ABSTRACT
The Caenorhabditis elegans genome sequence was published over a decade ago; this was the first published genome of a multi-cellular organism and now the WormBase project has had a decade of experience in curating this genome's sequence and gene structures. In one of its roles as a central repository for nematode biology, WormBase continues to refine the gene structure annotations using sequence similarity and other computational methods, as well as information from the literature- and community-submitted annotations. We describe the various methods of gene structure curation that have been tried by WormBase and the problems associated with each of them. We also describe the current strategy for gene structure curation, and introduce the WormBase 'curation tool', which integrates different data sources in order to identify new and correct gene structures. Database URL: http://www.wormbase.org/.
Subject(s)
Caenorhabditis elegans/genetics , Computational Biology/methods , Databases, Genetic , Genes, Helminth/genetics , Molecular Sequence Annotation/methods , Animals , Base Sequence , DNA, Intergenic/genetics , High-Throughput Nucleotide Sequencing , Open Reading Frames/geneticsABSTRACT
Bimatoprost is the ethyl amide derivative of 17-phenyl-trinor prostaglandin F(2alpha). Here, we show that bimatoprost (K(i)=9250+/-846nM) and bimatoprost free acid (17-phenyl-trinor prostaglandin F(2alpha); K(i)=59+/-6nM) bind to the FP receptor and displace [(3)H]-travoprost acid, a selective FP agonist. Bimatoprost (EC(50)=3070+/-1330nM), Lumigan((R)) (bimatoprost 0.03% ophthalmic solution; EC(50)=1150+/-93nM) and bimatoprost acid (EC(50)=15+/-3nM) mobilized intracellular Ca(2+) ([Ca(2+)](i)) in <5s in HEK-293 cells expressing the cloned human ciliary body FP receptor on a fluorometric imaging plate reader (FLIPR). Furthermore, agonist effects of bimatoprost and bimatoprost acid were blocked by AL-8810 (11beta-fluoro-15-epi-15-indanyl prostaglandin F(2alpha); K(i)=0.7-2.1 MicroM), an FP receptor-selective antagonist. Therefore, the prodrug bimatoprost and its hydrolytic product, bimatoprost free acid, bind to and activate the human ocular FP prostaglandin receptor to mobilize [Ca(2+)](i), thus behaving as FP receptor agonists.
Subject(s)
Calcium/metabolism , Ciliary Body/metabolism , Cloprostenol/analogs & derivatives , Lipids/pharmacology , Receptors, Prostaglandin/agonists , Amides , Bimatoprost , Cloning, Molecular , Cloprostenol/agonists , Cloprostenol/blood , Dose-Response Relationship, Drug , Eye/metabolism , Fluorometry , Humans , Lipid Metabolism , Receptors, Prostaglandin/genetics , Receptors, Prostaglandin/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Signal Transduction , TravoprostABSTRACT
Natural prostaglandins (PGs) such as PGD2, PGE2, PGF2(2alpha), and PGI2 exhibited the highest affinity for their respective cognate receptors, but were the least selective agents when tested in receptor binding assays. Travoprost acid ([+]-fluprostenol) was the most FP-receptor-selective compound, exhibiting a high affinity (Ki = 35 +/- 5 nM) for the FP receptor, and minimal affinity for DP (Ki = 52,000 nM), EP1 (Ki = 9540 nM), EP3 (Ki = 3501 nM), EP4 (Ki = 41,000 nM), IP (Ki > 90,000 nM), and TP (Ki = 121,000 nM) receptors. Travoprost acid was the most potent PG analog tested in FP receptor functional phosphoinositide turnover assays in the following cell types: human ciliary muscle (EC50 = 1.4 nM), human trabecular meshwork (EC50 = 3.6 nM), and mouse fibroblasts and rat aortic smooth muscle cells (EC50 = 2.6 nM). Although latanoprost acid exhibited a relatively high affinity for the FP receptor (Ki = 98 nM), it had significant functional activity at FP (EC50 = 32-124 nM) and EP1 (EC50 = 119 nM) receptors. Bimatoprost acid was less selective, exhibiting a relatively high affinity for the FP (Ki = 83 nM), EP1 (Ki = 95 nM), and EP3 (Ki = 387 nM) receptors. Bimatoprost acid exhibited functional activity at the EP1 (EC50 = 2.7 nM) and FP (EC50 = 2.8-3.8 nM in most cells) receptors. Bimatoprost (nonhydrolyzed amide) also behaved as an FP agonist at the cloned human FP receptor (EC50 = 681 nM), in h-TM (EC50 = 3245 nM) and other cell types. Unoprostone and S-1033 bound with low affinity (Ki = 5.9 microM to > 22 microM) to the FP receptor, were not selective, but activated the FP receptor. In conclusion, travoprost acid has the highest affinity, the highest FP-receptor-selectivity, and the highest potency at the FP receptor as compared to the other ocular hypotensive PG analogs known so far, including free acids of latanoprost, bimatoprost, and unoprostone isopropyl ester.
