The peripheral benzodiazepine receptor (PBZ) population in the rat liver has been kinetically and pharmacologically characterized. These data revealed a receptor with a dissociation constant (Kd) of 5.67 nM and a receptor density of 758 fmoles/ mg protein in a crude membrane preparation. Subcellular distribution studies demonstrated that PBZs were detected in highly purified nuclear, mitochondrial and microsomal fractions. Porphyrins were shown to interact at the PBZ with protoporphyrin IX and hemin having the highest affinity. Preliminary studies on the subnuclear distribution of PBZs are reported. In summary, the study has (1) characterized liver PBZs, (2) shown the subcellular distribution of these receptors and (3) pharmacologically characterized nuclear, mitochondrial and microsomal PBZs in the liver.

Thesis (Ph.D.)--Hong Kong University of Science and Technology, 2005. / On t.p. final "A" of GABAA is subscript. Includes bibliographical references (leaves 148-167). Also available in electronic version.

Clobazam (CLBZ) is a 1,5-benzodiazepine with potent anticonvulsant activity. The metabolism of this drug was investigated in the rat and in vitro with the aid of stable isotope-labelled analogues and gas chromatography-mass spectroscopy (GCMS). Pentadeuteriophenyl CLBZ [²H₅]CLBZ was synthesized in essentially quantitative isotopic purity, and characterized by ¹H-NMR and GCMS. Of the five steps involved in the synthesis of [²H₅]CLBZ, the most susceptible to deuterium exchange was the nucleophilic substitution of 2,4-dichloronitrobenzene by aniline-d₇ to form N-(5-chloro-2-nitrophenyl)pentadeuteriophenylamine 18. In this step, the isotopic impurity aniline-2,3,4,5,-d₅ introduced protons from nitrogen into the ortho and para positions of the deuteriophenyl ring of 18. Phenol and catechol metabolites of CLBZ and N-desmethylclobazam (DMC) were synthesized according to the method used for [²H₅]CLBZ using 4-methoxyaniline and 3,4-dimethoxyaniline as starting materials. The methyl ether protecting group was well-suited for the synthetic procedure and was cleanly removed under mild conditions with BBr₃ to afford the phenols and catechols in good yield. The 0-methylated catechols of CLBZ and DMC were enzymatically synthesized from the catechol analogues of CLBZ and DMC using rat liver cytosol as a source of catechol O-methyltransferase (COMT) and S-adenosyl-L-methionine as the methyl donor. A meta/para O-methylation ratio of 2 was obtained from the CLBZ catechol. Condensation of formaldehyde with DMC catalyzed by K₂CO₃ produced a compound whose GCMS (EI) spectral properties were consistent with the carbinolamide, however, this compound could not be isolated because of its marginal stability. A major product for this reaction was 3-hydroxymethyl DMC which was characterized by ¹H-NMR and LCMS. Formaldehyde condensation catalyzed by KOH also afforded 3-hydroxymethyl DMC. Whether formaldehyde addition at the 3-position occurs as a kinetic product or whether it arises as a result of an equilibrium process subsequent to N-hydroxymethylation remains to be resolved. Rats were administered CLBZ:[²H₅]CLBZ as an approximate 50:50 mixture. Isotope shifts detected by GCMS allowed the following CLBZ metabolites to be identified. In bile, decreasing levels of 4'-hydroxy CLBZ, 4'-hydroxy DMC, O-methylated CLBZ catechols, 4'-hydroxy-3'-methoxy DMC and 3',4'-dihydroxy CLBZ appeared as both glucuronide and sulfate conjugates. In urine decreasing levels of the following sulfate conjugates were observed: 4'-hydroxy-3'-methoxy CLBZ, 4'-hydroxy CLBZ, 4'-hydroxy DMC and 3',4'-dihydroxy CLBZ. In bile, the glucuronides were predominant over the sulfates, whereas in urine only sulfate conjugates were detected. There was no detectable evidence of unconjugated CLBZ metabolites in bile or urine. As the biliary glucuronide and urinary sulfate, 3'-hydroxy-4'-methoxy CLBZ constituted ≤2 % of the O-methylated CLBZ catechols, whereas in bile, the sulfate conjugate of this metabolite constituted 30 %. One possible explanation for the higher levels of this metabolite as a biliary sulfate could be the low regiospecific methylation of dihydroxy CLBZ by COMT combined with arylsulfatase activity present in the kidney. Neither the CLBZ carbinolamide nor the N-hydroxy DMC (hydroxamic acid) was present in detectable levels in the conjugated or unconjugated fractions of bile or urine. Isotope effects in the metabolism of CLBZ and DMC in the rat were marginal for phenyl ring oxidation as measured for phenol metabolites (kH/kD =1.103 ± .090) and for catechols (kH/kD. = 1.088 ± .207). The metabolism of CLBZ and DMC in vitro was examined using control and phenobarbital-induced rat liver microsomes. CLBZ metabolism resulted in N-demethylation to DMC and phenyl ring hydroxylation at the 4'-position. There was no detectable evidence for the carbinolamide intermediate. Induction appeared to increase N-demethylation compared to phenyl ring hydroxylation. DMC was metabolized to 4'-hydroxy DMC with no detectable production of the hydroxamic acid. The isotope effect associated with CLBZ N-demethylation was determined by incubating an approximate 50:50 mixture of pentadeuteriophenyl CLBZ:trideuteriomethyl CLBZ with control and phenobarbital-induced rat liver microsomes. In this way, the source of DMC derived from demethylation ([²H₅]DMC) and dedeuteriomethylation (DMC) could be determined. The values of kH/kD, determined by protio/deuterio product ratios, were calculated at 3.65 ± 0.27 (n = 3) and 2.80 ± 0.16 (n = 4) for control and induced microsomes respectively. / Pharmaceutical Sciences, Faculty of / Graduate

