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Sugammadex, marketed as Bridion™, is an approved cyclodextrin (CD) based drug for the reversal of neuromuscular blockade in adults undergoing surgery. Sugammadex forms an inclusion complex with the neuromuscular blocking agent (NMBA) rocuronium, allowing rapid reversal of muscle paralysis. In silico methods have been developed for studying CD inclusion complexes, aimed at accurately predicting their structural, energetic, dynamic, and kinetic properties, as well as binding constants. Here, a computational study aimed at characterizing the sugammadex-rocuronium system from the perspective of docking calculations, free molecular dynamics (MD) simulations, and biased metadynamics simulations with potential of mean force (PMF) calculations is presented. The aim is to provide detailed information about this system, as well as to use it as a model system for validation of the methods. This method predicts results in line with experimental evidence for both the optimal structure and the quantitative value for the binding constant. Interestingly, there is a less profound preference for the orientation than might be assumed based on electrostatic interactions, suggesting that both orientations may exist in solution. These results show that this technology can efficiently analyze CD inclusion complexes and could be used to facilitate the development and optimization of novel applications for CDs., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: AMA, RGF, AP, and MSO report financial support was provided by Cyclarity Therapeutics Inc. AMA, RGF, AP, and MSO report a relationship with Cyclarity Therapeutics Inc. that includes: employment, consultation or advisory, equity or stocks, and travel reimbursement. RGF and AP report a relationship with the company MD.USE that includes: employment, and equity or stocks., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Rocuronium is widely used in surgery as a neuromuscular relaxant, but it has been difficult to accurately control its specific dosage in clinical operation. Therefore, the development of fast and instant rocuronium detection methods has important application value for reducing risks and safeguarding health. In this study, N, N, N-trimethyl-4-(pyrene-1-butyl)-ammonium bromide (PyBTA) was designed as a probe to detect rocuronium rapidly. The method relied on replacing PyBTA in sugammadex with rocuronium to induce changes in fluorescence intensity of PyBTA, thereby realizing quantitative detection. Its sensing performance and detection mechanism were explored systematically by spectroscopy. The linear range of this method was 0.5-10 μM and the detection limit of it was 0.3 μM. In addition, we confirmed that the host-guest interaction among PyBTA, sugammadex, and rocuronium was mainly driven by electrostatic and hydrophobic interactions., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The crystal structures of 3β,19-dihydroxyandrost-5-en-17-one, 5, and its monohydrate, [((5).H 2 O]], are reported. The monohydrate, isolated from a solution of 5 in 50% aqueous methanol, recrystallizes in the orthorhombic space group, P2 1 2 1 2 1 , while that of the anhydrous compound, isolated from solutions of THF, Me 2 CO, EtOAc or dry MeOH, recrystallizes in the monoclinic space group, P2 1 . Apart from the different orientation of the 3-HO group, the conformations of the steroid molecules in 5 and [(5).H 2 O]: are similar. The two-dimensional structure of 5 consists of sheets of molecules formed from strong classical O3-H3⋯O19 (OH) and O19-H19···O3 (OH) , augmented by weaker C-H⋯O hydrogen bonds. Noticeably the presence of the C19 hydroxyl group results in the replacement of the O3-H3⋯O17 (one) hydrogen bonds as the strongest intermolecular interaction found in the stable polymorphs of the mono-hydroxylated compound, 3β-hydroxyandrost-5-en-17-one (dehydroepiandrosterone). An additional structural difference between 3β-hydroxyandrost-5-en-17-one and 3β,19-dihydroxyandrost-5-en-17-one is that the steroid molecules are most strongly linked head-to-tail in the former but head-to-middle fashion in the latter. In the three dimensional structure of [((5).H 2 O]], each steroid molecule is directly connected to another by a O19-H19⋯O3 (OH) hydrogen bond and indirectly to two others via the water molecule. Each water molecule forms three hydrogen bonds, namely O3-H3⋯Ow, Ow-Hw1⋯O17 and Ow-Hw2⋯O19. As found for most hydrated steroids, the hydrate molecules have strong influences on the structure. In addition to the reporting of the crystal structures of 5 and [(5).H 2 O], we report on a survey of the crystal structures of related di- and tri- hydroxy-17-one derivatives as well as solvated compounds., (Copyright © 2020 Elsevier Inc. All rights reserved.)

2α-Methyl-4,5α-dihydrotestosterone 17β-propionate, known as drostanolone propionate or masteron, is a synthetic anabolic-androgenic steroid derived from dihydrotestosterone. The crystal structures of two polymorphs of drostanolone propionate have been determined by single crystal X-ray diffraction and both crystallizes in the monoclinic crystal system. One is belonging to the P2 1 space group, Z = 2, and has one molecule in the asymmetric unit while the second belongs to the I2 space group, Z = 4, and contains two molecules in the asymmetric unit. Another polymorph has been investigated by an X-ray powder diffraction method and solved by Parallel tempering/Monte Carlo technique and refined with the Rietveld method. This polymorph crystallizes in the orthorhombic P2 1 2 1 2 1 space group, Z = 4 having one molecule in the asymmetric unit. The structural configuration analysis shows that the A, B, and C steroid rings exist as chair geometry, while ring D adopts a C13 distorted envelope configuration in all structures. For all polymorphs, the lattice energy has been computed by CLP (Coulomb-London-Pauli), and tight-binding density functional theory methods. Local electron correlation methods were used to estimate the role of electron correlation in the magnitude of the dimer energies. The nature of the intermolecular interactions has been analyzed by the SAPT0 energy decomposition methods as well as by Hirshfeld surfaces.