Subject(s)
Binding, Competitive/drug effects , Cloprostenol/analogs & derivatives , Dinoprost/analogs & derivatives , Intraocular Pressure/drug effects , Prostaglandins F, Synthetic/pharmacology , Receptors, Prostaglandin/drug effects , Receptors, Prostaglandin/physiology , Amides , Animals , Aorta/cytology , Aorta/drug effects , Bimatoprost , Binding, Competitive/physiology , Cattle , Cell Line , Ciliary Body/cytology , Ciliary Body/drug effects , Clinical Trials as Topic , Cloprostenol/chemistry , Cloprostenol/metabolism , Cloprostenol/pharmacology , Dinoprost/pharmacology , Drug Evaluation, Preclinical , Fibroblasts/drug effects , Humans , Intraocular Pressure/physiology , Kidney/cytology , Latanoprost , Lipid Metabolism , Lipids/pharmacology , Mice , Prodrugs/chemistry , Prodrugs/metabolism , Prodrugs/pharmacology , Prostaglandins/pharmacology , Prostaglandins F, Synthetic/chemistry , Prostaglandins, Synthetic/chemistry , Prostaglandins, Synthetic/metabolism , Prostaglandins, Synthetic/pharmacology , Radioligand Assay , Rats , Receptors, Prostaglandin/agonists , Receptors, Prostaglandin/classification , Receptors, Prostaglandin E/drug effects , Receptors, Prostaglandin E, EP1 Subtype , Receptors, Prostaglandin E, EP3 Subtype , Second Messenger Systems/drug effects , Second Messenger Systems/physiology , Signal Transduction/drug effects , Signal Transduction/physiology , Stereoisomerism , Trabecular Meshwork/cytology , Trabecular Meshwork/drug effects , TravoprostABSTRACT
Bimatoprost (17-phenyl-prostaglandin F(2alpha) ethyl amide) has been reported not to exert its actions via prostaglandin receptors. Here, bimatoprost displaced [3H]prostaglandin F(2alpha) from FP receptors (K(i)=6310+/-1650 nM). Bimatoprost rapidly mobilized intracellular Ca(2+) ([Ca(2+)](i)) via cloned human FP receptors expressed in human embryonic kidney cells (EC(50)=2940+/-1663 nM) and via native FP receptors in 3T3 mouse fibroblasts (EC(50)=2200+/-670 nM). Furthermore, AL-8810 ((5Z, 13E)-(9S,11S,15R)-9,15-dihydroxy-11-fluoro-15-(2-indanyl)-16,17,18,19,20-pentanor-5,13-prostadienoic acid), an FP receptor antagonist, blocked the bimatoprost-induced [Ca(2+)](i) mobilization.
