Physiologically Based Pharmacokinetic and Pharmacodynamic Modeling of Diazepam: Unbound Interstitial Brain Concentrations Correspond to Clinical End Points.

Benzodiazepines induce a series of clinical effects by modulating subtypes of γ-aminobutyric acid type A receptors in the central nervous system. The brain concentration-time profiles of diazepam that correspond to these effects are unknown, but can be estimated with physiologically based pharmacokinetic (PBPK) modeling. In this study, a PBPK model for the 1,4-benzodiazepines diazepam and nordiazepam was developed from plasma concentration-time courses with PK-Sim software to predict brain concentrations. The PBPK model simulations accurately parallel plasma concentrations from both an internal model training data set and an external data set for both intravenous and peroral diazepam administrations. It was determined that the unbound interstitial brain concentration-time profiles correlated with diazepam pharmacodynamic end points. With a 30-mg intravenous diazepam dose, the peak unbound interstitial brain concentration from this model is 160 nM at 2 minutes and 28.9 nM at 120 minutes. Peak potentiation of recombinant γ-aminobutyric acid type A receptors composed of α1ß2γ2s, α2ß2γ2s, and α5ß2γ2s subunit combinations that are involved in diazepam clinical endpoints is 108%, 139%, and 186%, respectively, with this intravenous dose. With 10-mg peroral administrations of diazepam delivered every 24 hours, steady-state peak and trough unbound interstitial brain diazepam concentrations are 22.3 ± 7.5 and 9.3 ± 3.5 nM. Nordiazepam unbound interstitial brain concentration is 36.1 nM at equilibrium with this diazepam dosing schedule. Pharmacodynamic models coupled to the diazepam unbound interstitial brain concentrations from the PBPK analysis account for electroencephalographic drug effect, change in 13- to 30-Hz electroencephalographic activity, amnesia incidence, and sedation score time courses from human subjects.

GABA(C) rho(1) subunits form functional receptors but not functional synapses in hippocampal neurons.

The ability to control the physiological and pharmacological properties of synaptic receptors is a powerful tool for studying neuronal function and may be of therapeutic utility. We designed a recombinant adenovirus to deliver either a GABA(C) receptor rho(1) subunit or a mutant GABA(A) receptor beta(2) subunit lacking picrotoxin sensitivity [beta2(mut)] to hippocampal neurons. A green fluorescent protein (GFP) reporter molecule was simultaneously expressed. Whole cell patch-clamp recordings demonstrated somatic expression of both bicuculline-resistant GABA(C) receptor-mediated and picrotoxin-resistant GABA(A) receptor-mediated GABA-evoked currents in rho(1)- and beta(2)(mut)-transduced hippocampal neurons, respectively. GABAergic miniature inhibitory postsynaptic currents (mIPSCs) recorded in the presence of 6-cyano-7-nitroquinoxalene-2,3-dione, Mg(2+), and TTX revealed synaptic events with monoexponential activation and biexponential decay phases. Despite the robust expression of somatic GABA(C) receptors in rho(1)-neurons, no bicuculline-resistant mIPSCs were observed. This suggested either a kinetic mismatch between the relatively brief presynaptic GABA release and slow-activating rho(1) receptors or failure of the rho(1) subunit to target properly to the subsynaptic membrane. Addition of ruthenium red, a presynaptic release enhancer, failed to unmask GABA(C) receptor-mediated mIPSCs. Short pulse (2 ms) application of 1 mM GABA to excised outside-out patches from rho(1) neurons proved that a brief GABA transient is sufficient to activate rho(1) receptors. The simulated-IPSC experiment strongly suggests that if postsynaptic GABA(C) receptors were present, bicuculline-resistant mIPSCs would have been observed. In contrast, in beta(2)(mut)-transduced neurons, picrotoxin-resistant mIPSCs were observed; they exhibited a smaller peak amplitude and faster decay compared with control. Confocal imaging of transduced neurons revealed rho(1) immunofluorescence restricted to the soma, whereas punctate beta(2)(mut) immunofluorescence was seen throughout the neuron, including the dendrites. Together, the electrophysiological and imaging data show that despite robust somatic expression of the rho(1) subunit, the GABA(C) receptor fails to be delivered to the subsynaptic target. On the other hand, the successful incorporation of beta(2)(mut) subunits into subsynaptic GABA(A) receptors demonstrates that viral transduction is a powerful method for altering the physiological properties of synapses.

Dominant gating governing transient GABA(A) receptor activity: a first latency and Po/o analysis.