The crystal structures, Hirshfeld surface analyses and electrostatic potential surfaces of the di- and tri-hydrates of (5α,17E)-17-hydrazonoandrostan-3-ol, 3, namely [3·(H 2 O) 2 ] and [3·(H 2 O) 3 ], are reported. The trihydrate, isolated from a solution of 3 in moist methanol, recrystallizes in the orthorhombic space group, P2 1 2 1 2 1 , while that of the dihydrate, isolated from a 1:1 aqueous methanol solution, recrystallizes in the monoclinic space group, P2 1 . The asymmetric unit of the trihydrate involves one steroid and three water molecules, while that of the dihydrate has two similar but independent steroid molecules and four hydrate molecules. Very similar conformations are found for the steroid molecules in both hydrates. As expected, the different mole ratios of water: steroid have major influences on the structures. In both cases, complex crystal structures are constructed from various classical hydrogen bonds, involving the hydrate molecules and the hydroxy and hydrazonyl moieties of the steroid. In the trihydrate, there are no direct connections between the steroid molecules, instead the water molecules link the steroid molecules, with only weak van der Waals forces between the steroid molecules. There are some direct links between the steroid molecules in the dihydrate, involving OH(steroid hydroxyl)⋯O(steroid oxo) hydrogen bonds, in a head to head fashion, and OH⋯N(hydrazonyl) hydrogen bonds, in a head to tail fashion. However, the major occurrence throughout the structure is of steroid molecules linked by water molecules., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background: Sugammadex is a relatively new molecule that reverses neuromuscular block induced by rocuronium. The particular structure of sugammadex traps the cyclopentanoperhydrophenanthrene ring of rocuronium in its hydrophobic cavity. Dexamethasone shares the same steroidal structure with rocuronium. Studies in vitro have demonstrated that dexamethasone interacts with sugammadex, reducing its efficacy. In this study, we investigated the clinical relevance of this interaction and its influence on neuromuscular reversal., Methods: In this retrospective case-control study, we analyzed data from 45 patients divided into 3 groups: dexamethasone after induction group (15 patients) treated with 8 mg dexamethasone as an antiemetic drug shortly after induction of anesthesia; dexamethasone before reversal group (15 patients) treated with dexamethasone just before sugammadex injection; and control group (15 patients) treated with 8 mg ondansetron. All groups received 0.6 mg/kg rocuronium at induction, 0.15 mg/kg rocuronium at train-of-four ratio (TOF) 2 for neuromuscular relaxation, and 2 mg/kg sugammadex for reversal at the end of the procedure at TOF2. Neuromuscular relaxation was monitored with a TOF-Watch® system., Results: The control group had a recovery time of 154 ± 54 seconds (mean ± SD), the dexamethasone after induction group 134 ± 55 seconds, and the dexamethasone before reversal group 131 ± 68 seconds. The differences among groups were not statistically significant (P = 0.5141)., Conclusions: Our results show that the use of dexamethasone as an antiemetic drug for the prevention of postoperative nausea and vomiting does not interfere with reversal of neuromuscular blockade with sugammadex in patients undergoing elective surgery with general anesthesia in contrast to in vitro studies that support this hypothesis.

Drostanolone is one of the most frequently detected anabolic androgenic steroids in doping control analysis. Here, we studied drostanolone urinary metabolic profiles using liquid chromatography quadruple time of flight mass spectrometry (LC-QTOF-MS) in full scan and targeted MS/MS modes with accurate mass measurement. The drug was administered to one healthy male volunteer and liquid-liquid extraction along with direct-injection were used to analyze urine samples. Chromatographic peaks for potential metabolites were identified with the theoretical [M-H](-) as a target ion in a full scan experiment and actual deprotonated ions were analyzed in targeted MS/MS mode. Eleven metabolites including five new sulfates, five glucuronide conjugates, and one free metabolite were confirmed for drostanolone. Due to the absence of useful fragment ions to illustrate the steroid ring structure of drostanolone phase II metabolites, gas chromatography mass spectrometry (GC-MS) was used to obtain structural details of the trimethylsilylated phase I metabolite released after enzymatic hydrolysis and a potential structure was proposed using a combined MS approach. Metabolite detection times were recorded and S4 (2α-methyl-5α-androstan-17-one-6β-ol-3α-sulfate) and G1 (2α-methyl-5α-androstan-17-one-3α-glucuronide) were thought to be new potential biomarkers for drostanolone misuse which can be detected up to 24days by liquid-liquid extraction and 7days by direct-injection analysis after intramuscular injection. S4 and G1 were also detected in two drostanolone-positive routine urine samples., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Rocuronium bromide is a non-depolarizing neuromuscular blocking agent that causes rapid muscle relaxation after intravenous injection. Regulatory authorities for registration of pharmaceuticals for human use require the evaluation of the stability of active compounds under various stress conditions. Forced degradation of rocuronium bromide was performed under hydrolytic, thermal, photolytic, and oxidative settings. HPLC-UV/vis analysis revealed an unknown degradation product under oxidative conditions (1% H2 O2 , reflux for 1 h). Investigation of the respective HPLC fraction by high resolution mass spectrometry indicated a formal loss of CH2 and an addition of one oxygen atom to the intact drug molecule. Additional multistage mass spectrometric structural elucidation experiments aided by complementary information from analysis of the intact drug and known rocuronium-related compounds showed that the morpholine moiety was unstable under oxidative stress. The data demonstrated that the morpholine ring was opened and transformed to an N-ethanoyl-formamide group. The structure was supported by appropriate mechanistic explanations., (Copyright © 2015 John Wiley & Sons, Ltd.)

A high-throughput method using solid-phase microextraction coupled to liquid chromatography-tandem mass spectrometry (SPME-LC-MS/MS) for determination of tranexamic acid and rocuronium bromide in human plasma was developed and validated. Standard analytical approaches employ acidification of the sample due to the instability of rocuronium bromide in collected plasma samples. However, acidification affects the binding equilibrium of the drug and consequently no information on the free/bound concentration can be obtained. Contrary to these protocols, the proposed method requires minimum sample handling and no ion pairing and/or derivatization procedure. A weak cation exchange coating was chosen as the best extracting phase for selected drugs, guaranteed a good recovery, minimum carry-over, reusability and reproducibility. SPME procedure met all Food and Drug Administration acceptance criteria for bioanalytical assays at three concentration levels, for both selected drugs. Post-extraction addition experiments showed that matrix effect was less than ±3%. Here, a weak cation exchange thin-film solid-phase microextraction (WCX TF-SPME) approach is presented, offering effective cleanup procedure and full quantitation of the drugs in plasma, undoubtedly one the most challenging matrices with regards to its complexity. In addition, the 96-well plate format of WCX TF-SPME system provides considerable advantages, such as high throughput analysis for up to 96 samples in 35min (22s/sample), requirement of small amounts of plasma samples (0.8mL), and a simple sample preparation protocol, all of which shows a promise for possible on-site application in hospitals to monitor concentrations of the drugs in close to real time., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Neuromuscular blockers (NMBs) provide good conditions for endotracheal intubation and surgery. NMBs have been associated with higher morbidity and mortality, mainly due to post-operative residual neuromuscular block. This may become history with the advent of sugammadex - an antidote to the NMB rocuronium - which within 1-3 minutes neutralizes the effects of rocuronium. High-dose rocuronium is now an alternative to suxamethonium in acute or short procedures and in a situation, where ventilation/intubating cannot be performed, sugammadex can reverse the rocuronium blockade within minutes.

Rocuronium, a non-depolarizing neuromuscular blocking drug has a rapid onset of action, a comparatively low potency and, with a more favourable side effects profile than succinylcholine, it has become a popular alternative to that drug for rapid sequence inductions in anaesthesia. The rocuronium-binding cyclodextrin derivative sugammadex, prepared by per-6 substitution of the primary hydroxyls of γ-cyclodextrin with thiol ether-linked propionic acid side chains to extend the hydrophobic cavity to accommodate rocuronium, is used to reverse neuromuscular blockade by encapsulating the drug as an inclusion complex and removing it from the neuromuscular junction to the plasma. It has recently been suggested that sugammadex might also be of value in the management of rocuronium-induced anaphylaxis and this has been potentially supported by recent case reports. However, before sugammadex can be recommended for this purpose, it is important to establish whether or not the allergenic substituted ammonium groups at each end of the rocuronium molecule in the inclusion complex are masked within the cavity or left exposed for interaction with rocuronium-reactive IgE antibodies in the sera of rocuronium-allergic patients. Detailed experimental strategies and experimental protocols to investigate the allergenic potential of the sugammadex-rocuronium inclusion complex are presented and a possible explanation of the apparently rapid and successful reversal of anaphylaxis by administration of sugammadex is advanced and discussed.