Subject(s)
Dinoprost/analogs & derivatives , Lipid Metabolism , Receptors, Prostaglandin/metabolism , 3T3 Cells , Amides , Animals , Bimatoprost , Binding, Competitive/drug effects , Calcium/metabolism , Cell Line , Cloprostenol/analogs & derivatives , Dinoprost/metabolism , Dinoprost/pharmacology , Dose-Response Relationship, Drug , Humans , Lipids/pharmacology , Mice , Receptors, Prostaglandin/agonists , Receptors, Prostaglandin/antagonists & inhibitors , TritiumABSTRACT
OBJECTIVE: The purpose of this study was to compare the efficacy and tolerability of a celecoxib 200 mg QD regimen with a 100 mg BID regimen in patients with osteoarthritis (OA) of the knee. METHODS: Patients enrolled in this prospective, double-blind, placebo-controlled, parallel-group, multicenter study were randomly assigned to receive celecoxib 100 mg BID, celecoxib 200 mg QD, or placebo for 6 weeks. Assessments of OA severity (Patient's and Physician's Global Assessments of Arthritis, Patient's Assessment of Arthritis Pain-Visual Analog Scale, Lequesne Osteoarthritis Severity Index, and the Western Ontario and McMaster Universities Osteoarthritis Index) were performed at baseline and at week 2 and/or 6. Patients who discontinued treatment underwent assessments at the time of withdrawal from the study. RESULTS: Of the 718 patients enrolled, 243 received celecoxib 100 mg BID, 231 received celecoxib 200 mg QD, and 244 received placebo. For all measures of efficacy, at all assessments, improvements from baseline in both celecoxib groups were superior to that seen in the placebo group (P < 0.05). No significant differences in efficacy between the celecoxib groups were observed. The overall incidence of adverse events was similar in the 2 celecoxib treatment groups. CONCLUSIONS: Dosing regimens of celecoxib 200 mg QD and 100 mg BID are equally effective and well tolerated in patients with OA of the knee. The availability of 2 effective regimens provides patients and physicians with increased flexibility in the selection of an appropriate dosing regimen for celecoxib therapy.
Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/administration & dosage , Osteoarthritis, Knee/drug therapy , Sulfonamides/administration & dosage , Aged , Anti-Inflammatory Agents, Non-Steroidal/therapeutic use , Celecoxib , Double-Blind Method , Drug Administration Schedule , Female , Humans , Male , Middle Aged , Pyrazoles , Sulfonamides/therapeutic useABSTRACT
1. A potent and highly selective DP prostanoid receptor antagonist radioligand, [(3)H]-cyclohexyl-N-BWA868C (3-benzyl-5-(6-carboxyhexyl)-1-(2-cyclohexyl-2-hydroxyethyl-amino) hydantoin, ([(3)H]-BWA868C)), has been generated for receptor binding and autoradiographic studies. 2. Specific [(3)H]-BWA868C binding to human platelet membranes achieved equilibrium within 60 min at 23 degrees C and constituted up to 95% of the total binding. The association (K(+1)) and dissociation (K(-1)) rate constants of binding were 0.758+/-0.064 min(-1), mmol and 0.0042+/-0.0002 min(-1), respectively, yielding dissociation constants (K(D)s) of 5.66+/-0. 44 nM (n=4). 3. Specific [(3)H]-BWA868C bound to DP receptors with a high affinity (K(D)=1.45+/-0.01 nM, n=3) and to a finite, saturable number of binding sites (B(max)=21.1+/-0.6 nmol g(-1) wet weight). 4. DP receptor class prostanoids (e.g. ZK118182, BW245C, BWA868C, PGD(2)) exhibited high (nanomolar) affinities for [(3)H]-BWA868C binding, while prostanoids selective for EP, FP, IP and TP receptors showed a low (micromolar) affinity. 5. Specific DP receptor binding sites were autoradiographically localized on the ciliary epithelium/process, longitudinal and circular ciliary muscles, retinal choroid and iris in human eye sections using [(3)H]-BWA868C. While [(3)H]-PGD(2) yielded similar quantitative distribution of DP receptors as [(3)H]-BWA868C, the level of non-specific binding observed with [(3)H]-PGD(2) was significantly greater than that observed with [(3)H]-BWA868C. 6. It is concluded that [(3)H]-BWA868C is a high-affinity and very specific DP receptor radioligand capable of selectively labelling the DP receptor. [(3)H]-BWA868C may prove useful for future homogenate-based and autoradiographic studies on the DP receptor.