Steady-state, single-channel gating of GABA(A) receptors (GABARs ) is complex. Simpler gating may dominate when triggered by rapid GABA transients present during fast inhibitory synaptic transmission and is critical to understanding the time course of fast IPSCs. We studied the single-channel activity of expressed alpha1beta1gamma2 GABARs in outside-out patches from human embryonic kidney 293 cells triggered by rapidly applied GABA (10-2000 microm) pulses (2-300 msec). Activation was analyzed with the time to first channel opening after GABA presentation, or first latency (FL). FL distributions are monoexponential at low GABA concentrations and biexponential above 30 microm GABA. The fast rate increases supralinearly to a plateau of approximately 1100 sec(-1), the apparent activation rate. The slow rate and amplitude are insensitive to GABA concentration. The results argue that doubly liganded receptors can rapidly desensitize before opening. Gating after the first opening was quantified with analysis of open probability conditioned on the first opening (P(o/o)). P(o/o) functions are biexponential, dominated by a fast component, and insensitive to GABA concentration. This suggests that open channels convert primarily to fast but also to slow desensitized states. Furthermore, dual modes of fast desensitization may influence IPSC amplitude and thereby synaptic efficacy. The findings provided for the construction of a mathematical gating model that accounts for FL and P(o/o) functions. In addition, the model predicts the time course of macroscopic current responses thought to mimic IPSCs. The results provide new insights into dominant gating that is likely operational during fast GABAergic synaptic transmission.

Zn2+ inhibition of recombinant GABAA receptors: an allosteric, state-dependent mechanism determined by the gamma-subunit.

1. The gamma-subunit in recombinant gamma-aminobutyric acid (GABAA) receptors reduces the sensitivity of GABA-triggered Cl- currents to inhibition by Zn2+ and transforms the apparent mechanism of antagonism from non-competitive to competitive. To investigate underlying receptor function we studied Zn2- effects on macroscopic and single-channel currents of recombinant alpha 1 beta 2 and alpha 1 beta 2 gamma 2 receptors expressed heterologously in HEK-293 cells using the patch-clamp technique and rapid solution changes. 2. Zn2+ present for > 60 s (constant) inhibited peak, GABA (5 microM)-triggered currents of alpha 1 beta 2 receptors in a concentration-dependent manner (inhibition equation parameters: concentration at half-amplitude (IC50) = 0.94 microM; slope related to Hill coefficient, S = 0.7) that was unaffected by GABA concentration. The gamma 2 subunit (alpha 1 beta 2 gamma 2 receptor) reduced Zn2+ sensitivity more than fiftyfold (IC50 = 51 microM, S = 0.86); increased GABA concentration (100 microM) antagonized inhibition by reducing apparent affinity (IC50 = 322 microM, S = 0.79). Zn2+ slowed macroscopic gating of alpha 1 beta 2 receptors by inducing a novel slow exponential component in the activation time course and suppressing a fast component of control desensitization. For alpha 1 beta 2 gamma 2 receptors, Zn2+ accelerated a fast component of apparent desensitization. 3. Zn2+ preincubations lasting up to 10 s markedly increased current depression and activation slowing of alpha 1 beta 2 receptors, but had little effect on currents from alpha 1 beta 2 gamma 2 receptors. 4. Steady-state fluctuation analysis of macroscopic alpha 1 beta 2 gamma 2 currents (n = 5) resulted in control (2 microM GABA) power density spectra that were fitted by a sum of two Lorentzian functions (relaxation times: 37 +/- 5.6 and 1.41 +/- 0.15 ms, means +/- S.E.M.). Zn2+ (200 microM) reduced the total power almost sixfold and accelerated the slow (23 +/- 2.8 ms, P < 0.05) without altering the fast (1.40 +/- 0.16 ms) relaxation time. The ratio (fast/slow) of Lorentzian areas was increased by Zn2+ (control, 3.39 +/- 0.55; Zn2+, 4.9 +/- 0.37, P < 0.05). 5. Zn2+ (500 microM) depression of previously activated current amplitudes (% control) for alpha 1 beta 2 gamma 2 receptors was independent of GABA concentration (5 microM, 13.2 +/- 0.72%; 100 microM, 12.2 +/- 2.9%, P < 0.8, n = 5). Both onset and offset inhibition time courses were biexponential. Onset rates were enhanced by Zn2+ concentration. Inhibition onset was also biexponential for preactivated alpha 1 beta 2 receptors with current depression more than fourfold less sensitive (5 microM GABA, IC50 = 3.8 microM, S = 0.84) relative to that in constant Zn2+. 6. The results lead us to propose a general model of Zn2+ inhibition of GABAA receptors in which Zn2+ binds to a single extracellular site, induces allosteric receptor inhibition involving two non-conducting states, site affinity is state-dependent, and the features of state dependence are determined by the gamma-subunit.