Diss. (sammanfattning) Stockholm : Karol. inst., 2002. / Härtill 5 uppsatser.

No description available.

Thesis (doctoral)--Lund University, 1995. / Added t.p. with thesis statement inserted.

Thesis (doctoral)--Lund University, 1995. / Added t.p. with thesis statement inserted.

No description available.

Benzodiazepine (BZ) drugs, such as diazepam (Valium®) and chlordiazepoxide (Librium®), are widely prescribed for their sedative/anxiolytic properties but also impair mnemonic processes in both humans and animals. In the Morris water maze, an aversively motivated spatial learning task, BZs impair spatial learning but spare retention/performance. This spatial learning deficit cannot be attributed to sedation, gross sensorimotor impairments, hypothermia, state-dependent learning, or reductions of escape motivation (anxiolysis). The following series of experiments sought to further characterize the neurochemical, neuroanatomical, and electrophysiological substrates of BZ-induced impairments of spatial learning. In Experiment I, the role of endogenous BZs in spatial learning was assessed. The BZ receptor antagonists flumazenil (Ro 15-1788) and CGS 8216, as well as the BZ receptor inverse-agonist β-carboline, enhanced spatial learning in an inverted-U dose-dependent manner, suggesting that endogenously released BZs impede optimal learning. In Experiment II, the role of the BZ ω1 receptor subtype in spatial learning was assessed. CL 218,872, a selective agonist for the BZ ω1 receptor subtype, impaired spatial learning in a dose-dependent and flumazenil-reversible manner, thereby implicating the ω1 receptor subtype in BZ-induced amnesia. Together these results suggest that endogenous BZs activity, like BZ drugs, is detrimental to spatial learning and that specific BZ receptors mediate this impairment. Several neurochemical systems are important for spatial learning in the MWM and arc influenced by BZs. The contributions of two of these neurochemical systems, the opioids and acetylcholine (ACh), to the spatial learning deficit produced by BZs were assessed. In Experiment III, a better understanding of the role of opioid systems in spatial learning was sought. Morphine, a prototypical opioid, impaired spatial learning in a dose-dependent and naloxone-reversible manner. However, morphine also impaired performance and escape to a visible platform and its effects on spatial learning could be attenuated by increasing the escape incentive (colder water). This impairment pattern suggests that morphine impairs spatial learning by reducing escape motivation. Because both BZs and cold water immersion increase endogenous opioid activity, it seemed possible that the combination of drug- and water-induced opioid release might mediate the spatial learning deficit produced by BZs. In Experiment IV, naloxone, an opioid receptor antagonist, completely blocked the spatial learning deficit produced by morphine but failed, even at a higher dose, to block the spatial learning deficit produced by diazepam . Conversely, flumazenil, a BZ receptor antagonist, completely blocked the spatial learning deficit produced by diazepam but failed to affect the amnesic effects of morphine. Together, these findings strongly suggest that the spatial learning deficit produced by BZs is not due to enhanced opioid activity. There is also biochemical evidence that BZs interact with ACh systems. In Experiment V, flumazenil attenuated the spatial learning deficit produced by scopolamine, an ACh (muscarinic) antagonist, but physostigmine, an acetylcholinesterase inhibitor, failed to attenuate the spatial learning deficit produced by chlordiazepoxide, even at doses that completely reversed the spatial learning deficit produced by scopolamine. Together these results fail to support the notion that BZs impair spatial learning by reducing ACh activity but suggest that scopolamine impairs spatial learning by enhancing