Bradford D. Harris

Isoflurane anesthesia is stereoselective

The anesthetic effects of the (+) and (-) isomers of isoflurane were determined in mice. Like the clinically important racemate, both isomers produced dose-dependent increases in anesthetic sleep time. A statistically significant (P less than 0.005, ANOVA) difference in the potencies of these isomers ((+) greater than (-)) was observed. To our knowledge, this is the first demonstration of a stereospecific action of a volatile anesthetic in vivo.

Volatile anesthetics bidirectionally and stereospecifically modulate ligand binding to GABA receptors

Pharmacologically relevant concentrations of volatile anesthetics can bidirectionally modulate radioligand binding to GABAA receptors. In mouse cerebral cortex, halothane (a prototypic volatile anesthetic) increased [3H]muscimol (a GABA receptor agonist) binding while inhibiting the binding of a GABA receptor antagonist ([3H]SR 95531). These bidirectional effects of inhalational anesthetics on ligand binding to GABA receptors are effected through changes in the Bmax with no significant alterations in the KD of these radioligands. Moreover, the concentration dependent, bidirectional modulation of radioligand binding to GABA receptors by volatile anesthetics exhibited stereoselectivity. Thus, (+)-isoflurane was about twice as potent as the (-)-enantiomer in enhancing [3H]muscimol binding and approximately 50% more potent as an inhibitor of [3H]SR 95531 binding, respectively. The demonstration of a bidirectional, stereospecific modulation of radioligand binding to GABA receptors by inhalational agents is consistent with the presence of specific recognition sites for inhalational anesthetics on the GABAA receptor complex.

Stereospecific actions of the inhalation anesthetic isoflurane at the GABAA receptor complex.

The inhalation anesthetic isoflurane stereoselectively modulates ligand binding to the GABAA receptor complex. The (+)-isomer of isoflurane was more potent and efficacious than the (-)-isomer in enhancing [3H]flunitrazepam binding to benzodiazepine receptors. For example, concentration effect curves for Cl- enhancement of [3H]flunitrazepam binding were significantly different (P < 0.001) in the presence of (+)- and (-)-isoflurane (0.44 and 0.88 mM). At the higher anesthetic concentration, they potency of Cl- to increase [3H]flunitrazepam binding was increased 3.2- and 1.45-fold by (+)- and (-)-isoflurane, respectively (P < 0.05). Likewise, concentration-effect curves for (+) isoflurane-enhanced [3H]flunitrazepam binding were significantly different (P < 0.05-P < 0.001) from the (-)-isomer in the presence of 0-200 mM Cl-. Stereoselectivity was not observed with respect to the potencies of these enantiomers as inhibitors of [35S]t-butylbicyclophosphorothionate (TBPS) binding to sites within the Cl- ionophore. In this measure, the isomers had similar potencies (P > 0.05), although at higher concentrations (> 0.1 mM) (+)-isoflurane produced significantly more inhibition than (-)-isoflurane. While the absolute potency differences between isomers were modest (< or = 2-fold) and measure dependent, these effects were manifested at clinically relevant concentrations of isoflurane and are consistent with in vivo studies demonstrating (+)-isoflurane is a more effective anesthetic than the (-)-isomer. This is the first demonstration of an inhalation anesthetic producing a stereoselective perturbation of the GABAA receptor complex.

The potential for safer anaesthesia using stereoselective anaesthetics

The molecular mechanisms by which inhalational agents produce anaesthesia remains a subject of controversy, despite a history of clinical use spanning two centuries. The demonstration of a significant difference in the anaesthetic potencies of (+)- and (-)-isoflurane provides compelling evidence for the hypothesis that proteins, rather than lipids, are the primary sites of anaesthetic action. Moreover, the optically active isomers of volatile anaesthetics provide new tools to discriminate among putative molecular targets of anaesthesia. A difference in the anaesthetic potencies of (+)- and (-)-isoflurane, together with an apparent lack of stereoselectivity in their myocardial suppression, raises the possibility that an optically active volatile agent may have clinical advantages over currently available racemic mixtures.

