Dexamethasone and transdehydroandrosterone significantly reduce pulmonary epithelial cell injuries associated with mechanical ventilation.

Many patients who suffer from pulmonary diseases cannot inflate their lungs normally, as they need mechanical ventilation (MV) to assist them. The stress associated with MV can damage the delicate epithelium in small airways and alveoli, which can cause complications resulting in ventilation-induced lung injuries (VILIs) in many cases, especially in patients with acute respiratory distress syndrome (ARDS). Therefore, efforts were directed to develop safe modes for MV. In our work, we propose a different approach to decrease injuries of epithelial cells (EpCs) upon MV. We alter EpCs' cytoskeletal structure to increase their survival rate during airway reopening conditions associated with MV. We tested two anti-inflammatory drugs dexamethasone (DEX) and transdehydroandrosterone (DHEA) to alter the cytoskeleton. Cultured rat L2 alveolar EpCs were exposed to airway reopening conditions using a parallel-plate perfusion chamber. Cells were exposed to a single bubble propagation to simulate stresses associated with mechanical ventilation in both control and study groups. Cellular injury and cytoskeleton reorganization were assessed via fluorescence microscopy, whereas cell topography was studied via atomic force microscopy (AFM). Our results indicate that culturing cells in media, DEX solution, or DHEA solution did not lead to cell death (static cultures). Bubble flows caused significant cell injury. Preexposure to DEX or DHEA decreased cell death significantly. The AFM verified alteration of cell mechanics due to actin fiber depolymerization. These results suggest potential beneficial effects of DEX and DHEA for ARDS treatment for patients with COVID-19. They are also critical for VILIs and applicable to future clinical studies.NEW & NOTEWORTHY Preexposure of cultured cells to either dexamethasone or transdehydroandrosterone significantly decreases cellular injuries associated with mechanical ventilation due to their ability to alter the cell mechanics. This is an alternative protective method against VILIs instead of common methods that rely on modification of mechanical ventilator modes.

A- and D-Ring Structural Modifications of an Androsterone Derivative Inhibiting 17ß-Hydroxysteroid Dehydrogenase Type 3: Chemical Synthesis and Structure-Activity Relationships.

Decreasing the intratumoral androgen biosynthesis by using an inhibitor of 17ß-hydroxysteroid dehydrogenase type 3 (17ß-HSD3) is a strategy to treat prostate cancer. The androsterone (ADT) derivative 1 (RM-532-105) has shown strong inhibitory activity on 17ß-HSD3, but needs to be improved. Herein, we describe the chemical synthesis and characterization of two series of analogues to address the impact of A- and D-ring modifications on 17ß-HSD3 inhibitory activity, androgenic effect, and metabolic stability. Structure-activity relationships were generated by adding different groups at C16/C17 (D-ring diversification) or replacing the ADT backbone by a nor-androstane or an estrane backbone (A-ring diversification). D-ring derivatives were less potent inhibitors than lead compound 1, whereas steroidal backbone (A-ring) change led to identifying promising novel estrane derivatives. This culminated with potent 17ß-HSD3 inhibitors 23, 27, 31, and 33 (IC50 = 0.10, 0.02, 0.13, and 0.17 µM, respectively), which did not stimulate LAPC-4 cell proliferation and displayed higher plasma concentration in mice than lead compound 1.

Glucose-6-phosphate dehydrogenase is the target for the trypanocidal action of human steroids.

Steroids such as dehydroepiandrosterone (DHEA) and epiandrosterone (EA) exert multiple effects in mammals including the inhibition of glucose-6-phosphate dehydrogenase (G6PDH). Initially, the inhibition was considered specific for the mammalian enzyme. The beneficial effect of these steroids on infections by protists and nematodes was attributed to stimulation of the immune system. However, we showed previously that DHEA and EA also inhibit Trypanosoma brucei and T. cruzi G6PDH, with low micromolar K(i)' values, but not the enzyme from Leishmania species, and kill in vitro cultured trypanosomes. We report here that, contrary to wild-type trypanosomes, mutant bloodstream-form T. brucei cells expressing L. mexicana G6PDH are not susceptible to the steroids, proving that G6PDH is the in situ target. Moreover, bromo-derivatives of the steroids show 50-100 fold lower K(i)' values for the enzyme and display an increased potency to kill the parasites. Therefore, the compounds offer promise for use in development of parasite-selective drugs.