Subject(s)
Blood Platelets/metabolism , Eye/metabolism , Hydantoins/metabolism , Platelet Aggregation Inhibitors/metabolism , Prostaglandin D2/metabolism , Receptors, Immunologic , Receptors, Prostaglandin/metabolism , Autoradiography , Cell Membrane/metabolism , HumansABSTRACT
The prostanoid receptor-subtype binding affinities, selectivities, potencies, and intrinsic activities of four natural prostanoids and six synthetic DP class prostanoids were determined using binding and functional assays with endogenous receptors. SQ27986 exhibited the highest affinity for the human platelet DP receptor and the best DP receptor selectivity profile. Prostaglandin (PG)D(2) was the least DP receptor-selective. The rank order of compound affinities at the DP receptor was SQ27986 (K(i) = 10 +/- 2 nM) > RS93520 = ZK110841 = BW245C (K(i) = 23-26 nM) > ZK118182 (K(i) = 50 +/- 9 nM) > PGD(2) (K(i) = 80 +/- 5 nM). DP receptor agonists produced cAMP in embryonic bovine tracheal fibroblasts with different potencies (EC(50) values in nM): ZK118182 (18 +/- 6), RS93520 (28 +/- 6), SQ27986 (29 +/- 7), ZK110841 (31 +/- 7), BW245C (53 +/- 16), and PGD(2) (98 +/- 10). BW245C was more efficacious and RS93520 was less efficacious than PGD(2). ZK110841 and ZK118182 exhibited a relatively high potency at the adenylyl cyclase-coupled EP(2) receptor in human nonpigmented ciliary epithelial cells but were partial agonists. None of the DP class agonists showed any EP(4) receptor functional activity in Chinese hamster ovary cells. The DP receptor antagonist BWA868C competitively antagonized the PGD(2)-induced cAMP accumulation in embryonic bovine tracheal fibroblast cells (pA(2) = 7.83 +/- 0.08). The dissociation constants for BWA868C antagonizing PGD(2)-, BW245C-, and ZK118182-induced cAMP production were quite similar (apparent -log K(b) = 7.9-8.2, n = 5-9). The pharmacological properties of some natural and numerous DP class synthetic prostanoids have been determined using endogenous receptors.
Subject(s)
Prostaglandins D/metabolism , Prostaglandins, Synthetic/pharmacology , Prostaglandins/pharmacology , Receptors, Prostaglandin/drug effects , 3T3 Cells , Adenylyl Cyclases/metabolism , Alprostadil/analogs & derivatives , Alprostadil/metabolism , Animals , Blood Platelets/drug effects , Blood Platelets/metabolism , CHO Cells , Cell Line , Cricetinae , Epoprostenol/metabolism , Humans , In Vitro Techniques , Mice , Phosphatidylinositols/metabolism , Prostaglandin D2/metabolism , Prostaglandins/metabolism , Prostaglandins D/pharmacology , Prostaglandins F/metabolism , Prostaglandins, Synthetic/metabolismABSTRACT
A previous study has shown celecoxib 100 mg b.i.d. to be comparably effective to naproxen 500 mg b.i.d. in treating osteoarthritis (OA). The primary objective of this study was to compare the efficacy of a once-daily regimen of celecoxib (200 mg q.d.) to the 100 mg b.i.d. regimen in treating the signs and symptoms of OA. In this double-blind, placebo-controlled, parallel-group, multicenter study, 686 patients with OA of the knee in a flare state were enrolled. Patients were randomly assigned to receive celecoxib 100 mg b.i.d. (N = 231), celecoxib 200 mg q.d. (N = 223), or the placebo (N = 232) for 6 weeks. Arthritis assessments were performed at baseline and at weeks 2 and 6, or at early termination. In all measurements of efficacy, at all assessments, improvements from baseline in both celecoxib groups were statistically superior to those in the placebo group (p = 0.001 for all post-baseline comparisons of celecoxib vs the placebo), whereas the results were quite similar and statistically indistinguishable between celecoxib 100 mg b.i.d. and 200 mg q.d.. As a representative measure, 43% of patients in each celecoxib group met the definition of "improved" in Physician's Global Assessment at week 6, versus 25% of the placebo patients. The incidence of withdrawal as a result of treatment failure was 8% for celecoxib 100 mg b.i.d., 9% for celecoxib 200 mg q.d., and 24% for the placebo. Both regimens of celecoxib were well tolerated. We conclude that celecoxib 200 mg q.d. is efficacious and safe in treating patients with OA. Furthermore, no difference in efficacy or safety can be discerned between celecoxib 100 mg b.i.d. and 200 mg q.d., providing flexibility to both patients and physicians in choosing a dosing regimen.