endogenous BZ activity. Several neuroanatomical regions possess a high density of BZ receptors and are also integral for spatial learning in the MWM. In Experiment VI, infusions of chlordiazepoxide into the medial septum, but not frontal cortex, nucleus basalis magnocellularis, amygdala, hippocampus, or cerebellum, impaired spatial learning but had little effect on anxiety. Conversely, infusions of chlordiazepoxide into the amygdala reduced anxiety but had little effect on spatial learning. These results suggest that the medial septum mediates the amnesic effects of BZs and that the amygdala mediates the anxiolytic effects. In Experiment VII, intraseptal infusions of chlordiazepoxide were additionally found to impair spatial learning in a dose-dependent and flumazenil-reversible manner. However, infusions of flumazenil into the medial septum failed to block the amnesic effects of systemically administered chlordiazepoxide, suggesting that the amnesic to effects of BZs are not mediated by the medial septum exclusively. Tetrahydroaminoacridine, an acetylcholinesterase inhibitor, failed to attenuate the spatial learning deficit produced by intraseptal infusions of chlordiazepoxide, suggesting that the deficit was not due to a disruption of the septohippocampal ACh projection. Together, these results suggest that chlordiazepoxide impairs spatial learning by interacting with the septohippocampal GABAergic projection. The septohippocampal GABAergic projection regulates the excitability of hippocampal afferents (e.g., perforant path). Experiment VIII assessed the effects of systemically administered BZs on the induction of long-term potentiation (LTP) in the perforant path. CL 218,872, but not chlordiazepoxide or diazepam , significantly suppressed long-term potentiation. However, all drugs impaired spatial learning. These findings suggest that CL 218,872 impairs spatial learning by suppressing LTP but that BZ-induced spatial learning deficits can occur in the absence of perforant path LTP suppression. Taken together, the above results suggest that endogenous BZ systems, particularly those in the septohippocampal system, are important modulators of mnemonic processes. These findings are discussed in the context of understanding information storage processes and the implications for clinical populations. / Graduate

Flavanone (2,3-dihydro-2-phenyl-4H-benzopyran-4-one) and a series of 4'- and 7-halogeno derivatives were prepared from the corresponding 2'-hydroxychalcones [1-(2-hydroxyphenyl)-3-phenyl-2-propen-l-ones], which, in turn, were synthesized by aldol condensation of substituted 2'-hydroxacetophenones with various benzaldehydes. A series of 2,3-dihydro-2-phenyl-l,4-benzoxazepin-5(4H)-ones were prepared by ring expansion of the corresponding flavanones, via the Schmidt reaction, using trimethylsilylazide and trifluoroacetic acid. A series of tetrazoles {2,3-dihydro-2-phenyl-tetrazolo[1,5-d]-1,4-benzoxazepines} were also isolated as by-products of the Schmidt reaction. Flavanone oxime was synthesized for use in Beckmann reactions, and its molecular structure was determined by x-ray crystallography. Attempts to prepare 1,4-benzoxazepinone or its 1,5-analogue via Beckmann rearrangement of flavanone oxime, with polyphosphoric acid or phosphorus pentachloride catalysts, however, were unsuccessful. Several methods for introducing Δ²-unsaturation into the benzoxazepinone system were also examined. High resolution ¹H n.m.r., computer modelling, and molecular mechanics techniques were used to determine the conformations of the heterocycles of the benzoxazepinones and tetrazoles and results are compared with earlier studies in this field. Certain trends in the fragmentation patterns were observed in the low resolution mass spectra of the benzoxazepinones and tetrazoles, and high resolution mass spectrometric data were used to explore the major fragmentation patterns of these compounds.