16-bromoepiandrosterone, an activator of the mammalian immune system, inhibits glucose 6-phosphate dehydrogenase from Trypanosoma cruzi and is toxic to these parasites grown in culture.

Glucose 6-phosphate dehydrogenase (G6PDH) catalyzes the first step of the pentose-phosphate pathway which supplies cells with ribose 5-phosphate (R5P) and NADPH. R5P is the precursor for the biosynthesis of nucleotides while NADPH is the cofactor of several dehydrogenases acting in a broad range of biosynthetic processes and in the maintenance of the cellular redox state. RNA interference-mediated reduction of G6PDH levels in bloodstream-form Trypanosoma brucei validated this enzyme as a drug target against Human African Trypanosomiasis. Dehydroepiandrosterone (DHEA), a human steroidal pro-hormone and its derivative 16α-bromoepiandrosterone (16BrEA) are uncompetitive inhibitors of mammalian G6PDH. Such steroids are also known to enhance the immune response in a broad range of animal infection models. It is noteworthy that the administration of DHEA to rats infected by Trypanosoma cruzi, the causative agent of Human American Trypanosomiasis (also known as Chagas' disease), reduces blood parasite levels at both acute and chronic infection stages. In the present work, we investigated the in vitro effect of DHEA derivatives on the proliferation of T. cruzi epimastigotes and their inhibitory effect on a recombinant form of the parasite's G6PDH (TcG6PDH). Our results show that DHEA and its derivative epiandrosterone (EA) are uncompetitive inhibitors of TcG6PDH, with K(i) values of 21.5 ± 0.5 and 4.8 ± 0.3 µM, respectively. Results from quantitative inhibition assays indicate 16BrEA as a potent inhibitor of TcG6PDH with an IC50 of 86 ± 8 nM and those from in vitro cell viability assays confirm its toxicity for T. cruzi epimastigotes, with a LD50 of 12 ± 8 µM. In summary, we demonstrated that, in addition to host immune response enhancement, 16BrEA has a direct effect on parasite viability, most likely as a consequence of TcG6PDH inhibition.

16alpha-Bromoepiandrosterone (HE2000) limits non-productive inflammation and stimulates immunity in lungs.

16alpha-Bromoepiandrosterone (HE2000) is a synthetic steroid that limits non-productive inflammation, enhances protective immunity and improves survival in clinical studies of patients with human immunodeficiency virus (HIV), malaria and tuberculosis infections. We now show that HE2000 decreased nitric oxide production by lipopolysaccharide (LPS)-stimulated RAW264.7 cells. Treatment with HE2000 also reduced non-productive inflammation associated with carrageenan-induced pleurisy and LPS-induced lung injury in mice. In the hapten-carrier reporter antigen popliteal lymph node assay, HE2000 increased absolute numbers of lymphocytes, antigen-presenting cells, hapten-specific immunoglobulin (Ig)M antibody-forming cells and shifted the interferon (IFN)-gamma/interleukin (IL)-4 balance towards IFN-gamma production. In the cystic fibrosis transmembrane conductance regulator (CFTR(-/-)) mouse model of acute Pseudomonas aeruginosa infection, treatment with HE2000 consistently reduced bacterial burden in lungs. All HE2000 effects were dose-dependent. In H1N1 infection in mice, HE2000 was safe but not effective as a monotherapy, as treatment did not effect survival. HE2000 reduced mortality related to excessive inflammation and opportunistic lung infections in animals and patients, and this might extend to those with H1N1 influenza infection.

Effects of androsterone on convulsions in various seizure models in mice.