ABSTRACT
AL-5848 (5Z,13E)-(9 S,11R,15S)-9,11,15-trihydroxy-5,13-prostadienoic acid) is the carboxylic acid of travoprost (AL-6221), a single (+)-isomer of (+/-)-fluprostenol, an FP-class prostaglandin agonist which lowers intraocular pressure. We have prepared a radioligand from this selective prostaglandin and demonstrated its utility for studying the pharmacology and autoradiographic location of the FP-receptor. Specific [3H]AL-5848 binding (84% of total) was linearly related to bovine corpus luteum tissue concentration and reached equilibrium within 275 min at 23 degrees C. Scatchard analysis of saturation isotherms indicated interaction of [3H]AL-5848 with a single class of high-affinity (dissociation constant, Kd, = 33.8+/-2.9 nM, n = 4) and saturable (Bmax = 37.3+/-3.0 pmol (g wet weight tissue)(-1)) FP receptor-binding sites in bovine corpus luteum. Specific [3H]AL-5848 binding was potently inhibited by the FP-receptor ligands 16-phenoxyPGF2alpha (inhibition constant Ki = 17.3 nM); cloprostenol (Ki = 56.8 nM); 17-phenyl PGF2alpha (Ki = 87.0 nM); AL-5848 (Ki = 52.1 nM); PGF2alpha (Ki = 195 nM); PHXA85 (Ki = 223 nM); (n = 3-11) but very weakly by PGD2, ZK118182, BW245C, PGE2, PGI2 and U-46619. The pharmacology of specific [3H]AL-5848 binding correlated well with the pharmacology of [3H]PGF2alpha binding in the bovine corpus luteum preparation (r = 0.98, n = 14, P<0.0001) and also with functional responses in Swiss 3T3 and rat vascular smooth muscle cells (A7r5) (r = 0.96) expressing FP receptors. Autoradiographic studies revealed high levels of specific FP-receptor binding with [3H]AL-5848 on granulosa cells in the bovine corpus luteum sections, and on longitudinal ciliary muscle, the ciliary process, the iris sphincter and the retina in eye sections from man. These studies show [3H]AL-5848 to be a high-affinity agonist radioligand capable of selectively labelling the FP prostaglandin receptor.
Subject(s)
Prostaglandins F, Synthetic/metabolism , Receptors, Prostaglandin/analysis , 3T3 Cells , Adult , Aged , Animals , Autoradiography , Binding Sites , Cattle , Dinoprost/metabolism , Dose-Response Relationship, Drug , Humans , Mice , Middle Aged , Rats , Receptors, Prostaglandin/metabolism , StereoisomerismABSTRACT
Specific binding of [3H]prostaglandin (PG) E1, [3H]PGE2 and [3H]PGF2alpha to washed total particulate homogenates of bovine corpus luteum comprised 60 to 82% of total binding. Scatchard analysis of competition data revealed the presence of an apparent single population of binding sites for [3H]PGE1 and [3H]PGE2 with dissociation constants (Kds) of 2.76 to 3.39 nM and apparent receptor density (Bmax) of 1.5 to 1.56 pmol/g wet weight (n = 3-4). However, [3H]PGF2alpha appeared to interact with two classes/states of binding sites (Kd1 = 6.51 +/- 0.65 nM, Bmax1 = 2.33 +/- 0.26 pmol/g wet weight; Kd2 = 986 +/- 269 nM; Bmax2 = 44.8 +/- 11.3 pmol/g wet weight, n = 11). Specific [3H]PGE1 and [3H]PGE2 binding was most potently (nanomolar affinity) inhibited by PGs with high selectivity for the EP3 receptor subtype (e.g., GR63799, sulprostone, enprostil) but was weakly (Kis > 1 microM) influenced by EP1-selective (SC-19220), FP-selective (fluprostenol, PHXA85), DP-selective (BWA868C; ZK118182), IP-selective (iloprost) and TP-selective (U46619) PGs. Specific [3H]PGF2alpha binding was potently displaced by FP-selective agents such as fluprostenol, PHXA85 and cloprostenol with nanomolar affinities (n = 3-25), but weakly (Kis > 1 microM) by other PGs showing high selectivity for other PG receptor subtypes mentioned above. The relative specificities and potencies of EP3- and FP-selective PGs tested in the binding assays were confirmed using various functional assays. These studies have provided strong pharmacological evidence for the similarity of [3H]PGE1 and [3H]PGE2 binding to EP3 receptors and for [3H]PGF2alpha binding to FP receptors in washed bovine corpus luteum homogenates.
