Effect of estrogen receptor ß agonists on proliferation and gene expression of ovarian cancer cells.

BACKGROUND: Estrogen receptor (ER) ß has been suggested to affect ovarian carcinogenesis. We examined the effects of four ERß agonists on proliferation and gene expression of two ovarian cancer cell lines. METHODS: OVCAR-3 and OAW-42 ovarian cancer cells were treated with the ERß agonists ERB-041, WAY200070, Liquiritigenin and 3ß-Adiol and cell growth was measured by means of the Cell Titer Blue Assay (Promega). ERß expression was knocked down by transfection with specific siRNA. Additionally, transcriptome analyses were performed by means of Affymetrix GeneChip arrays. To confirm the results of DNA microarray analysis, Western blot experiments were performed. RESULTS: All ERß agonists tested significantly decreased proliferation of OVCAR-3 and OAW-42 cells at a concentration of 10 nM. Maximum antiproliferative effects were induced by flavonoid Liquiritigenin, which inhibited growth of OVCAR-3 cells by 31.2% after 5 days of treatment, and ERB-041 suppressing proliferation of the same cell line by 29.1%. In OAW-42 cells, maximum effects were observed after treatment with the ERß agonist WAY200070, inhibiting cell growth by 26.8%, whereas ERB-041 decreased proliferation by 24.4%. In turn, knockdown of ERß with specific siRNA increased cell growth of OAW-42 cells about 1.9-fold. Transcriptome analyses revealed a set of genes regulated by ERß agonists including ND6, LCN1 and PTCH2, providing possible molecular mechanisms underlying the observed antiproliferative effects. CONCLUSION: In conclusion, the observed growth-inhibitory effects of all ERß agonists on ovarian cancer cell lines in vitro encourage further studies to test their possible use in the clinical setting.

Efectos de los inductores antiepilépticos en la neuropsicofarmacología: una cuestión ignorada. Parte II: cuestiones farmacológicas y comprensión adicional / The effects of antiepileptic inducers in neuropsychopharmacology, a neglected issue. Part II: pharmacological issues and further understanding

La literatura sobre los inductores en la epilepsia y el trastorno bipolar está contaminada por falsos negativos. Esta segunda parte de una revisión exhaustiva sobre los fármacos antiepilépticos (FAE) con propiedades inductoras aporta más material educativo a los clínicos acerca de la complejidad de interpretar sus interacciones farmacológicas. Se revisa la farmacología básica de la inducción incluyendo los citocromos P450 (CYP), las enzimas de glucuronización (UGT) y la glucoproteína P (P-gp). Los CYP2B6 y CYP3A4 son muy sensibles a la inducción. El CYP1A2 es moderadamente sensible. Los el CYP2C9 y el CYP2C19 son solo levemente sensibles. El CYP2D6 no puede ser inducida por los fármacos. La inducción de las enzimas metabólicas, los CYP o las UGT, y los transportadores como la P-gp, se debe a un incremento de la síntesis de estas proteínas mediado por los denominados receptores nucleares (receptores constitutivo de androstano, de los estrógenos, de los glucocorticoides y de pregnano X). Aunque la primera parte de este artículo describe los factores de corrección para los antiepilépticos inductores, la extrapolación de estos valores desde un paciente promedio a un individuo concreto está influenciada por la ruta de administración, la carencia de la enzima metabólica debida a razones genéticas, y la presencia de inhibidores, u otros inductores. También pueden ser importantes las interacciones farmacológicas de los FAE al nivel de los mecanismos farmacodinámicos. Se describen 6 pacientes con una sensibilidad extrema a los inductores antiepilépticos (AU)

The literature on inducers in epilepsy and bipolar disorder is seriously contaminated by false negative findings. Part II of this comprehensive review on antiepileptic drug (AED) inducers provides clinicians with further educational material about the complexity of interpreting AED drug-drug interactions. The basic pharmacology of induction is reviewed including the cytochrome P450 (CYP) isoenzymes, the Uridine Diphosphate Glucuronosyltransferases (UGTs), and P-glycoprotein (P-gp). CYP2B6 and CYP3A4 are very sensitive to induction. CYP1A2 is moderately sensitive while CYP2C9 and CYP2C19 are only mildly sensitive. CYP2D6 cannot be induced by medications. Induction of UGT and P-gp are poorly understood. The induction of metabolic enzymes such as CYPs and UGTs, and transporters such as P-gp, implies that the amount of these proteins increases when they are induced; this is almost always explained by increasing synthesis mediated by the so-called nuclear receptors (constitutive androstane, estrogen, glucocorticoid receptors and pregnane X receptors). Although part i provides correction factors for AEDs, extrapolation from an average to an individual patient may be influenced by administration route, absence of metabolic enzyme for genetic reasons, and presence of inhibitors or other inducers. AED pharmacodynamic DDIs may also be important. Six patients with extreme sensitivity to AED inductive effects are described (AU)

5α-Reduced neurosteroids sex-dependently reverse central prenatal programming of neuroendocrine stress responses in rats.