It is believed that a deficiency of androgens, including free testosterone, may promote the development of convulsions. The present study revealed differences in the action of androsterone (AND), a major excreted metabolite of testosterone and a neurosteroid, in three commonly used seizure models in mice. AND administered intraperitoneally exhibited dose-dependent protection against tonic-clonic convulsions caused by maximal electroshock (MES) with ED(50) (effective dose(50)) of 227 mg/kg. The compound also inhibited the convulsive action of pentylenetetrazole (PTZ), increasing its CD(50) (convulsive dose(50)) for clonic convulsions from 77.2 (PTZ + saline) to 93.9 (p < 0.05) for PTZ + AND 40 mg/kg and 113.9 mg/kg (p < 0.001) for PTZ + AND 60 mg/kg. In mice pretreated with 60 mg/kg AND, the CD(50) for PTZ-induced tonic convulsions increased from 102 to 127.6 mg/kg (p < 0.01). Surprisingly, doses of 50 and 100 mg/kg AND lowered the CD(50) for kainate (KA)-induced convulsions from 40.8 to 28.7 (p < 0.05) and 25.4 mg/kg (p < 0.001), respectively. In summary, for two of the mouse seizure models, our findings confirmed previous studies that demonstrated protective activity of AND. However, the potentiation of KA-induced convulsions by AND was somewhat unexpected and suggested that AND may also possess proconvulsant activity.

Anti-inflammatory effects and changes in prostaglandin patterns induced by 7beta-hydroxy-epiandrosterone in rats with colitis.

High dose levels of dehydroepiandrosterone and its 7-hydroxylated derivatives have been shown to reduce oxidative stress and inflammatory responses in dextran sodium sulfate (DSS)-induced colitis in rats. Another endogenous steroid, 7beta-hydroxy-epiandrosterone (7beta-hydroxy-EpiA) has been shown to exert neuroprotective effects at much smaller doses. Our aims were to evaluate whether 7beta-hydroxy-EpiA pre-treatment prevents DSS-induced colitis and to determine whether the effects involve changes in anti-inflammatory prostaglandin (PG) D(2) and 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)) levels. Rats were administered 0.01, 0.1 and 1mg/kg 7beta-hydroxy-EpiA i.p. once a day for 7 days. Thereafter, colitis was induced by administration of 5% DSS in drinking water for 7 days. Levels of the PGs and the expression of cyclooxygenase (COX-2) and PG synthases were assessed during the course of the experiment. Administration of 7beta-hydroxy-EpiA caused a transient increase in COX-2 and PGE synthase expression within 6-15h and augmented colonic tissue levels of 15d-PGJ(2) levels starting at day 2. Treatment with DSS resulted in shortened colon length, depleted mucus in goblet cells and induced oxidative stress. COX-2 and mPGES-1 synthase expression were enhanced and accompanied by increased PGE(2), D(2) and 15d-PGJ(2) production. Although all dose levels of 7beta-hydroxy-EpiA reduced PGE(2) production, only the lowest dose (0.01mg/kg) of the steroid completely prevented colitis damage and tissue inflammation. 7beta-Hydroxy-EpiA pre-treatment prevents the occurrence of DSS-induced colitis through a shift from PGE(2) to PGD(2) production, associated with an early but transient increase in COX-2 expression and a sustained increase in the production of the anti-inflammatory prostaglandin 15d-PGJ(2).

5,19-cyclo-9beta,10xi-androstane-3,17-dione promotes neurotrophic factor biosynthesis in 1321N1 human astrocytoma cells and improves passive avoidance learning impairment.

Since neurotrophic factors are essential for neurons to form neuronal networks and maintain neuronal functions, neurotrophic factor-like substances or inducers of neurotrophic factors can be useful for the treatment of serious neuronal diseases such as Alzheimer's and Parkinson's diseases. In the present study, we examined an effect of 5,19-cyclo-9beta,10xi-androstane-3,17-dione (CAD) on neurotrophic factor synthesis in glial cells and scopolamine-induced impairment of learning in mice. 1321N1 human astrocytoma cells promoted secretion of certain neurotrophic factors in response to CAD with no cytotoxicity, which caused dramatic neurite outgrowth in rat pheochromocytoma (PC12) cells. In fact, CAD significantly enhanced nerve growth factor (NGF) secretion and its gene expression in 1321N1 cells, in a time and concentration-dependent manner. Because second messengers such as cAMP, inositol 1,4,5-trisphosphates and Ca(2+) induce NGF gene expression, we measured activities of adenylyl cyclase and phospholipase C and intracellular Ca(2+) concentration in 1321N1 cells. However, CAD changed neither second messenger levels. CAD enhanced the gene expression of proto-oncogene, c-fos that is one of the components of transcription factor (AP-1). In addition to those above, the in vivo effects of CAD were also examined. Although injection of muscarinic receptor antagonist scopolamine impaired passive avoidance learning in mice, pretreatment with CAD significantly reversed the adverse effect in a dose-dependent manner. Taking these results together, CAD has enormous therapeutic potential for serious neuronal diseases.

