Unraveling the molecular dynamics of sugammadex-rocuronium complexation: A blueprint for cyclodextrin drug design.

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.

Rapid detection of rocuronium based on host/guest complex between a pyrene derivative and sugammadex.

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.

Exploring the Polymorphism of Drostanolone Propionate.

2α-Methyl-4,5α-dihydrotestosterone 17ß-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 P21 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 P212121 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.

Crystal structures of 3ß,19-dihydroxyandrost-5-en-17-one, 5, and its monohydrate, [(5).H2O]: A survey of the structures of related di- and tri-substituted hydroxy derivatives and their solvated compounds.

The crystal structures of 3ß,19-dihydroxyandrost-5-en-17-one, 5, and its monohydrate, [((5).H2O]], are reported. The monohydrate, isolated from a solution of 5 in 50% aqueous methanol, recrystallizes in the orthorhombic space group, P212121, while that of the anhydrous compound, isolated from solutions of THF, Me2CO, EtOAc or dry MeOH, recrystallizes in the monoclinic space group, P21. Apart from the different orientation of the 3-HO group, the conformations of the steroid molecules in 5 and [(5).H2O]: 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, 3ß-hydroxyandrost-5-en-17-one (dehydroepiandrosterone). An additional structural difference between 3ß-hydroxyandrost-5-en-17-one and 3ß,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).H2O]], 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).H2O], we report on a survey of the crystal structures of related di- and tri- hydroxy-17-one derivatives as well as solvated compounds.

Crystal structures and Hirshfeld surface analyses of the di- and tri-hydrates of (5α,17E)-17-hydrazonoandrostan-3-ol: Significant differences in the hydrogen bonding patterns and supramolecular arrangements.

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·(H2O)2] and [3·(H2O)3], are reported. The trihydrate, isolated from a solution of 3 in moist methanol, recrystallizes in the orthorhombic space group, P212121, while that of the dihydrate, isolated from a 1:1 aqueous methanol solution, recrystallizes in the monoclinic space group, P21. 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.

Dexamethasone Does Not Inhibit Sugammadex Reversal After Rocuronium-Induced Neuromuscular Block.

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.

New drostanolone metabolites in human urine by liquid chromatography time-of-flight tandem mass spectrometry and their application for doping control.

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-6ß-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.

Structural characterization of a degradation product of rocuronium using nanoelectrospray-high resolution mass spectrometry.

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.

Development of SPME method for concomitant sample preparation of rocuronium bromide and tranexamic acid in plasma.

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.

[Possible clinical potential in reverting muscular block with sugammadex in anaesthesia and surgery]. / Muligt klinisk potentiale ved revertering af neuromuskulær blokade med sugammadex ved anæstesi og kirurgi.

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.

The cyclodextrin sugammadex and anaphylaxis to rocuronium: is rocuronium still potentially allergenic in the inclusion complex form?

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.

Probing amphotericin B single channel activity by membrane dipole modifiers.

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-3ß-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-3ß-ol. These findings suggest that the processes of association/dissociation of channel forming molecules depend on the membrane fluidity.

Synthesis of novel D-ring fused 7'-aryl-androstano[17,16-d][1,2,4] triazolo[1,5-a]pyrimidines.

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 3ß-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.

Novel acylated steroidal glycosides from Caralluma tuberculata induce caspase-dependent apoptosis in cancer cells.

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-3ß,12ß,14ß,20ß-tetrahydroxy-pregnan-3-ylO-ß-D-glucopyranosyl-(1→4)-ß-D-glucopyranosyl-(1 → 4)-3-methoxy-ß-D-ribopyranoside, and as compound 2: 7-O-acetyl-12-O-benzoyl-3ß,7ß,12ß,14ß-tetrahydroxy-17ß-(3-methylbutyl-O-acetyl-1-yl)-androstan-3-ylO-ß-D-glucopyranosyl-(1 → 4)-6-deoxy-ß-D-allopyranosyl-(1 → 4)-ß-D-cymaropyranosyl-(1 → 4)-ß-D-cymapyranosyl-(1→ 4)-ß-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.

Drug-specific cyclodextrins with emphasis on sugammadex, the neuromuscular blocker rocuronium and perioperative anaphylaxis: implications for drug allergy.

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.

Ionic liquid-promoted Wagner-Meerwein rearrangement of 16α,17α-epoxyandrostanes and 16α,17α-epoxyestranes.

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.

[Indications and clinical use of sugammadex]. / Indications et utilisation clinique du sugammadex.

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.

Radiolabeled 5-iodo-3'-O-(17beta-succinyl-5alpha-androstan-3-one)-2'-deoxyuridine and its 5'-monophosphate for imaging and therapy of androgen receptor-positive cancers: synthesis and biological evaluation.

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.

Hydrogen bonding between the 17beta-substituent of a neurosteroid and the GABA(A) receptor is not obligatory for channel potentiation.

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.

17beta-Nitro-5alpha-androstan-3alpha-ol and its 3beta-methyl derivative: neurosteroid analogs with potent anticonvulsant and anxiolytic activities.

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.