The preparation of novel steroidal heterocycles containing the 7-aryl-substituted 1,2,4-triazolo[1,5-a]pyrimidine moiety fused to the 16,17-positions of the steroid nucleus is described. The Aldol reaction of 4-aza-androst-3,17-dione (1a) and dehydroepiandrosterone (DHEA, 1b) with aromatic aldehydes was catalyzed by KF/Al(2)O(3) to give the corresponding 3-oxo-4-aza-5α- and 3β-hydroxy-5-en-16-arylidene-17-ketosteroids (2a-r). Subsequently, the intermediates 2a-r reacted with dinucleophilic 3-amino-1,2,4-triazole in presence of t-BuOK to afford the title compounds (3a-r). All the synthesized heterosteroids are new and are currently being evaluated for their biological activities., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The effects of dipole modifiers and their structural analogs on the single channel activity of amphotericin B in sterol-containing planar phosphocholine membranes are studied. It is shown that the addition of phloretin in solutions bathing membranes containing cholesterol or ergosterol decreases the conductance of single amphotericin B channels. Quercetin decreases the channel conductance in cholesterol-containing bilayers while it does not affect the channel conductance in ergosterol-containing membranes. It is demonstrated that the insertion of styryl dyes, such as RH 421, RH 237 or RH 160, in bilayers with either cholesterol or ergosterol leads to the increase of the current amplitude of amphotericin B pores. Introduction of 5α-androstan-3β-ol into a membrane-forming solution increases the amphotericin B channel conductance in a concentration-dependent manner. All the effects are likely to be attributed to the influence of the membrane dipole potential on the conductance of single amphotericin B channels. However, specific interactions of some dipole modifiers with polyene-sterol complexes might also contribute to the activity of single amphotericin B pores. It has been shown that the channel dwell time increases with increasing sterol concentration, and it is higher for cholesterol-containing membranes than for bilayers including ergosterol, 6-ketocholestanol, 7-ketocholestanol or 5α-androstan-3β-ol. These findings suggest that the processes of association/dissociation of channel forming molecules depend on the membrane fluidity.

Cyclodextrins, oligosaccharides linked in a circular arrangement around a central cavity, are used extensively in the pharmaceutical industry to improve drug delivery. Their usefulness depends on their capacity to form a drug inclusion, or host-guest, complex within the cavity. In an attempt to improve the delivery of the widely used neuromuscular blocking drug (NMBD) rocuronium, a rocuronium inclusion complex was formed with a chemically modified γ-cyclodextrin. The high binding affinity and specificity of the modified carrier (named sugammadex) for rocuronium (and other aminosteroid NMBDs) led to its use in anaesthesia as an innovative and useful agent for rapid reversal of rocuronium-induced neuromuscular block by sequestering the drug as an inclusion complex. This, in turn, led to the suggestion that sugammadex might be useful to remove the NMBD from the circulation of patients experiencing rocuronium-induced anaphylaxis, a suggestion subsequently supported in case reports where traditional treatment had failed. Successful resuscitations suggested that sugammadex might be a valuable new treatment for such intractable cases but, given the inappropriateness of clinical trials, confirmation or refutation will have to await the slow accumulation of results of individual case reports. Important questions related to antibody accessibility of drug allergenic structures on the rocuronium-sugammadex inclusion complex, and the competition between sugammadex and IgE antibodies (both free and cell bound) for rocuronium, also remain and can be investigated in vitro. The sugammadex findings indicate that the use of carrier molecules such as the cyclodextrins to improve drug delivery will sometimes give rise to changed immunologic and allergenic behaviour of some drugs and this will have to be taken into account in preclinical drug safety assessments of drug-carrier complexes. The possibility of encapsulating and removing other allergenic drugs, e.g., penicillins and cephalosporins, in cases of difficult-to-reverse anaphylaxis to these drugs is discussed., (© 2011 Blackwell Publishing Ltd.)

Aim of the Study: Pregnane glycosides are potent cytotoxic agents which may represent new leads in the development of anti-tumour drugs, particularly in the treatment of breast cancer, because of the structural similarity to estrogenic agonists. Caralluma species are natural sources of a wide variety of pregnane glycosides. The aim of the study was to isolate, using an activity-guided fractionation approach, novel pregnane glycosides for testing on breast cancer and other tumour lines., Materials and Methods: The effect of crude extracts, specific organic fractions and isolated compounds from Caralluma tuberculata was tested on the growth and viability of MCF-7 estrogen-dependent, and MDA-MB-468 estrogen-independent breast cancer cells, Caco-2 human colonic cells, HUVECs and U937 cells. Neutral red uptake and MTT assays were used. Apoptosis was detected by Western blot of poly-(ADP ribose) polymerase (PARP) as were other markers of nuclear fragmentation (DNA ladder assay, staining of cells with nuclear dye DAPI). The involvement of caspases was investigated using the pan-caspase inhibitor Z-VAD-FMK., Results: The ethyl acetate fraction of Caralluma tuberculata was found to be the most potent anti-proliferative fraction against all three cancer cell lines. Two novel steroidal glycosides were isolated from the active fraction after a series of chromatographic experiments. The structure of the isolated compounds was elucidated solely based on 2D-NMR (HMBC, HETCOR, DQF-COSY) and MS spectral analysis as compound 1: 12-O-benzoyl-20-O-acetyl-3β,12β,14β,20β-tetrahydroxy-pregnan-3-ylO-β-D-glucopyranosyl-(1→4)-β-D-glucopyranosyl-(1 → 4)-3-methoxy-β-D-ribopyranoside, and as compound 2: 7-O-acetyl-12-O-benzoyl-3β,7β,12β,14β-tetrahydroxy-17β-(3-methylbutyl-O-acetyl-1-yl)-androstan-3-ylO-β-D-glucopyranosyl-(1 → 4)-6-deoxy-β-D-allopyranosyl-(1 → 4)-β-D-cymaropyranosyl-(1 → 4)-β-D-cymapyranosyl-(1→ 4)-β-D-cymaropyranoside. Compound 1 (pregnane glycoside) and compound 2 (androstan glycoside) induced apoptosis at <25 μM after 48 h as assessed by cell shrinkage, PARP cleavage, DNA fragmentation, and reversal with the caspase inhibitor., Conclusions: Two novel steroid glycosides isolated from Caralluma tuberculata possess moderate, micromolar cytotoxic activity on breast cancer and other cells in vitro, which may indicate a source of activity in vivo of interest to future drug design., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Ionic liquids 1-butyl-3-methylimidazolium hexafluorophosphate ([bmim](+)[PF(6)](-)) and 1-butyl-3-methylimidazolium tetrafluoroborate ([bmim](+)[BF(4)](-)) were found to promote an unusual Wagner-Meerwein rearrangement of steroidal 16α,17α-epoxides leading to unnatural 13-epi-18-nor-16-one derivatives as the main products. These compounds were isolated in good to excellent yields. 16α-Hydroxy-Δ(13)-18-norsteroids, the results of the usual rearrangement, were obtained as minor components of the reaction mixtures. The ionic liquid [bmim](+)[PF(6)](-) was shown to induce C-ring aromatization of 16α,17α-epoxyestranes due to the formation of HF, the hydrolysis product of [PF(6)](-). Increasing amounts of HF and [PO(2)F(2)](-) were detected by (19)F and (31)P NMR when the ionic liquid was reused. The structures of the steroidal products, 16-oxo-18-nor-13α-steroid derivatives, 16α-hydroxy-Δ(13)-18-norsteroids, and C-aromatic compounds were determined by two-dimensional NMR techniques and high-resolution mass spectrometry (HRMS). The ionic liquids were recirculated efficiently.