Maternal social stress during late pregnancy programs hypothalamo-pituitary-adrenal (HPA) axis hyper-responsiveness to stressors, such that adult prenatally stressed (PNS) offspring display exaggerated HPA axis responses to a physical stressor (systemic interleukin-1ß; IL-1ß) in adulthood, compared with controls. IL-1ß acts via a noradrenergic relay from the nucleus tractus solitarii (NTS) to corticotropin releasing hormone neurons in the paraventricular nucleus (PVN). Neurosteroids can reduce HPA axis responses, so allopregnanolone and 3ß-androstanediol (3ß-diol; 5α-reduced metabolites of progesterone and testosterone, respectively) were given subacutely (over 24 h) to PNS rats to seek reversal of the "programmed" hyper-responsive HPA phenotype. Allopregnanolone attenuated ACTH responses to IL-1ß (500 ng/kg, i.v.) in PNS females, but not in PNS males. However, 3ß-diol normalized HPA axis responses to IL-1ß in PNS males. Impaired testosterone and progesterone metabolism or increased secretion in PNS rats was indicated by greater plasma testosterone and progesterone concentrations in male and female PNS rats, respectively. Deficits in central neurosteroid production were indicated by reduced 5α-reductase mRNA levels in both male and female PNS offspring in the NTS, and in the PVN in males. In PNS females, adenovirus-mediated gene transfer was used to upregulate expression of 5α-reductase and 3α-hydroxysteroid dehydrogenase mRNAs in the NTS, and this normalized hyperactive HPA axis responses to IL-1ß. Thus, downregulation of neurosteroid production in the brain may underlie HPA axis hyper-responsiveness in prenatally programmed offspring, and administration of 5α-reduced steroids acutely to PNS rats overrides programming of hyperactive HPA axis responses to immune challenge in a sex-dependent manner.

Libraries of 2ß-(N-substituted piperazino)-5α-androstane-3α, 17ß-diols: chemical synthesis and cytotoxic effects on human leukemia HL-60 cells and on normal lymphocytes.

Libraries of steroid derivatives with two levels of molecular diversity were prepared to optimize the antiproliferative activity on leukemia HL-60 cells by first varying the amino acid (AA) at R(1) (libraries A, B, C, and D: with 45, 45, 20, and 20 members, respectively) and, subsequently, the capping group at R(2) (library E: 168 members). The screening of these aminosteroids revealed interesting structure-activity relationships. In library A, the compounds bearing a tetrahydroisoquinolone residue as the first element of diversity showed potent cytotoxicity, principally when isovaleric or cyclohexyl acetic acid was used as a capping group (>40% of cell growth inhibition at 1 µM). In library B, the phenylalanine (Phe) derivatives bearing a cyano group induced a higher growth inhibition than the other Phe derivatives. The screening of library C indicated the increase of hydrophobicity of proline (Pro) seems to preserve the cytotoxic effect achieved by the lead compound. However, the synthesis of structural Pro variants (library D) clearly shows weaker activities when compared to L-Pro building blocks. Finally, by incorporating some of the most active AA of libraries A-D in library E, we observed that the amide coupling functionality gave stronger cytotoxic activity compared to the corresponding sulfonamides or benzylamines. Six of the most active amide derivatives (E-37P, E-41P, E-42P, E-46P, E-48F, and E-12T) were selected and IC(50) determined on HL-60 cells as well as on normal human lymphocytes. Among this series of new anticancer agents, good to high selectivity indices (SI = IC(50) (lymphocytes)/IC(50) (HL-60 cells) = 5 - 55) were obtained.

Neurosteroids: endogenous role in the human brain and therapeutic potentials.