Aminoglycoside neurotoxicity involves NMDA receptor activation

Previous studies have led to the hypothesis that the ototoxicity produced by aminoglycoside antibiotics involves the excitotoxic activation of cochlear NMDA receptors. If this hypothesis is correct, then these antibiotics should also injure neurons within the brain. Because aminoglycosides do not readily penetrate the blood brain barrier, we examined the effects of the aminoglycoside neomycin following intrastriatal injection. Neomycin (10-250 nmol) produced dose-dependent striatal damage manifested as an increased gliosis as measured by: (1) [3H]PK-11195 binding, (2) staining for the astrocytic marker glial fibrillary acidic protein (GFAP) and (3) staining for OX-6, an MHC class II antigen expressed by microglia and macrophages. Co-injection of subthreshhold doses of NMDA potentiates the striatal damage produced by neomycin (10 nmol). Moreover, neomycin-induced striatal damage is attenuated by a combination of the NMDA antagonists ifenprodil and 5, 7-dichlorokynurenic acid. Intrastriatal administration of compounds structurally related to neomycin, but devoid of modulatory actions at NMDA receptors (paromamine and 2-deoxystreptamine), fail to produce neuronal damage. These data support the hypothesis that aminoglycoside-induced ototoxicity is, in part, an excitotoxic process involving the activation of NMDA receptors. Moreover, aminoglycosides may damage the central nervous system in individuals with compromised blood brain barriers.

Stereoselective actions of halothane at GABAA receptors

Isoflurane anesthesia exhibits stereoselectivity, and a corresponding stereoselectivity ((+)->(-)-isomer) has been reported at GABA(A) receptors in vitro. The objective of the present study was to determine if the positive modulatory actions of halothane at GABA(A) receptors exhibited a similar stereoselectivity. Both (R)- and (S)-halothane ((+)- and (-)- isomers, respectively) enhanced [3H]flunitrazepam binding to brain membranes in a concentration dependent manner without a significant difference in either potency (EC50) or efficacy (Emax). While both (R)- and (S)-halothane enhanced [3H]muscimol binding, the potency of the (+)-isomer was slightly greater than the corresponding (-)-isomer (0.91 +/- 0.17 versus 1.45 +/- 0.04% atmospheres, respectively (P < 0.02)). Thus, subtle structural differences between inhalational anesthetics can have a significant impact on the degree of stereoselectivity at the receptor level and may provide insights for the development of more specific drugs.

Syntheses of 5-thienyl and 5-furyl-substituted benzodiazepines: probes of the pharmacophore for benzodiazepine receptor agonists

Summary The synthesis of 5-thienyl and 5-furyl-substituted benzodiazepines is described. These compounds were employed to probe the lipophilic pocket ( L 3 ) of the benzodiazepine receptor (BzR) and to determine the effect of occupation of L 3 on biological activity. Of the new analogs synthesized, the 5-(2-thienyl)-benzodiazepines 6a and 7a displayed high affinity for the BzR (IC 50 28 and 18 nM, respectively) and exhibited both anticonvulsant (ED 50 ~ 9 and 3 mg/kg) and muscle relaxant (ED 50 ~ 10 and 7 mg /kg) activity. The 5-(3-thienyl)benzodiazepines 6d and 7d displayed only moderate affinity for the BzR (IC 50 140 and 110 nM) and exhibited no biological activity (no anticonvulsant or muscle relaxant activity) at doses up to 40 mg/kg. The 5-(2-furyl)benzodiazepines ( 6b, 7b, 19b and 20b ) exhibit low affinities for the BzR. These in vitro and in vivo findings are consistent with our model suggesting that pocket L 3 is very sensitive to lipophilic effects. Thus, decreasing the lipophilicity of functional groups which occupy this region decreases ligand affinity at BzR. The 2′-halogen (F or Cl) substituent of the 5-phenylbenzodiazepines increases ligand affinity in vitro because the active conformation of the phenyl N(4)=C(5)-C(1′)=C(2′) moiety is syn rather than anti . The syn conformation permits the 2′-halogen (F or Cl) atom to interact at the hydrogen bonding site H 2 and form a stable three-centered hydrogen bond in the proposed ligand binding cleft. The 3-thienyl and 2-furyl groups decrease the lipophilicity of the substituent which occupies L 3 but do not form a hydrogen bond at H 2 , thus resulting in a diminished affinity at BzR.