Background and Purpose: Potentiating neurosteroids are some of the most efficacious modulators of the mammalian GABA(A) receptor. One of the crucial interactions may be between the C20 ketone group (D-ring substituent at C17) of the neurosteroid, and the N407 and Y410 residues in the M4 domain of the receptor. In this study, we examined the contribution of hydrogen bonding between 17beta-substituents on the steroid D-ring and the GABA(A) receptor to potentiation by neurosteroids., Experimental Approach: Whole-cell and single-channel recordings were made from HEK 293 cells transiently expressing wild-type and mutant alpha1beta2gamma2L GABA(A) receptors., Key Results: A steroid with a 17beta-carbonitrile group (3alpha5alpha18nor17betaCN) was a potent and efficacious potentiator of the GABA(A) receptor. Potentiation was also shown by a cyclosteroid in which C21 and the C18 methyl group of (3alpha,5alpha)-3-hydroxypregnan-20-one are connected within a six-membered ring containing a double bond as a hydrogen bond acceptor (3alpha5alphaCDNC12), a steroid containing a 17beta-ethyl group on the D-ring (3alpha5alpha17betaEt) and a steroid lacking a 17beta-substituent on the D-ring (3alpha5alpha17H). Single-channel kinetic analysis indicates that the kinetic mechanism of action is the same for the neurosteroid 3alpha5alphaP, 3alpha5alpha18nor17betaCN, 3alpha5alphaCDNC12, 3alpha5alpha17betaEt and 3alpha5alpha17H. Interestingly, 3alpha5alpha17betaEt, at up to 3 microM, was incapable of potentiating the alpha1N407A/Y410F double mutant receptor., Conclusions and Implications: Hydrogen bonding between the steroid 17beta-substituent and the GABA(A) receptor is not a critical requirement for channel potentiation. The alpha1N407/Y410 residues are important for neurosteroid potentiation for reasons other than hydrogen bonding between steroid and receptor.

Many 17-substituted androstan-3alpha-ol analogs act as positive allosteric modulators of GABA(A) receptors and exert anticonvulsant and anxiolytic-like activity actions in animal models. The endogenous neurosteroid allopregnanolone (17beta-acetyl; 1) is among the most potent of these. Here we demonstrate that 3alpha-hydroxy-17beta-nitro-5alpha-androstane (2b) and its 3beta-methyl analog (3alpha-hydroxy-3beta-methyl-17beta-nitro-5alpha-androstane; 2c) modulate GABA(A) receptors as assessed by [(35)S]t-butylbicyclo-phosphorothionate and [(3)H]flunitrazepam binding with potencies equivalent to or greater than 1. These compounds also had potencies equivalent to or greater than 1 in the pentylenetetrazol and 6Hz seizure models in the mouse. Furthermore, 2b exhibited anxiolytic-like activity in the elevated zero maze. The 3beta-hydroxy, 3alpha-desmethyl analog (2a) was devoid of activity on GABA(A) receptors in vitro but had moderate activity in the seizure models, possibly as a result of epimerization in vivo at the 3-position. This conclusion was supported by the lack of in vivo activity of the 3beta-hydroxy, 3alpha-methyl analog (2d), which is not expected to undergo epimerization. We conclude that nitro can serve as a bioisostere for acetyl at the 17beta-position of 5alpha-androstan-3alpha-ol, such that the nitro analog fully retains the bioactivity of the endogenous neurosteroid at GABA(A) receptors.

Sugammadex, a cyclodextrin, is a novel agent designed to encapsulate selectively steroidal neuromuscular blocking agents such as rocuronium and vecuronium as well. One molecule of sugammadex is able to encapsulate only one molecule of muscle relaxant. This original pharmacological property allows a very rapid reversal of muscle paralysis. After sugammadex injection, a train of four ratio higher than 0.9 is obtained in less than 5 minutes in all the patients whatever the degree of muscle paralysis at the time of reversal and even when anesthesia is maintained with halogenated agents. However, in order to preserve this efficacy, the dose of sugammadex needs to be adjusted to the degree of muscle paralysis at the time of reversal : 2 mg/kg after obtaining 2 responses at the adductor pollicis muscle after a train of four stimulation, 4 mg/kg with a post-tetanic count between 1 and 3 responses, and 12 to 16 mg/kg in case of rescue reversal (3 to 15 minutes after 0.6 to 1.2 mg/kg rocuronium). Even if the original property of sugammadex lets us think that per-operative neuromuscular transmission monitoring would not be furthermore useful, the assessment of the exact degree of muscle paralysis before reversal is mandatory for choosing the right dose of sugammadex.

High levels of androgen receptor (AR) are often indicative of recurrent, advanced, or metastatic cancers. These conditions are also characterized by a high proliferative fraction. 5-Radioiodo-3'-O-(17beta-succinyl-5alpha-androstan-3-one)-2'-deoxyuridine 8 and 5-radioiodo-3'-O-(17beta-succinyl-5alpha-androstan-3-one)-2'-deoxyuridin-5'-yl monophosphate 13 target AR. They are also degraded intracellularly to 5-radioiodo-2'-deoxyuridine 1 and its monophosphate 20, respectively, which can participate in the DNA synthesis. Both drugs were prepared at the no-carrier-added level. Precursors and methods are readily adaptable to radiolabeling with various radiohalides suitable for SPECT and PET imaging, as well as endoradiotherapy. In vitro and in vivo studies confirm the AR-dependent interactions. Both drugs bind to sex hormone binding globulin. This binding significantly improves their stability in serum. Biodistribution and imaging studies show preferential uptake and retention of 8 and 13 in ip xenografts of human ovarian adenocarcinoma cells NIH:OVCAR-3, which overexpress AR. When these drugs are administered at therapeutic dose levels, a significant tumor growth arrest is observed.

Cyclodextrins consist of rings of sugar molecules with a lipophilic core and a hydrophilic periphery. Thus they are well soluble in water and possess the ability to bind (encapsulate) steroid molecules. Cyclodextrins have now been modified (sugammadex) in such a way a perfect complex formation with rocuronium results. In this way an alternative to the classical indirect antagonists has been discovered. It is now possible to terminate a neuromuscular blockade via an intravasal encapsulation of rocuronium far distant from the neuromuscular endplates and avoiding the side effects associated with acetylcholinestase inhibitors instead of by an intervention in the acetylcholine system. Furthermore, it has been found that even deep neuromuscular blockades can be reversed within 2 minutes by means of this novel mechanism.

