 × 104 M-1 and 3.9 ± .05 × 104 M-1, and their binding free energies were found to be -6.4 and -6.16 kcal/mol, respectively. The displacement studies confirmed that lidocaine 1.4 ± .05 × 104 M-1 replaced Dhc, and phenylbutazone 1.5 ± .05 × 104 M-1 replaced by DhcA, which explains domain I and domain II are the binding sites for Dhc and DhcA. Further, FT-IR, synchronous spectroscopy, and CD results revealed that the secondary structure of HSA was altered in the presence of Dhc and DhcA. Furthermore, the atomic force microscopy and transmission electron microscopy showed that the dimensions like height and molecular size of the HSA-Dhc and HSA-DhcA complex were larger compared to HSA alone. Detailed analysis through molecular dynamics simulations also supported greater stability of HSA-Dhc and HSA-DhcA complexes, and root-mean-square-fluctuation interpreted the binding site of Dhc as domain IB and domain IIA for DhcA. This information is valuable for further development of steroid derivative with improved pharmacological significance as novel anti-cancer drugs.

Cationic lipid-conjugated hydrocortisone as selective antitumor agent.

Hydrocortisone, the endogenously expressed steroidal, hormonal ligand for glucocorticoid receptor (GR), is body's natural anti-inflammatory and xenobiotic metabolizing agent. It has both palliative as well as adverse effects in different cancer patients. Herein, we show that conjugation product of C16-carbon chain-associated cationic lipid and hydrocortisone (namely, HYC16) induces selective toxicity in cancer (e.g. melanoma, breast cancer and lung adenocarcinoma) cells with least toxicity in normal cells, through induction of apoptosis and cell cycle arrest at G2/M phase. Further, significant tumor growth inhibition was observed in syngeneic melanoma tumor model with considerable induction of apoptosis in tumor-associated cells. In contrast to hydrocortisone, significantly higher anti-angiogenic behavior of HYC16 helped in effective tumor shrinkage. This is the first demonstration to convert natural hormone hydrocortisone into a selective bioactive entity possessing anti-tumor effect.

New applications of carbonylmetalloimmunoassay (CMIA): a non-radioisotopic approach to cortisol assay.

A non-isotopic heterogeneous competitive immunoassay procedure, carbonylmetalloimmunoassay (CMIA) has been applied to the assay of cortisol. The organometallic tracers employed were two stereoisomers (Z and E) of certain cobalt carbonyl complexes of cortisol which have strong, characteristic v(CO) absorptions in the infrared, detectable by Fourier transform infrared (FT-IR) spectroscopy at the femtomole level. The separation of the free and bound organometallic-labelled fractions was achieved by solvent extraction with isopropyl ether. Complete characterization (i.e., dilution, competition and standard curves) of two different polyclonal anticortisol antibodies was possible using the CMIA method. Identical titre values were obtained for the two different stereoisomers used as tracers. In terms of the standard curves, however, the isomers behaved differently depending on which batch of antibody was used. When the best antibody/tracer pair (30 pmol of E isomer; anticortisol 1 antibody) was employed, we obtained a B/B(o)value at 50% of 42 +/- 2.12 pmol and a coefficient of variation of 5%. Finally, preliminary results of a CMIA analysis of plasma cortisol from a patient indicated that reliable and reproducible assays are possible for amounts as small as 50 microliters of serum.

Synthesis of a deuterium-labeled cortisol for the study of its rate of 11 beta-hydroxy dehydrogenation in man.