Subject(s)
Alprostadil/metabolism , Corpus Luteum/metabolism , Dinoprost/metabolism , Dinoprostone/metabolism , Receptors, Prostaglandin E/drug effects , Receptors, Prostaglandin E/metabolism , Receptors, Prostaglandin/drug effects , Receptors, Prostaglandin/metabolism , Animals , Autoradiography , Cattle , Corpus Luteum/drug effects , Female , In Vitro Techniques , Kinetics , Membranes/drug effects , Membranes/metabolism , Phosphatidylinositols/metabolismABSTRACT
A detailed pharmacological characterization of the prostaglandin (PG) receptor coupled to phosphoinositide (PI) turnover and intracellular calcium mobilization in Swiss 3T3 mouse fibroblast cells was undertaken. The pharmacological profile of this functional receptor was compared with the pharmacological profile of specific [3H]PGF2 alpha binding to bovine corpus luteum membranes, which are known to contain a bona fide FP receptor. PGs that were potent stimulators and full agonists in the PI turnover assay in the 3T3 cells were the following (for all, n = 3-45): 16-phenoxy-PGF2 alpha (EC50 = 0.61 +/- 0.1 nM), cloprostenol (EC50 = 0.73 +/- 0.04 nM), 17-phenyl-PGF2 alpha (EC50 = 2.71 +/- 0.35 nM), fluprostenol (EC50 = 3.67 +/- 0.61 nM), PhXA85 (EC50 = 27.3 +/- 5.63 nM) and PGF2 alpha (EC50 = 28.5 +/- 5.26 nM). However, PGD2 (EC50 = 155 +/- 29.9 nM; Emax = 49% of cloprostenol), PGE2 (EC50 = 2570 +/- 566 nM; Emax = 59%) and U46619 (EC50 = 1060 +/- 310 nM; Emax = 63%) were less potent and were partial agonists, and iloprost and BW245C were inactive. Although the PGs tested exhibited lower affinities in the 3[H]PGF2 alpha binding assay than their functional potencies in the PI turnover assay, the rank orders of potencies and affinities were well correlated (r = 0.94; n = 15 compounds). However, the PI turnover assay was more sensitive than the calcium mobilization assay for rank ordering PG agonists. In conclusion, the Swiss 3T3 cells express an FP receptor coupled to PI turnover and intracellular Ca+2 mobilization signal transduction pathways. The pharmacological profile of this receptor was similar to that of the FP receptor found in the bovine corpus luteum, a tissue previously used to clone the first pharmacologically defined FP receptor.
Subject(s)
Calcium/metabolism , Inositol Phosphates/biosynthesis , Receptors, Prostaglandin/physiology , Xanthones , 3T3 Cells , Animals , Dibenz(b,f)(1,4)oxazepine-10(11H)-carboxylic acid, 8-chloro-, 2-acetylhydrazide/pharmacology , Hydantoins/pharmacology , Inositol Phosphates/metabolism , Mice , Prostaglandin Antagonists/pharmacology , Protein Binding , Receptors, Prostaglandin/antagonists & inhibitors , Receptors, Prostaglandin/metabolism , Xanthenes/pharmacologyABSTRACT
A new method for measuring cellular rubidium (Rb+) uptake activities based on cation chromatography was developed and compared with the standard technique, uptake of the radioisotope 86Rb+, using cultured bovine retinal pigment epithelial (RPE) cells. The Rb+ response was strictly linear from 0.25 nmol (detection limit) to 25 nmol. The Na+/K(+)-ATPase inhibitor ouabain inhibited Rb+ uptake with IC50 values of 128.7 +/- 23.5 nM (n = 8; radioactive method) and 56.6 +/- 9.3 nM (n = 9; non-radioactive method, p < 0.01). The latter value is identical to the IC50 value of 54.4 +/- 16.2 nM (n = 3) for ouabain binding to the intact RPE cells. Ouabain and bumetanide, an inhibitor of the Na+/K+/Cl(-)-cotransporter, each inhibited Rb+ uptake maximally by 66 and 30%, respectively. This new technique allows sensitive measurement of intracellular Rb+, as well as K+ and Na+, and, thus, should prove useful for studying the effects of pharmacologic agents and simulated disease conditions on cation transport and cation balance in RPE and other cell types.