Methyldrostanolone (2alpha,17alpha-dimethyl-17beta-hydroxy-5alpha-androstan-3-one) was synthesized from drostanolone (17beta-hydroxy-2alpha-methyl-5alpha-androstan-3-one) and identified in commercial products. Cultures of cryopreserved human hepatocytes were used to study the biotransformation of drostanolone and its 17-methylated derivative. For both steroids, the common 3alpha- (major) and 3beta-reduced metabolites were identified by GC-MS analysis of the extracted culture medium and the stereochemistry confirmed by incubation with 3alpha-hydroxysteroid dehydrogenase. Structures corresponding to hydroxylated metabolites in C-12 (minor) and C-16 were proposed for other metabolites based upon the evaluation of the mass spectra of the pertrimethylsilyl (TMS-d(0) and TMS-d(9)) derivatives. Finally, on the basis of the GC-MS and (1)H NMR data and through chemical synthesis of the 17-methylated model compounds, structures could be proposed for metabolites hydroxylated in C-2. All the metabolites extracted from hepatocyte culture medium were present although in different relative amounts in urines collected following the administration to a human volunteer, therefore confirming the suitability of the cryopreserved hepatocytes to generate characteristic metabolites and study biotransformation of new steroids.

In this study, we synthesized some new substituted steroidal derivatives using 3beta-hydroxyandrosten-17-one (dehydroepiandrosterone) as starting material. The synthesized steroidal derivatives 1-11 were evaluated for their androgenic-anabolic activities compared to testosterone as positive control. Details of the synthesis, spectroscopic data and toxicity (LD50) of synthesized compounds are reported.

The interaction of a potent percutaneous penetration enhancer, 1,8-cineole, with the stratum corneum (SC) and DPPC membranes was investigated by electron paramagnetic resonance spectroscopy (EPR) of spin-labeled analogs of stearic acid (5-DSA) and androstanol (ASL). The EPR spectra of lipid derivatives spin probes structured in stratum corneum tissue of neonatal rat containing of 0.1-10% (v/v) 1,8-cineole in the solvent indicate an abrupt increase in membrane fluidity at around 1% 1,8-cineole. These spectra of stratum corneum membranes are characterized by the presence of two spectral components differing in mobility. Component 1 was attributed to the spin labels H-bonded to the headgroups, while component 2 possibly arose from spin labels H-bonded to water molecules or temporally non-hydrogen-bonded. With the addition of 1,8-cineole, the spin probes were transferred from the motionally more restricted component 1 to the more mobile component 2, suggesting that 1,8-cineole causes ruptures in the hydrogen-bonded network of the membrane-water interface, with consequent displacements of spin probes towards the hydrophobic core. 1,8-Cineole increased the rotational diffusion rates of component 2, whereas no significant mobility changes were observed in component 1. The EPR spectra of maleimide derivative spin label (6-MSL) covalently attached to stratum corneum proteins indicate that 1,8-cineole does not alter the dynamics of protein backbones. Instead, this terpene only increases the solvent's ability to 'dissolve' and mobilize the nitroxide side chain, which is in agreement with its low irritation response.

The knowledge of the structure of a molecular crystal is frequently a prerequisite for the understanding of its solid state properties. Even though single-crystal diffractometry is the method of choice when it comes to crystal structure determination, methods using powder diffraction data become more and more competitive. There has been much recent interest in the development of a new generation of "direct-space" approaches that are particularly suited for molecular crystals. The crystallographic structure of a steroid derivative molecule (17,17-di-n-propyl-17a-aza-D-homo-5alpha-androstan-3beta-ol) was obtained in two independent ways: from a single crystal by laboratory X-rays and from a polycrystalline powder by high-resolution synchrotron powder diffraction. The molecule crystallizes in the orthorhombic space group P2(1)2(1)2(1) (a = 6.5346, b = 17.6006 and c = 19.6978 A). Hydrogen bonds form infinite chains of molecules parallel to the c axis.

For many years, much effort has been done to develop non-depolarizing neuromuscular blocking agents of rapid onset and short duration. It has been shown that rocuronium bromide is a nondepolarizing, steroidal muscle relaxant with fast onset and short-to-intermediate duration of action. In addition, rocuronium-induced neuromuscular blockade can be readily and consistently reversed following the administration of reversal agents. The pharmacology and pharmacodynamics of rocuronium bromide are summarized in this review.

Background: Binding of the steroidal molecule of rocuronium by a cyclodextrin is a new concept for reversal of neuromuscular block. The present study evaluated the ability of Sugammadex Org 25969, a synthetic gamma-cyclodextrin derivative, to reverse constant neuromuscular block of about 90% induced by rocuronium or the non-steroidal neuromuscular blocking drugs, mivacurium or atracurium, in the anaesthetized Rhesus monkey., Methods: After a bolus injection of rocuronium, mivacurium or atracurium, a continuous infusion of these drugs was started to maintain the first twitch contraction of the train-of-four at approximately 10% of its baseline value. After a steady state block of at least 10 min the infusion was stopped and the preparation was allowed to recover spontaneously. This process was repeated, but at the time the infusion was stopped, either sugammadex 0.5 or 1.0 mg kg(-1) was given in the rocuronium-induced blockade and sugammadex 1.0 mg kg(-1) was given in the mivacurium- and atracurium-induced blockade., Results: Sugammadex caused a rapid and complete reversal of rocuronium-induced neuromuscular block. The recovery time to train of four ratio=0.9 after spontaneous recovery was 14.4 min (sd=3.4 min; n=14). This was reduced significantly (P<0.001) to 3.7 min (sd=3.3 min; n=4) with sugammadex 0.5 mg kg(-1) and to 1.9 min (sd=1.0 min; n=4) with sugammadex 1.0 mg kg(-1). Signs of residual blockade or re-curarization were not observed. Reversal of mivacurium- or atracurium-induced neuromuscular block (n=2 in each experiment) by sugammadex (1.0 mg kg(-1)) was not effective. In all experiments, injection of sugammadex had no effects on blood pressure or heart rate., Conclusions: Sugammadex is effective in reversing rocuronium, but not mivacurium- or atracurium-induced neuromuscular block.