11 beta-Hydroxy dehydrogenation of cortisol to cortisone is specifically impaired in the syndrome of apparent mineralocorticoid excess. This defect bears on the pathogenesis of the disorder by unmasking the potential mineralocorticoid agonism of unmetabolized cortisol at or near mineralocorticoid target tissues. A specific index of this defect is provided by measurement of the formation of tritiated water following the administration of [3H]11 alpha-cortisol. We have explored the use of a non-radioactive tracer to follow this unidirectional dehydrogenation reaction but because of the relatively lower sensitivity of measurement of 2H2O compared to 3H2O in body fluids, use of the corresponding [2H]11 alpha-cortisol was not feasible. We have devised instead a method incorporating additional deuterium atoms into cortisol to measure unidirectional 11 beta-hydroxy dehydrogenation not by the formation of labeled water but by the determination of the dehydrogenated cortisol product from its residual deuterium content. Cortisol-d4 metabolized to cortisone-d3 is conveniently measured by the techniques of organic mass spectrometry. The synthesis of cortisol-9 alpha, 11 alpha, 12 alpha 12 beta-d4 and the validation of its isotopic distribution by mass spectrometry and nuclear magnetic resonance is described.

The synthesis of [1,2,3,4-13C] cortisol.

The preparation of [1,2,3,4-13C] cortisol 21 acetate by total synthesis is described. The C labels are introduced in a way analogous to the one described by us previously for the synthesis of testosterone and estradiol. The cortisol dihydroxyacetone side chain was elaborated using known methods. The 11 beta-hydroxyl function was introduced by addition of hypobromous acid to a 9-double bond followed by reductive debromination. 13C-labeled cortisol can be used as a tracer for the determination of cortisol production rates.

Preparation of europium-labeled derivatives of cortisol for time-resolved fluoroimmunoassays.

Cortisol 3-(O-carboxymethyl)oxime, 6- and 21-hemisuccinoxycortisol, and cortisol 7-carboxyethyl thio-ether were synthesized. These carboxyl derivatives were labeled using a described general labeling method with a europium chelate. The labeled steroids were tested in a competitive time-resolved fluoroimmunoassay using antibodies raised against cortisol. Only the site-homologous antigen-antibody pairs underwent immunoreaction and gave satisfactory calibration curves.

Convenient synthesis of 18-hydroxylated cortisol and prednisolone.

18,20-Epoxy-11 beta,17 alpha,20 beta,21-tetrahydroxypregn-4-en-3-one was synthesized by the application of hypoiodite reaction to the cortisol acetonide. The intermediary 18-iodo derivative was converted to the 11-oxo steroid by chromic acid prior to silver ion-assisted solvolysis. Removal of the protective group with hydrochloric acid was finally carried out to give the desired 11 beta,17 alpha,18,21-tetrahydroxypregn-4-ene-3,20-dione as the hemiacetal form. 18,20-Epoxy-11 beta-17 alpha,20 beta,21- tetrahydroxypregna-1,4-dien-3-one was also prepared from prednisolone through a similar reaction sequence.

Synthesis of multi-labeled cortisols and cortisones with (2)H and (13)C for study of cortisol metabolism in humans.

A method is described for the preparation of multi-labeled cortisol and cortisone with (13)C and (2)H via the indan synthon method, starting from chiral 11-oxoindanylpropionic acid. [1, 3-(13)C(2)]Acetone was used for the syntheses of [1,2,4, 19-(13)C(4)]cortisol (cortisol-(13)C(4)) and [1,2,4, 19-(13)C(4)]cortisone (cortisone-(13)C(4)), and [1,3-(13)C(2),1,1,1, 3,3,3-(2)H(6)]acetone was for [1,2,4,19-(13)C(4),1,1,19,19, 19-(2)H(5)]cortisol (cortisol-(13)C(4),(2)H(5)) and [1,2,4, 19-(13)C(4),1,1,19,19,19-(2)H(5)]cortisone (cortisone-(13)C(4), (2)H(5)). The chemical shifts for the (13)C and (1)H NMR spectra of cortisol and cortisone were fully assigned.

Enzyme immunoassay for cortisol in serum using cortisol 21-amine.