Subject(s)
Chromatography, Ion Exchange/methods , Pigment Epithelium of Eye/metabolism , Potassium/metabolism , Rubidium Radioisotopes/pharmacokinetics , Sodium/metabolism , Animals , Bumetanide/pharmacology , Cattle , Cell Membrane/drug effects , Cell Membrane/metabolism , Cells, Cultured , Diuretics/pharmacology , Enzyme Inhibitors/pharmacology , Intracellular Fluid/metabolism , Ion Transport , Ouabain/pharmacology , Pigment Epithelium of Eye/cytology , Pigment Epithelium of Eye/drug effects , Sodium-Potassium-Exchanging ATPase/antagonists & inhibitors , Sodium-Potassium-Exchanging ATPase/metabolismABSTRACT
Inhibition of masculine copulatory behavior was previously demonstrated following systemic injections of hydroxyflutamide (OHF). In the present study, we examined the localization of the effects of this androgen receptor blocker by direct intracranial implantation of OHF into the medial preoptic area (MPOA), ventromedial nucleus of hypothalamus (VMN), medial amygdala (AME), and lateral septum (SEPT). Animals were implanted intracranially with crystalline OHF or cholesterol, and at the same time received two 10-mm testosterone-filled Silastic capsules SC. Tests for restoration of copulatory behavior were initiated 3 days later, and conducted twice weekly for 2 weeks. Implants of OHF into the MPOA were effective in preventing restoration of male sexual behavior. However, the most effective site was the VMN. Implants of OHF into the AME were only partially effective in stimulating male sexual behavior, whereas implants into the SEPT had no effect. The OHF was discontinued and 1 week later males were retested for sexual behavior. The majority of these animals ejaculated, indicating, that the effects of OHF are reversible. The result of this study demonstrate that the functional integrity of androgen receptors in some, but not all, androgen-concentrating brain loci is necessary for the expression of the complete pattern of male sexual behavior. These data lend support to the view that androgen receptor populations in specific brain loci differentially express proteins involved in mediating the masculine copulatory response.
Subject(s)
Androgen Antagonists/pharmacology , Flutamide/analogs & derivatives , Hypothalamus/drug effects , Preoptic Area/drug effects , Sexual Behavior, Animal/drug effects , Amygdala/drug effects , Animals , Flutamide/pharmacology , Male , Rats , Septal Nuclei/drug effectsABSTRACT
This systematic approach to low back pain limits the use of imaging studies and surgery. The natural history of this condition and its resolution show that such modalities are best restricted to a minority of patients.
Subject(s)
Low Back Pain/diagnosis , Low Back Pain/therapy , Lumbar Vertebrae , Cost-Benefit Analysis , Diagnosis, Differential , Diagnostic Imaging/statistics & numerical data , Humans , Low Back Pain/economics , Low Back Pain/etiologyABSTRACT
The relative affinities of various muscarinic drugs in the antagonist ([3H]N-methyl scopolamine ([3H]NMS)) and agonist ([3H]Oxotremorine-m ([3H]OXO-M)) binding assays using a mixture of tissues containing M1-M4 receptor subtypes have been determined. [3H]NMS bound with high affinity (Kd = 25 +/- 5.9 pM; n = 3) and to a high density Bmax = 11.8 +/- 0.025 nmol/g wet weight) of muscarinic receptors. [3H]OXO-M appeared to bind to two binding sites with differing affinities (Kd1 = 2.5 +/- 0.1 nM; Kd2 = 9.0 +/- 4.9 microM; n = 4) and to a different population of binding sites (Bmax1 = 5.0 +/- 0.26 nmol/g wet weight; Bmax2 = 130 +/- 60 nmol/g wet weight). Well known antagonists exhibited high affinity for [3H]NMS binding but a lower affinity for [3H]OXO-M binding. The opposite was true for acetylcholine and other known agonists. However, pilocarpine and McN-A-343 had similar affinities for sites labeled by both radioligands. Using the ratios of antagonist-to-agonist binding affinities, it was possible to group compounds into apparently distinct full agonist (ratios of 180-665; e.g. carbachol, muscarine, OXO-M, OXO-S and arecoline), partial agonist (ratios of 14-132; e.g. McN-A-343, pilocarpine, aceclidine, bethanechol, OXA-22 and acetylcholine) and antagonist (ratios of 0.22-1.9; e.g. atropine, NMS, pirenzepine, methoctramine, 4-DAMP and p-fluorohexahydrosialo-difenidol) classes. These data suggest that the NMS/OXO-M affinity ratios using a mixture of M1-M4 muscarinic receptors may be a useful way to screen and group a large number of compounds into apparent agonist, partial agonist, and antagonist classes of cholinergic agents.