Experimental evidence and observations in humans strongly support an interactive role of mutated alpha-adducin, sodium (Na(+))/potassium (K(+))-adenosine triphosphatase (ATPase) activity and endogenous ouabain in Na(+) homeostasis and the pathogenesis of hypertension. The Ouabain and Adducin for Specific Intervention on Sodium in HyperTension (OASIS-HT) trial is an early Phase II dose-finding study, which will be conducted across 39 European centers. Following a run-in period of 4 weeks without treatment, eligible patients will be randomized to one of five oral doses of rostafuroxin consisting of 0.05, 0.15, 0.5, 1.5, or 5.0 mg/day. Each dose will be compared to a placebo in a double-blind crossover experiment with balanced randomization. Treatment will be initiated with the active drug and continued with placebo or vice versa. Each double-blind period will last 5 weeks. The primary end point is the reduction in systolic blood pressure defined as the average of three clinic readings with the patient in the sitting position. Secondary end points include the reduction in diastolic blood pressure on clinic measurement, the decrease in the 24-h blood pressure, and the incidence of end points related to safety. Secondary objectives are to investigate the dependence of the blood pressure-lowering activity on the plasma concentration of endogenous ouabain and the genetic variation of the enzymes involved in the metabolism of this hormone, and the adducin cytoskeleton proteins. Eligible patients will have Grade I or II systolic hypertension without associated conditions and no more than two additional risk factors. In conclusion, OASIS-HT is a combination of five concurrent crossover studies, one for each dose of rostafuroxin to be studied. To our knowledge, OASIS-HT is the first Phase II dose-finding study in which a genetic hypothesis is driving primary and secondary end points.

Ligand-dependent signal transduction by nuclear receptors (NRs) includes dynamic exchanges of coactivator (CoA) and corepressor (CoR) proteins. Here we focused on the structural determinants of the antagonist- and inverse agonist-enhanced interaction of the endocrine NR vitamin D receptor (VDR) and the adopted orphan NR constitutive androstane receptor (CAR) from two species with the CoR NR corepressor. We found that the pure VDR antagonist ZK168281 and the human CAR inverse agonist clotrimazole are both effective inhibitors of the CoA interaction of their respective receptors, whereas ZK168281 resembled more the mouse CAR inverse agonist androstanol in its ability to recruit CoR proteins. Molecular dynamics simulations resulted in comparable models for the CoR receptor interaction domain peptide bound to VDR/antagonist or CAR/inverse agonist complexes. A salt bridge between the CoR and a conserved lysine in helix 4 of the NR is central to this interaction, but also helix 12 was stabilized by direct contacts with residues of the CoR. Fixation of helix 12 in the antagonistic/inverse agonistic conformation prevents an energetically unfavorable free floatation of the C terminus. The comparable molecular mechanisms that explain the similar functional profile of antagonist and inverse agonists are likely to be extended from VDR and CAR to other members of the NR superfamily and may lead to the design of even more effective ligands.

The goal of this study was to develop a series of allopregnanolone analogues substituted by conformationally constrained 17beta side chains to obtain additional information about the structure-activity relationship of 5alpha-reduced steroids to modulate GABA(A) receptors. Specifically, we introduced alkynyl-substituted 17beta side chains in which the triple bond is either directly attached to the 17beta-position or to the 21-position of the steroid skeleton. Furthermore, we investigated the effects of C22 and C20 modification. The in vitro binding affinity for the GABA(A) receptor of the new analogues was measured by allosteric displacement of the specific binding of [(3)H]4'-ethynyl-4-n-propyl-bicycloorthobenzoate (EBOB) to GABA(A) receptors on synaptosomal membranes of rat cerebellum. An allosteric binding model that has been successfully applied to ionotropic glycine receptors was employed. The most active derivative is (20R)-17beta-(1-hydroxy-2,3-butadienyl)-5alpha-androstane-3-ol (20), which possesses low nanomolar potency to modulate cerebellar GABA(A) receptors and is 71 times more active than the control compound allopregnanolone. Theoretical conformational analysis was employed in an attempt to correlate the in vitro results with the active conformations of the most potent of the new analogues.

Due to the potential for misuse of a wide range of anabolic steroids in horse racing, a screening test to detect multiple compounds, via a common class of metabolites, would be a valuable forensic tool. An enzyme-linked immunosorbent assay (ELISA) has been developed to detect 17alpha-alkyl anabolic steroid metabolites in equine urine. 16beta-Hydroxymestanolone (16beta,17beta-dihydroxy-17alpha-methyl-5alpha-androstan-3-one) was synthesised in six steps from commercially available epiandrosterone (3beta-hydroxy-5alpha-androstan-17-one). Polyclonal antibodies were raised in sheep, employing mestanolone (17beta-hydroxy-17alpha-methyl-5alpha-androstan-3-one) or 16beta-hydroxymestanolone conjugated to human serum albumin, via a 3-carboxymethyloxime linker, as antigens. Antibody cross-reactivities were determined by assessing the ability of a library of 54 representative steroids to competitively bind the antibodies. Antibodies raised against 16beta-hydroxymestanolone showed excellent cross-reactivities for all of the 16beta,17beta-dihydroxy-17alpha-methyl steroids analysed and an ELISA has been developed to detect these steroid metabolites. Using this 16beta-hydroxymestanolone assay, urine samples from horses administered with stanozolol (17alpha-methyl-pyrazolo[4',3':2,3]-5alpha-androstan-17beta-ol), were analysed raw, following beta-glucuronidase hydrolysis, and following solid-phase extraction (SPE) procedures. The suppressed absorbances observed were consistent with detection of the metabolite 16beta-hydroxystanozolol. Positive screening results were confirmed by comparison with standard LCMS analyses. Antibodies raised against mestanolone were also used to develop an ELISA and this was used to detect metabolites retaining the parent D-ring structure following methandriol (17alpha-methylandrost-5-ene-3beta,17beta-diol) administration. The ELISA methods developed have application as primary screening tools for detection of new and known anabolic steroid metabolites.

The binding of rocuronium bromide to 6-perdeoxy-6-per(4-carboxyphenyl)thio-gamma-cyclodextrin sodium salt, displays biphasic behaviour characteristic of the formation of a binary and 2 : 1 ternary guest-host complex in aqueous solution. Thermodynamic and structural data on this sequential complexation process can be rationalised within a single model involving switching of the conformational equilibria of both the rocuronium bromide and cyclodextrin molecules. Isothermal titration calorimetry (ITC), NMR and fluorescence experiments in solution, together with X-ray crystallography and molecular modelling, suggest that in order to induce encapsulation both rocuronium bromide and the modified cyclodextrin undergo conformational changes. Ring A of rocuronium bromide 'switches' from the more sterically encumbered chair to the sterically less demanding twist-boat, whilst the modified cyclodextrin "opens" its cavity to allow the steroid to enter. The recognition and mutual induced fit between cyclodextrin and steroid represents a classic example of dynamic host-guest chemistry.

A series of per-6-substituted cyclodextrin derivatives was synthesized as synthetic host molecules for rocuronium, a steroidal muscle relaxant. By forming host-guest complexes with rocuronium, these cyclodextrin derivatives reverse the muscle relaxation induced by rocuronium in vitro and in vivo. The isothermal microcalorimetry data are consistent with the biological data supporting the encapsulation mechanism of action. Binary and biphasic complexes are reported with NMR experiments clearly showing free and bound rocuronium. [structure: see text]

Two orphan nuclear receptors, constitutive active (or androstane) receptor (CAR) and pregnane X receptor (PXR), are among the most important mediators of ligand-activated transcriptional induction of liver microsomal cytochrome P450 drug-metabolizing enzymes. CAR and PXR belong to the same NR1I receptor subfamily and show high sequence homology to each other. The vitamin D receptor (VDR) also belongs to the NR1I subfamily and has the second highest homology to CAR in the ligand binding domain. A 3D model of the ligand binding domain of human CAR (hCAR) was constructed based on the available X-ray structures of human PXR (hPXR) and VDR (hVDR). The model shows that the size of the ligand binding cavities of hCAR and hPXR are similar, but larger than that of hVDR. hPXR's capability of binding to extremely large ligands, such as rifampicin, implies that its binding cavity may be able to expand further through the flexibility of a surface loop. In contrast, hCAR does not have this loop so that its cavity cannot expand, suggesting that hCAR would not bind to the largest hPXR ligands. Docking calculations of selected ligands to hCAR, based on the structural model, are consistent with previously reported receptor binding data. The results from this study indicate that structural modeling will be a useful tool for understanding ligand binding to hCAR and for design of drugs free of hCAR-mediated enzyme induction.