Cortisol 21-amine (21-amino-11beta,17-dihydroxy-4-pregnene-3,20-dione) was prepared and an enzyme immunoassay for cortisol in serum was established using cortisol 21-amine conjugated with alkaline phosphatase. The minimal amount of cortisol detected was 1ng/tube and the measurable range was from 1 to 80 microgram/d1, using 10 mu 1 of serum sample. This enzyme immunoassay satisfied the standard criteria of dilution, accuracy and precision. The values correlated well with those obtained by radioimmunoassay. This enzyme immunoassay is applicable to the routine determination of serum cortisol in any clinical laboratory. Cortisol 21-amine was found to be a useful derivative for preparing cortisol-enzyme conjugate in enzyme immunoassay.

A simple synthesis of 11,19-oxidosteroids.

The photochemical hypoiodination of cortisol acetonide gave a mixture of 18-iodocortisol acetonide and of the 11 beta,19-oxidoderivative. The proportion of the two products was slightly modified by the reaction temperature. Deprotection of the acetonide group of the 11 beta,19-oxidoderivative gave 11 beta,19-oxido-17 alpha,21-dihydroxy-4-pregnen-3,20-dione which led to the formation of 11 beta,19-oxido-4-androsten-3,17-dione upon treatment with sodium bismutate.

Syntheses of stable isotope-labeled 6 beta-hydroxycortisol, 6 beta-hydroxycortisone, and 6 beta-hydroxytestosterone.

A method is described for the preparation of two types of multi-labeled 6 beta-hydroxycortisol containing either five deuterium atoms at C-19 methyl and C-1 methylene or four 13C atoms at C-1, C-2, C-4, and C-19 in addition to the five deuterium atoms for use as analytical internal standards for gas chromatography-mass spectrometry (GC-MS). BMD derivatives of [1,1,19,19,19-2H(5)]cortisone and [1,2,4,19-13C(4),1,1,19,19,19-2H(5)]cortisone (cortisone-2H(5)-BMD and cortisone-13C(4),2H(5)-BMD) were first synthesized via indan synthon method starting from optical active 11-oxoindanylpropionic acid and labeled isopropenyl anion ([1,1,3,3,3-2H(5)]- or [1,3-13C(2),1,1,3,3,3-2H(5)]isopropenyl anion). The labeled isopropenyl anion was prepared from commercially available [1,1,1,3,3,3-2H(6)]- or [1,3-13C(2),1,1,1,3,3,3-2H(6)]acetone. Ultraviolet (UV) irradiated autoxidation at C-6 position of 3-ethyl-3,5-dienol ether derivatives of the labeled cortisone-BMDs gave 6 beta-hydroxy-[1,1,19,19,19-2H(5)]cortisone-BMD and 6 beta-hydroxy-[1,2,4,19-13C(4),1,1,19,19,19-2H(5)]cortisone-BMD, respectively, as a mixture of 6 beta- and 6 alpha-epimers in a ratio of 4:1. Separation of 6 beta- and 6 alpha-epimers by thin-layer chromatography (TLC) and subsequent hydrolysis of the BMD group at C-17 gave pure labeled 6 beta-hydroxycortisone. After protecting the keto group at C-3 of the labeled 6 beta-hydroxycortisone-BMD as semicarbazone, reduction of 11-keto group with NaBH(4) and subsequent removal of the C-3 and C-17 protecting groups gave 6beta-hydroxy-[1,1,19,19,19-2H(5)]cortisol (6 beta-hydroxycortisol-2H(5)) and 6 beta-hydroxy-[1,2,4,19-13C(4),1,1,19,19,19-2H(5)]cortisol (6 beta-hydroxycortisol-13C(4),2H(5)), respectively, as a mixture of 6 beta- and 6 alpha-epimers (6 beta:6 alpha=4.4:1). The isotopic compositions of 6 beta-hydroxycortisol-2H(5) and 6 beta-hydroxycortisol-13C(4),2H(5) were 90.9 and 92.1 at.%, respectively. Furthermore, 6 beta-hydroxy-[1 alpha,16,16,17 alpha-2H(4)]testosterone was synthesized by the UV irradiated autoxidation at C-6 position of 3-ethyl-3,5-dienol ether derivative of deuterium-labeled testosterone ([1 alpha,16,16,17 alpha-2H(4)]testosterone) obtained by using catalytic deuteration and hydrogen-deuterium exchange reactions.