Reduction of parasite levels in patients with uncomplicated malaria by treatment with HE2000.

16alpha-Bromoepiandrosterone (HE2000) is a synthetic androstane steroid that has immune effects in pre-clinical models of malaria, tuberculosis, and infection with human immunodeficiency virus. In pilot studies, 42 patients with confirmed uncomplicated Plasmodium falciparum malaria were treated with a seven-day course of HE2000 by either buccal administration or intramuscular injection. Of the 42 patients, 41 showed a 50% reduction in blood levels of parasites, the primary endpoint of the study. Of these, 32 (76%) cleared malaria parasites below detectable levels. All febrile patients became afebrile by the end of treatment. There was no reduction in gametocyte forms. Adverse events were transient and mild to moderate in intensity. The anti-malarial response was generally similar with either the intramuscular or buccal routes of administration. HE2000 shows a safety profile and pharmacologic activity worthy of further investigation to understand its role in the treatment of malaria, perhaps in combination with anti-malarial agents.

Improvement in immune parameters and human immunodeficiency virus-1 viral response in individuals treated with 16alpha-bromoepiandrosterone (HE2000).

A randomised, double-blind, placebo-controlled study examined the safety, tolerance, immunological effect and anti-human immunodeficiency virus (HIV) activity of sub-cutaneously administered HE2000 (16alpha-bromoepiandrosterone) as monotherapy in treatment-naïve patients with HIV-1. Twenty-four patients received five sequential daily doses of 50 or 100 mg of HE2000 or placebo every 6 weeks for up to three courses, and were followed thereafter for 3 months. HE2000 was safe, with transient injection site reactions being the main side-effect. Peripheral blood samples, collected serially, were analysed for changes in immune cell phenotypes. Significant increases were observed in the numbers of circulating dendritic cells, early activated (CD69+ CD25-) CD8 T-cells and T-NK cells after administration of 50-mg doses of HE2000 (p < 0.05). Gene expression in peripheral blood mononuclear cells was analysed by real-time RT-PCR. Before treatment, HIV-1-infected patients had significantly elevated transcripts for a number of inflammatory mediators (p < 0.012). After 50 mg or 100 mg HE2000, but not after placebo, there were significant sustained decreases in IL-1beta, TNF-alpha, IL-6 and Cox-2 transcripts (p < 0.05). There were no significant differences in CD4 cell numbers, although patients receiving 50-mg doses demonstrated a significant decrease in viral load (- 0.6 log; p < 0.01). Anti-HIV-1 T-cell responses were analysed serially using GAG-peptides to stimulate cytoplasmic IFN-gamma responses. After three courses, the 50-mg dose group demonstrated a significant increase in CD8 T-cell response against two distinct GAG peptide pools (p < 0.03). These findings suggest that immune-based therapies may be able to impact viral load by decreasing inflammation and/or stimulating CD8 T-cells.

Lactandrate: a D-homo-aza-androsterone alkylator in the treatment of breast cancer.

The sensitivity of breast neoplasms to hormonal control provides the basis of novel investigational treatments with steroidal alkylators. An androsterone D-lactam steroidal ester, the 3beta-hydroxy-13alpha-amino-13,17-seco- 5alpha-androstan-17-oic-13,17-lactam, p-bis(2-chloroethyl)amino phenyl acetate (lactandrate) was synthesized and tested for antitumor activity against six human breast cancer cell lines in vitro and against two murine and one xenograft mammary tumors in vivo. A docking study on the binding interactions of lactandrate with the ligand-binding domain (LBD) of estrogen receptor-alpha (ERalpha) was inquired. In vitro testing of lactandrate cytostatic and cytotoxic activity was performed on T47D, MCF7, MDA-MB-231, BT-549, Hs578T, MDA-MB-435 breast adenocarcinoma human cell lines. In vivo testing was performed on two murine mammary tumors, the MXT tumor and CD8F1 adenocarcinoma, as well as on human mammary carcinoma MX-1 xenograft. Molecular modeling techniques were adopted to predict a possible location and interaction mode of the molecule into LBD. Lactandrate induced significantly high antitumor effect against all tested in vitro and in vivo models. The cell lines with positive ER expression found to be significantly more sensitive to lactandrate. Moreover, lactandrate found to be positioned inside the binding cavity with its steroidal moiety, whilst the alkylating moiety protrudes out of receptor's pocket. Lactandrate produced important anticancer activity on breast cancer in vitro and in vivo. Some correlation between ER and lactandrate effect was demonstrated. Docking studies provide the basis for the structure-based design of improved steroidal alkylating esters for the treatment of estrogen-related cancers.