The ability of a genus of cephalosporium-like fungus isolated from soil, Acremonium strictum PTCC 5282, for hydrocortisone biotransformation has been investigated. This potential had not been previously examined. The fermentation yielded 11beta,17beta-dihydroxyandrost-4-en-3-one, 11beta,17alpha,20beta,21-tetrahydroxypregn-4-en-3-one and 21-acetoxy-11beta,17alpha,20-trihydroxypregn-4-en-3-one. Each microbial metabolite was purified and characterized using spectroscopic methods.

The first use of neuromuscular blocking agents (muscle relaxants) in clinical practice (1942) revolutionised the practice of anaesthesia and started the modern era of surgery. Since 1942 introduction of tubocurarine (18) neuromuscular blocking agents have been used routinely to provide skeletal muscle relaxation during surgical procedures allowing access to body cavities without hindrance from voluntary or reflex muscle movement. After the introduction of tubocurarine and the depolarizing suxamethonium chloride (4) (1949) several nondepolarizing steroidal and nonsteroidal neuromuscular blocking agents with different onset time and duration of effect were introduced e.g. gallamine triethiodide (1) (1949), methocurine (2) (1949), alcuronium chloride (3) (1963), pancuronium bromide (9) (1968), vecuronium bromide (11) (1982), pipecuronium bromide (10) (1982), atracurium besylate (5) (1982), doxacurium chloride (6) (1991), mivacurium chloride (8) (1992), rocuronium bromide (12) (1994) cisatracurium besylate (7) (1996), and rapacuronium bromide (13) (2000). SZ 1677 (14) a steroid type nondepolarizing neuromuscular blocking agent under development (preclinical phase). This review article deals with a comprehensive survey of the progress in chemical, pharmacological and, in some respects, of clinical studies of neuromuscular blocking agents used in the clinical practice and under development, including the synthesis, structure elucidation, pharmacological actions, structure activity relationships studies of steroidal and nonsteroidal derivatives.

A series of mono- and per-6-substituted cyclodextrin derivatives were synthesized as synthetic receptors (or host molecules) of rocuronium bromide, the most widely used neuromuscular blocker in anaesthesia. By forming host-guest complexes with rocuronium, these cyclodextrin derivatives reverse the muscle relaxation induced by rocuronium in vitro and in vivo and therefore can be used as reversal agents of the neuromuscular blocker to assist rapid recovery of patients after surgery. Because this supramolecular mechanism of action does not involve direct interaction with the cholinergic system, the reversal by these compounds, e.g., compound 14 (Org 25969), is not accompanied by cardiovascular side effects usually attendant with acetylcholinesterase inhibitors such as neostigmine. The structure-activity relationships are consistent with this supramolecular mechanism of action and are discussed herein. These include the effects of binding cavity size and hydrophobic and electrostatic interaction on the reversal activities of these compounds.

A new convergent synthesis of the antitumor steroid formestane (4-OHA) 5 has been performed from the easily available epimeric mixture of 5 alpha- and 5 beta-androst-3-en-17-one 1a and 1b in order to attempt a yield improvement. A two-step oxidative route followed by base-catalyzed isomerization was applied to the 5 alpha- and 5 beta-epimers 1a and 1b, either as a mixture or separately, leading to the title compound 5. From epimer 1a an efficient process was attained to prepare the desired aromatase inhibitor formestane. Epimer 1b led to the formation of the same compound 5. Additionally, 1b have also been converted in 5 beta-hydroxyandrostane-3,17-dione 12 and androst-4-ene-3,17-dione 13, revealing an unexpected reactivity of the 3 beta,4 beta-epoxy-5 beta-androstan-17-one intermediate 6 formed from 1b during the first oxidative step with performic acid. Cleavage of the epoxide 6 led to the trans-diaxial and the trans-diequatorial vic-diols 7 and 8 and to the 1,3-diol 9. The formation of the abnormal products 8 and 9 were investigated through X-ray and deuterium labeling studies. Diol 8 was formed through a trans-diequatorial epoxide ring opening and the 1,3-diol 9 was formed through an intramolecular rearrangement involving a 1,2-hydride shift. All the vic-diols 3, 7 and 8 formed, proved to be good precursors for the synthesis of the target compound 5.

Type 3 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) transforms 4-androstene-3,17-dione (Delta(4)-dione) into the androgen testosterone. To produce potent inhibitors of this key steroidogenic enzyme, we performed parallel liquid-phase synthesis of 3beta-substituted androsterone (ADT) libraries (A-D) in good yields and average high-performance liquid chromatography (HPLC) purities of 92-94%. The first library (A) of 3 beta-amidomethyl-ADT derivatives (168 members), including two levels of molecular diversity on the amide (R(1) and R(2)), was synthesized with a parallel liquid-phase method (method I) in less time than with the classic chemistry method. The screening of library A revealed that relatively small hydrophobic chains at R(1) (5-8 carbons) and small hydrophobic substituents at R(2) (1-4 carbons) provided the most potent inhibitors. In accordance with these inhibition results, a second library (B) of 3 beta-amidomethyl-ADT derivatives (56 members) was generated in a very short time using an improved method based on scavenger resins and liquid-phase parallel chemistry. Library B produced more potent inhibitors than library A and provided useful structure-activity relationships that directed the design of a third library (C) of 49 members. Once again, very potent inhibitors were identified from library C and 3 beta-[(N-adamantylmethyl-N-butanoyl)aminomethyl]-3 alpha-hydroxy-5 alpha-androstan-17-one (C-7-3) was identified as the most potent inhibitor of the three libraries with an inhibitory activity (IC(50) = 35 nM) 18-fold higher than that of the natural substrate of the enzyme, Delta(4)-dione, (IC(50) = 650 nM) used itself as inhibitor. Finally, we designed a library (D) of 3-carbamate-ADT derivatives (25 members) using the efficient parallel liquid-phase method III, which allowed the synthesis of more rigid molecules with two levels of molecular diversity (R(1)/R(2) and R(3)) in the local area occupied by the adamantane group of C-7-3. Interestingly, one of the most potent inhibitors of library D, the 3R-spiro-[3'-[3' '-N-morpholino-2' '-(3' "-cyclopentyl-propionyloxy)propyl]-2'-oxo-oxazolidin-5'-yl]-5 alpha-androstan-17-one (D-5-4), showed an inhibitory activity on type 3 17 beta-HSD similar to that of compound C-7-3, while exhibiting a nonandrogenic profile.