Synthesis and analgesic effects of kyotorphin-steroid linkers.

Kyotorphin (KTP, H-Tyr-Arg-OH) was covalently bonded with hydrocortisone or estrone to form the corresponding hydrocortisone-21-O-yl-succinyl-Tyr-ArgOH or estrone-3-O-yl-acyl-Tyr-Arg-OH. Their analgesic activities were investigated using the tail flick test. The potency of the two linkers were significantly higher than that of KTP and the mixture of KTP and hydrocortisone or estrone in the CNS and/or the periphery administration.

Solution kinetics of a water-soluble hydrocortisone prodrug: hydrocortisone-21-lysinate.

Hydrocortisone-21-lysinate was synthesized as an amino acid prodrug of hydrocortisone to serve as a substrate for brush border aminopeptidases. This strategy was developed to demonstrate that an improvement in oral absorption could be obtained through reconversion in vivo. The aqueous stability of hydrocortisone-21-lysinate was studied over the pH range 3-8 at 25 degrees C. Reversible acyl migration of the lysine group between the 21- and 17-position hydroxyl groups was observed as well as hydrolysis. The observed half-life for direct hydrolysis of hydrocortisone-21-lysinate is 40 d at pH 3 and 30 min at pH 7. The relative instability at pH 7 is probably due to electrostatic stabilization of the negatively charged tetrahedral intermediate by the protonated amino groups.

[The synthesis and immunosuppressive effects of steroid-peptide linkers].

Hydrocortisone was coupled with urotoxin tripeptide UTP-A, UTP-B and UTP-C respectively yielding 4 linkers. Their bioactivities such as prolongation of heterotopic transplanted cardiac tissue survival, inhibitory effects on phagocytosis of mouse peritoneal macrophages and the influence on Con A induced proliferation of spleen lymphocytes of mouse were observed. Compared with UTP-A, UTP-B, UTP-C or hydrocortisone the linkers were more potent immunosuppressants. The results suggest that the linker of steroid-peptide may simulate the permissive action.

Detección de cortisol en pelo como biomarcador de estrés crónico en perros de trabajo de las FAS / Hair cortisol detection as biomarker of chronic stress in military working dogs

JUSTIFICACIÓN: La medición de cortisol en pelo, es una técnica no invasiva que proporciona una imagen retrospectiva de su acumulación durante un periodo de tiempo prolongado. Objetivo: Determinar los niveles de cortisol en pelo en perros de trabajo como biomarcador del estrés crónico, así como el efecto de factores como la estacionalidad y el despliegue en operaciones internacionales. Diseño: El estudio se realizó en el Laboratorio de Investigación Aplicada, con un total de 24 perros del ET sometidos a distintas condiciones ambientales. Metodología: Las muestras de pelo se analizaron mediante un ensayo tipo ELISA (Enzyme-LinkedImmuno-SorbentAssay) para medir la concentración de cortisol, previa extracción alcohólica. Resultados: La concentración media de cortisol fue significativamente superior en primavera respecto al verano y no se encontraron efectos significativos debidos al despliegue. Conclusión: El presente trabajo desvela una concentración diferencial en los niveles de cortisol en pelo de perros de trabajo debido a la estación del año

JUSTIFICACIÓN: The measurement of hair cortisol, is a noninvasive technique that provides a retrospective of its accumulation over a period of time. OBJECTIVE: To investigate the levels of cortisol in dogs as a biomarker of chronic stress, and the effect of factors such as season and deployment. DESIGN: The study was conducted in the LIA, with a total of 24 dogs subjected to differential environmental conditions. METHODS: Hair samples were analyzed by ELISA for measuring cortisol concentration after alcoholic extraction. RESULTS: The mean concentration of cortisol in dogs was significantly higher in spring than summer, and no significant effects of deployment were found. CONCLUSION: This study reveals different levels of cortisol concentration in hair dog's between seasons