Subject(s)
Brain/metabolism , Cholinergic Agents/metabolism , Oxotremorine/metabolism , Receptors, Muscarinic/metabolism , Scopolamine Derivatives/metabolism , Animals , Binding, Competitive , Cholinergic Agents/classification , Kinetics , Lung/metabolism , Muscarinic Agonists , Muscarinic Antagonists , Myocardium/metabolism , N-Methylscopolamine , Rabbits , Radioligand Assay , Rats , TritiumSubject(s)
Hypokalemia/etiology , Liver Transplantation/adverse effects , Acute Disease , Blood Glucose/metabolism , Blood Transfusion , Diuresis , Electrolytes/blood , Erythrocyte Transfusion , Female , Humans , Hyperkalemia/etiology , Hyperkalemia/prevention & control , Hypokalemia/blood , Infant , Liver Failure/surgery , Liver Transplantation/physiology , Male , Osmolar ConcentrationABSTRACT
The pharmacokinetics of AL03152 (RS) and its enantiomers, AL03802 (R) and AL03803 (S), were studied in the Sprague-Dawley rat following intravenous bolus administration. The enantiomers had differing pharmacokinetic profiles, while the racemic compound exhibited pharmacokinetic parameters approximating the mean values of the individual enantiomers. The total clearance (CLT) values of the two enantiomers were similar, but the intrinsic clearance (Cl(int)) was much greater for the S-enantiomer than for the R-enantiomer. The volume of distribution (Vss) for AL03802 (R) was threefold greater than that for AL03803 (S). The stereoselectivity in Vss could not be totally accounted for by the slight difference in serum protein binding of the isomers and resulted in a difference in the half-lives of the enantiomers. Only the R-isomer exhibited a persistent terminal elimination phase, consistent with more extensive tissue binding than the S-isomer. AL03152 enantiomers were equivalent in potency assessed from in vitro IC50 values toward rat lens aldose reductase and rat kidney L-hexonate dehydrogenase and lens EC50 values in diabetic rats.
Subject(s)
Aldehyde Reductase/antagonists & inhibitors , Fluorenes/pharmacokinetics , Hydantoins/pharmacokinetics , Animals , Carbohydrate Dehydrogenases/antagonists & inhibitors , Chromatography, Gas , Fluorenes/administration & dosage , Fluorenes/pharmacology , Half-Life , Hydantoins/administration & dosage , Hydantoins/pharmacology , In Vitro Techniques , Injections, Intravenous , Lens, Crystalline/drug effects , Lens, Crystalline/metabolism , Protein Binding , Rats , Rats, Sprague-Dawley , Sorbitol/metabolism , StereoisomerismABSTRACT
This paper illustrates aspects of data monitoring of clinical trials in the pharmaceutical industry. Formal interim analyses are performed at least in part to address the question of whether the trial should proceed or whether there should be an early termination of the trial. For formal interim analyses, frequently independent data and safety monitoring committees are utilized for monitoring clinical trials, and adjustments to nominal significance levels for test statistics are required. Various statistical methods developed during the last fifteen years are utilized. Administrative interim analyses are those analyses that are performed without any intention to stop the trial as a consequence of those analyses. For administrative interim analyses, adjustments to significance levels may not be required, but results must still be carefully interpreted. Regardless of the interim analyses performed, it is critical that the plans for interim analyses be identified in the study protocol, and the dissemination of interim results be carefully restricted. The following clinical trials sponsored by Merck Sharp and Dohme Research Laboratories (MSDRL) will illustrate these points: CONSENSUS; CONSENSUS II; 4S; Haemophilus influenza type b efficacy trial; famotidine in upper gastrointestinal haemorrhage, and a phase II analgesic study. It is anticipated that data monitoring and interim analysis activities will increase for future clinical trials due to the availability of appropriate statistical methods and improved data management systems.