The synthesis and binding affinities to the digitalis Na(+),K(+)-ATPase receptor of a series of 3 beta,14 beta-dihydroxy-5 beta-androstane and 3 beta-hydroxy-14-oxoseco-D-5 beta-androstane derivatives bearing a 17 alpha-(aminoalkoxy)imino chain are reported; some derivatives were also studied for their inotropic activity. Our recently proposed model of interaction of molecules with the digitalis receptor was used to design these compounds. On that basis, the possibility to design novel potent inhibitors of Na(+),K(+)-ATPase without being constrained by the stereochemistry of the classical digitalis skeleton in the D-ring region was predicted. The binding affinities of the most potent compounds in the two series, (EZ)-17 alpha-[2-[(2-aminoethoxy)imino]ethyl]-5 beta-androstane-3 beta,14 beta-diol (6f) and (EZ)-3 beta-hydroxy-17 alpha-[2-[(2-aminoethoxy)imino]ethyl]-14,15-seco-5 beta-androstan-14-one (24c) are higher than that of the potent natural compound digitoxigenin, despite the unusual alpha-exit of the substituent in position 17 of 6f or the disruption of the D-ring in 24c. These results further support the validity of our recently proposed model of binding at the digitalis receptor. Results of the inotropic tests on guinea pig atrium deserve further investigation on the pharmacological profile of these derivatives.

16alpha-Hydroxymethyl-5alpha-androstane-3beta,17beta-diol and 16beta-hydroxymethyl-5alpha-androstane-3beta,17beta-diol, were obtained from reduction of 16-acetoxymethylene-5alpha-androstan-17-one. The corresponding 16alpha,17alpha- and 16beta,17alpha-hydroxymethyl isomers were obtained by neighboring group participation of the 16- and 17-acetates, respectively. The reactions involving carbocation formation also led to ring D rearrangement products.

Various cyclic ether and other 3 alpha-hydroxyandrostane derivatives bearing a conformationally constrained hydrogen-bonding moiety were prepared. Their anesthetic potency and their binding affinity for GABA(A) receptors, measured by intravenous administration to mice and inhibition of [(35)S]TBPS binding to rat whole brain membranes, were compared with that of known anesthetic 3 alpha-hydroxypregnan-20-ones. Synthetic steroids with similar in vitro and in vivo activities to the endogenous 3 alpha-hydroxypregnan-20-ones all had an ether oxygen on the beta-face of the steroid D-ring. These results suggest that for optimal GABA(A) receptor modulation, the hydrogen bond-accepting substituent should be near perpendicular to the plane of the D-ring on the beta-face of the steroid.

A series of digitalis-like compounds with a 17-aminoalkoxyiminoalkyl or -alkenyl substituent was synthesized and evaluated for inhibition of Na(+),K(+)-ATPase and for inotropic activity. The highest inhibition was found with compounds having the substituent in configuration 17beta and the amino group at a distance of 6 or 7 bonds from C(17) of the digitoxigenin skeleton. The presence of the oxime function strengthens the interaction with the receptor, more if alpha,beta-unsaturated, thus mimicking the electronic situation of the unsaturated lactone in natural digitalis compounds. The most active compounds showed Na(+),K(+)-ATPase inhibitory potencies (IC(50)) 17-25 times higher than the standards digitoxigenin and digoxin and 3-11 times higher inotropic potencies (EC(50)) in isolated guinea pig left atria. These features are supported by a molecular model suggesting the possible interactions of the groups described above with particular amino acid residues in the H1-H2 domains of Na(+),K(+)-ATPase. Some interactions are the classical ones already described in the literature; a new, very strong interaction of the basic group with the Cys138 was found and adds new possibilities to design compounds interacting with this region of the receptor. The most interesting compounds were also studied in vivo in the anesthetized guinea pig for evaluating their inotropic effect versus the lethal dose. Compounds 9 and 12 showed a slightly higher safety ratio than digoxin and deserve further evaluation.

A wide range of xenobiotic compounds are metabolized by cytochrome P450 (CYP) enzymes, and the genes that encode these enzymes are often induced in the presence of such compounds. Here, we show that the nuclear receptor CAR can recognize response elements present in the promoters of xenobiotic-responsive CYP genes, as well as other novel sites. CAR has previously been shown to be an apparently constitutive transactivator, and this constitutive activity is inhibited by androstanes acting as inverse agonists. As expected, the ability of CAR to transactivate the CYP promoter elements is blocked by the inhibitory inverse agonists. However, CAR transactivation is increased in the presence of 1,4-bis[2-(3, 5-dichloropyridyloxy)]benzene (TCPOBOP), the most potent known member of the phenobarbital-like class of CYP-inducing agents. Three independent lines of evidence demonstrate that TCPOBOP is an agonist ligand for CAR. The first is that TCPOBOP acts in a dose-dependent manner as a direct agonist to compete with the inhibitory effect of the inverse agonists. The second is that TCPOBOP acts directly to stimulate coactivator interaction with the CAR ligand binding domain, both in vitro and in vivo. The third is that mutations designed to block ligand binding block not only the inhibitory effect of the androstanes but also the stimulatory effect of TCPOBOP. Importantly, these mutations do not block the apparently constitutive transactivation by CAR, suggesting that this activity is truly ligand independent. Both its ability to target CYP genes and its activation by TCPOBOP demonstrate that CAR is a novel xenobiotic receptor that may contribute to the metabolic response to such compounds.

Unlabelled: In an effort to determine the extent to which atracurium may represent an exception to the rule that molar potency predicts onset time, we studied the onset profile of atracurium after a dose selected to produce approximately 95% twitch depression. We compared these results with data obtained in a previous study after the administration of vecuronium, rocuronium, and cisatracurium. Eighteen ASA physical status I and II patients were studied. After the induction of anesthesia, tracheal intubation was accomplished without relaxants. The evoked electromyographic response to 0.10-Hz single stimuli was continuously recorded. After baseline stabilization, a single bolus of atracurium, averaging 0.21 mg/kg, was administered. If peak twitch depression did not fall within the range of 90%-98%, the patient was excluded. The time to 50% and 90% of peak effect was recorded. The time to 90% of maximal effect (192 +/- 23 s) was not different from that previously observed for vecuronium (201 +/- 20 s). The time to 50% of peak effect (110 +/- 15 s) was shorter (P < 0.05) after atracurium administration than after vecuronium (125 +/- 9 s). The onset times recorded for atracurium were slower than previously observed after rocuronium and more rapid than that which was seen after cisatracurium (P < 0.001). The observed onset profile of atracurium was considerably slower than anticipated, based on the drug's molar potency. The 95% effective dose (microM/kg) may not be a reliable predictor of a muscle relaxant's onset time, when the drug administered is a mixture isomers of varying potency., Implications: The speed of onset of atracurium is slower than predicted, based on its molar potency. Potency of a relaxant may not be a reliable predictor of its time to peak effect, when the drug administered is a mixture of isomers with widely different neuromuscular activities.