This chapter provides an overview of neurosteroids, especially their impact on the brain, sex differences and their therapeutic potentials. Neurosteroids are synthesized within the brain and rapidly modulate neuronal excitability. They are classified as pregnane neurosteroids, such as allopregnanolone and allotetrahydrodeoxycorticosterone, androstane neurosteroids, such as androstanediol and etiocholanolone, and sulfated neurosteroids such as pregnenolone sulfate. Neurosteroids such as allopregnanolone are positive allosteric modulators of GABA-A receptors with powerful anti-seizure activity in diverse animal models. Neurosteroids increase both synaptic and tonic inhibition. They are endogenous regulators of seizure susceptibility, anxiety, and stress. Sulfated neurosteroids such as pregnenolone sulfate, which are negative GABA-A receptor modulators, are memory-enhancing agents. Sex differences in susceptibility to brain disorders could be due to neurosteroids and sexual dimorphism in specific structures of the human brain. Synthetic neurosteroids that exhibit better bioavailability and efficacy and drugs that enhance neurosteroid synthesis have therapeutic potential in anxiety, epilepsy, and other brain disorders. Clinical trials with the synthetic neurosteroid analog ganaxolone in the treatment of epilepsy have been encouraging. Neurosteroidogenic agents that lack benzodiazepine-like side effects show promise in the treatment of anxiety and depression.

Antiseizure effects of 3alpha-androstanediol and/or 17beta-estradiol may involve actions at estrogen receptor beta.

Testosterone (T), the principal androgen secreted by the testes, can have antiseizure effects. Some of these effects may be mediated by T's metabolites. T is metabolized to 3alpha-androstanediol (3alpha-diol). T, but not 3alpha-diol, binds androgen receptor. We investigated effects of 3alpha-diol (1 mg/kg, SC) and/or an androgen receptor blocker (flutamide 10 mg, SC), 1 hour prior to administration of pentylenetetrazol (85 mg/kg, IP). Juvenile male rats administered 3alpha-diol had less seizure activity than those administered vehicle. Flutamide had no effects. T is aromatized to 17beta-estradiol (E(2)), which, like 3alpha-diol, acts at estrogen receptors (ERs). Selective estrogen receptor modulators that favor ERalpha (propyl pyrazole triol, 17alpha-E(2)) or ERbeta (diarylpropionitrile, coumestrol, 3alpha-diol), or both (17beta-E(2)), were administered (0.1 mg/kg, SC) to juvenile male rats 1 hour before pentylenetetrazol. Estrogens with activity at ERbeta, but not those selective for ERalpha, produced antiseizure effects. Actions at ERbeta may underlie some antiseizure effects of T's metabolites.

Antiseizure effects of 5*-androstane-3*,7beta-diol may be independent of actions at estrogen receptor beta.

Testosterone (T), the principal androgen secreted by the testes, can have antiseizure effects; however, the mechanism(s) underlying this action is not well understood. T is metabolized to dihydrotestosterone (DHT) by the enzyme 5*-reductase. DHT is then converted to 5*-androstane-3*,17beta-diol (3*-diol) by the enzyme 3*-hydroxysteroid dehydrogenase. T and DHT bind with high affinity to intracellular androgen receptors; however, 3*-diol does not. The mnemonic effects of 3*-diol are mediated in part through the beta isoform of estrogen receptors (ERbeta) in the hippocampus. As such, we investigated whether 3*-diol has antiseizure effects in mice that require action at ERbeta. 3*-Diol (2 mg/kg subcutaneously) was administered to wild-type C57/B6 mice and heterozygous and homozygous ERbeta knockout (betaERKO) mice 1 hour prior to administration of pentylenetetrazol (PTZ; 85 mg/kg intraperitoneally). Mice administered 3*-diol had significantly longer latencies to clonic seizure and death and lower seizure scores than did mice administered vehicle. This pattern of effects was observed in wild-type or betaERKO mice. Thus, for these mice, the antiseizure effects of 3*-diol for the chemoconvulsant PTZ occur independent of actions at ERbeta.

Testosterone derivatives are neuroprotective agents in experimental diabetic neuropathy.

In this study we have assessed the effect of testosterone (T), dihydrotestosterone (DHT) and 5alphaandrostan-3alpha, 17beta-diol (3alpha-diol) therapies on diabetic neuropathy. Diabetes was induced in adult male rats by the injection of streptozotocin and resulted in decreased T and increased 3alpha-diol levels in plasma and in decreased levels of pregnenolone and DHT in the sciatic nerve. Moreover, a reduced expression of the enzyme converting Tinto DHT (i.e., the 5alpha-reductase) also occurs at the level of sciatic nerve, suggesting that the decrease of DHT levels could be due to an impairment of this enzyme. Chronic treatment for 1 month with DHT or 3alpha-diol increased tail nerve conduction velocity and partially counteracted the increase of thermal threshold induced by diabetes. Treatment with DHT increased tibial Na(+),K(+)-ATPase activity and the expression of myelin protein P0 in the sciatic nerve.DHT, 3alpha-diol and T reversed the reduction of intra-epidermal nerve fiber density induced by diabetes. These observations indicate that T metabolites can reverse behavioral, neurophysiological, morphological and biochemical alterations induced by peripheral diabetic neuropathy.