Therapeutic effects of dehydroepiandrosterone metabolites in diabetes mutant mice (C57BL/KsJ-db/db).

Dehydroepiandrosterone (DHEA) fed at 0.4% in the diet is known to exert strong antihyperglycemic effects in C57BL/KsJ genetically diabetic (db/db) mice. Three of the major metabolic products of DHEA; DHEA sulfate, alpha-hydroxyetiocholanolone (alpha-ET), and beta-hydroxyetiocholanolone (beta-ET) when fed at 0.1% in the diet, and one putative product, 17 beta-estradiol, when fed at 0.005% also prevented the development of severe diabetes while having little effect on the amount of food eaten or the rate of weight gain. When suboptimal doses (5-20 micrograms/week) of estradiol were injected in combination with diets containing either alpha-ET or beta-ET, marked potentiating effect was noted, normalization of the hyperglycemia being produced with as little as 0.025% of beta-ET and 0.05% of alpha-ET. The ability of the etiocholanolones to maintain islet integrity and prevent the development of most diabetes symptoms suggests that these metabolites are not merely inactive end products of steroid metabolism, but are physiological effectors in their own right.

A study of the efficacy of 5 alpha- and 5 beta-androstanes in chronic experimental aplastic anemia in mice.

The efficacy of in vivo administration of a 5 alpha-androstane and two 5 beta-androstanes (3 alpha and 3 beta) on CFU/GM and CFU/E mouse bone marrow stem cells was compared. The subjects were two groups of Swiss Webster mice: one normal group and one group in whom chronic aplastic anemia was induced by irradiation followed by mesenteric node lymphocyte grafting from C57/Bl donor mice. In normal animals, the two types of androgens (5 alpha and 5 beta) had the same efficacy, and the injection of 5 beta-androstane, before that of 5 alpha, significantly increased its efficacy. In aplastic mice, 5 alpha and 5 beta-androstanes had the same efficacy on CFU/E but 5 beta-androstanes were more efficient on the granulopoietic committed stem cells. The efficacy of the association of the two compounds in aplastic mice was not superior to that of each drug alone, which can be explained by the depletion of the stem cell compartment induced by the stimulating effect of the first androgen.

Correlation of clinical and in vitro erythropoietic responses to androgens in renal failure.

To assess in vitro erythropoietic cultures as a tool to discriminate between patients whose anemia improves with and those whose anemia fails to improve with androgens, bone marrows of 24 anemic patients receiving maintenance hemodialysis in hormone-depleted plasma clots were cultured. While erythroid colony proliferation in the presence and absence of androgens by cells of seven patients with good clinical outcomes who were initially studied was similar to that of normal donors, it was reduced (P less than 0.001) in cultures of cells from seven patients whose clinical responses were ultimately poor. Fewer than 25 colonies/6 X 10(4) cells at optimal erythropoietin concentrations, and less than a 25% enhancement in colony growth by androgens were observed in only those cultures derived from clinical nonresponder marrows. Using these criteria, nine of ten prospectively classified patients (five clinical responders and four nonresponders) were identified correctly.

[Carcinoma of the breast: introduction (author's transl)]. / Mamma Carcinom. I.Einführung.

In the German Federal Republic, carcinoma of the breast and colon lie close together as the second und third most tumors. We owe to our surgical forefathers of 100 years ago, Dieffenbach, Rotter, and Halsted, the decisive therapeutic advance whereby as a result of a better understanding of the anatomy of lymphatic drainage, excision of axillary glands and pectoralis muscle was first undertaken. Neither increasing the extent of the operation (superradical excision) nor reducing it (enucleation, partial mastectomy) has so far with any certainly proved as effective as radical mastectomy. The prevalence of various operations used in the western world was ascertained from a survey of about 1000 surgical clinics. With less extensive operations (enucleation) there is the special danger that the understandable desire to strive for a cosmetic result may vitiate the chance of cure.