Androgen receptor mediated growth control of breast cancer and endometrial cancer modulated by antiandrogen- and androgen-like steroids.

Androgens are involved in many regulatory processes in mammary and endometrial epithelium, but their role in the development and progression of breast and endometrial carcinoma is poorly understood. Androgen receptors (AR) are found in normal epithelium as well as in more than 50% of specimen from both tumor types. The occurrence of AR is correlated with estrogen and progesterone receptors. Androgen receptor positive cell lines were established during the last few years in our laboratory from malignant mammary (MFM-223) and endometrial (MFE-296) tumors supplementing the small number of androgen-responsive cell lines published so far. In this paper some aspects of the role of androgens in these two types of hormone responsive female cancers are presented. The proliferation of ZR-75-1, MFM-223 and MFE-296 cells is inhibited by androgens. The progestin medroxyprogesterone acetate inhibits the proliferation of estrogen- and progesterone receptor negative MFM-223 cells via the androgen receptor. Some steroid metabolites with distinct estrogenic properties like androst-5-ene-3 beta,17 beta-diol possess androgenic properties in this model system. Androgens stimulate the in vitro secretion of gross cystic disease fluid proteins by human mammary cancer cells. These proteins are normally found in benign breast cysts in vivo. The occurrence of gross cystic disease is correlated with an increased risk of breast cancer. The AR is autoregulated in MFM-223 mammary cancer cells on the protein and mRNA level. In MFE-296 cells with endometrial origin AR protein was increased after incubation with androgens.

Newer neuromuscular blocking drugs. An overview of their clinical pharmacology and therapeutic use.

Four new nondepolarising muscle relaxants, pipecuronium bromide, doxacurium chloride, mivacurium chloride and Org 9426 (rocuronium) offer alternatives to the established agents atracurium besilate and vecuronium bromide. Pipecuronium and Org 9426 are steroidal compounds, the latter being a monoquaternary agent, whereas doxacurium and mivacurium are bisquaternary benzylisoquinolinium compounds. Pipecuronium and doxacurium have a relatively slow onset and a long duration of action. Pipecuronium produces maximum block in 3 to 6 min when given in a dose of 45 to 80 micrograms/kg, and a duration of clinical relaxation of between 40 and 110 min. Doxacurium is more potent, but is the least rapid and the longest acting relaxant currently available. When administered in doses of 37 to 80 micrograms/kg, it produces maximum block within 3.5 to 10 min, with a duration of clinical relaxation of 77 to 164 min. The advantage of both pipecuronium and doxacurium is their cardiovascular stability. Both agents are primarily eliminated via the kidneys and both have now been licensed for use in the US. Mivacurium is a muscle relaxant with a short duration of action. When administered in doses of 0.1 to 0.25 mg/kg it produces maximum block in 2 to 4 min, but the duration of clinical relaxation is less than 20 min. Higher doses which could induce a faster neuromuscular block are unfortunately associated with some histamine liberation. The drug is metabolised by plasma cholinesterase. Mivacurium has also been licensed for use in the US. Org 9426 is an agent with a rapid onset but an intermediate duration of action. A dose of 0.5 to 0.6 mg/kg induces maximum block in about 1.5 min and has a duration of clinical relaxation of about 30 min. The rapid onset of effect could be useful for early intubation as an alternative to suxamethonium chloride. However, much more clinical experience is needed with this agent, particularly with regard to duration of action of larger doses and occurrence of side effects. The drug is mainly eliminated via the liver.

Intubating conditions after pipecuronium bromide: the influence of dose and time.

STUDY OBJECTIVE: To determine the intubating conditions following the administration of pipecuronium bromide in doses of two (0.07 mg/kg) or three (0.1 mg/kg) times ED95 (average dose that gives 95% block of the first twitch). DESIGN: To compare intubating conditions at 11/2 and 21/2 minutes in 41 patients receiving balanced anesthesia. SETTING: Surgical patients at Thomas Jefferson University Hospital. PATIENTS: Forty-one patients undergoing surgical procedure who received general anesthesia. INTERVENTIONS: After obtaining a stable baseline of train-of-four (TOF), 41 patients randomly received either 0.07 mg/kg or 0.1 mg/kg of pipecuronium as a single intravenous (IV) bolus dose, and the trachea was intubated either at 11/2 or 21/2 minutes. MEASUREMENTS AND MAIN RESULTS: Intubating conditions at 21/2 minutes appeared significantly better than those at 11/2 minutes, regardless of the pipecuronium dose. The mean time for T1 (first twitch of TOF) to reach 50% and 90% suppression was 1.36 +/- 0.51 minutes and 2.29 +/- 0.8 minutes, respectively, for the 0.07 mg/kg dose and 1.07 +/- 0.27 minutes and 1.72 +/- 0.45 minutes, respectively, for the 0.1 mg/kg dose. This did not make a significant difference in intubating conditions at either time. The time to 25% recovery of T1 was 68.2 +/- 22 minutes for the 0.07 mg/kg dose and 121.5 +/- 49 minutes for the 0.1 mg/kg dose. In patients who had spontaneous recovery of T1 to between 10% and 25% of control, administration of neostigmine or edrophonium resulted in identical recovery in 10 minutes. However, in patients with less than 10% spontaneous recovery of T1, neostigmine appeared to be superior to edrophonium. CONCLUSION: Pipecuronium has a relatively rapid onset. The trachea could be intubated successfully in 11/2 minutes with a dose of either 0.07 mg/kg or 0.1 mg/kg. If the clinical situation requires perfect relaxation with no movement or bucking, we recommend waiting at least 21/2 minutes.

Arduan muscle relaxation prolonged with norcuron.

Norcuron injection has been applied for the prolongation of the duration of muscle relaxing action of Arduan in 104 patients. The patients underwent major surgery and 1/5 of them were of advanced age. Neuromuscular blockade could be prolonged for the required duration of operation with lower doses of Norcuron (2 mg, 1 mg) than the currently used ones. For avoiding eventual cumulation or synergism the repetition of lower doses to meet the requirements seems to be the most advantageous method.

Biochemical and histological studies on prostates in castrated dogs after treatment with androstanediol, oestradiol and cyproterone acetate.

The effect of cyproterone acetate (CA) on experimentally induced benign prostatic hyperplasia (BPH) in the castrated dog was investigated. BPH was induced by 6 months' treatment with 3 alpha-androstanediol (3 alpha-diol) alone and in combination with 17 beta-oestradiol (Oe2). RNA, DNA and zinc content of the glands were determined in addition to histological examination and measurement of the prostates. Two different types of prostatic enlargement were observed. First, 3 alpha-diol induced typical diffuse canine hyperplasia with replacement of functional activity. DNA, RNA and the zinc content of total glands were increased compared with intact controls. Second, 3 alpha-diol plus Oe2 produced on the one hand a more striking increase of prostatic weights, but on the other a loss of typical morphological structure and function. Histologically, transformation of simple glandular epithelium into stratified squamous metaplasia occurred in addition to stimulation of fibromuscular tissue. Biochemically, a relative decrease of DNA per mg tissue was measured with a fall in the RNA to DNA ratio and zinc to the values of castrates. Administration of CA resulted in an abolition of the 3 alpha-diol effect. Biochemical determinations and histological examinations revealed an effect similar to castration after treatment with 3 alpha-diol plus CA. After treatment with 3 alpha-diol plus Oe2 plus CA fibromuscular stimulation as an oestrogen effect predominated in addition to glandular atrophy and metaplastic changes, especially in prostatic ducts. Epithelial hyperplasia is an effect of 3 alpha-diol, whereas metaplastic proliferation only occurs in oestrogenized and androgenized dogs. In both types of prostatic enlargement CA prevents development of hyperplastic prostate.

[Effect of 5alpha-androstan-3beta, 17beta-diol on serum lipids and lipoproteins and the development of experimental atherosclerosis]. / Vliianie 5alpha-androstan-3beta, 17beta-diola na lipidy i lipoproteidy syvorotki krovi i na razvitie éksperimental'nogo ateroskleroza.

The author studied the influence of testosterone metabolite--5alpha-androstan-3beta,17beta-diol--on the development of hyperlipidemia and atherosclerosis in chinchilla rabbits given cholesterol-free semisynthetic atherogenic diet. The metabolite under study inhibited the development of hypercholesterolemia and hyperbetalipoproteinemia and decreased blood phospholipid content in the blood serum of experimental animals below the initial level. Lipid content in the sum total fraction of pre-beta and beta-lipoproteins decreased under the effect of the mentioned metabolite; there was also a fall in the amount of lipoproteins of low density and their greater saturation with cholesterol. Development of experimental atherosclerosis was intensified.