Benzodiazepine Compounds Research Articles

QSAR studies—potent benzodiazepine γ-secretase inhibitors

A series of benzodiazepine compounds, which act as γ-secretrase inhibitors were subjected to QSAR studies. A correlation between the physicochemical properties Q log P, SMR and the inhibitory activity was obtained and a model equation was generated to predict the best possible pharmacophore for treating Alzheimer’s disease. The inhibitory activity of the compound depends on the lipophilicity positively and is sensitive to small changes in its SMR. The compound with a high lipophilicity (around 9.31) and low SMR gives a potent activity.

Pharmacokinetic Studies of Intramuscular Midazolam in Guinea Pigs Challenged with Soman

Studies have demonstrated that benzodiazepine compounds are effective at antagonizing seizure activity produced by the organophosphate (OP) cholinesterase inhibitor soman. In this present study we have investigated the pharmacokinetics of midazolam and its associated effects on electroencephalographic (EEG) activity following intramuscular (im) injection to soman‐exposed guinea pigs (Crl:(HA)BR). Prior to experiments, the animals were surgically implanted with EEG leads to monitor seizure activity. For the study, animals were administered the following pretreatment/OP/treatment regimen. Pyridostigmine bromide (0.026 mg/kg, im) was given 30 min prior to soman (56 µg/kg, 2 × LD50; subcutaneously, sc), followed in one minute by atropine sulfate (2 mg/kg, im) and pralidoxime chloride (25 mg/kg, im). All animals receiving this regimen developed seizure activity. Midazolam 0.8 mg/kg, im, was administered 5 min after onset of seizure activity. Based on EEG data, animals were categorized as either seizure‐terminated or seizure not‐terminated at 30 min following anticonvulsant administration. Serial blood samples were collected for the plasma midazolam analysis; the assay was accomplished with a gas chromatograph/mass spectrometer. The mean time to seizure termination was 8.8 ± 1.6 min. The mean time‐plasma concentration data were fit to standard pharmacokinetic models. The following parameter estimates were determined from the model‐fit for seizure terminated and not‐terminated animals respectively: apparent volumes of distribution (Vd) were 1.4 and 1.7 l/kg; area under the time‐concentration curves (AUC), 15,990 and 15,120 ng · min/ml; times to maximal plasma concentration (Tmax), 1.66 and 2.91 min and maximal plasma concentrations (Cmax) 535.1 and 436.6 ng/ml. These data indicate that im injection of midazolam is effective at terminating ongoing soman‐induced seizure activity. Additionally, the relatively short Tmax and latency to seizure termination demonstrate the rapidity of drug absorption and action respectively.

Anxiolytic profile of HG1, a 5-HT-moduline antagonist, in three mouse models of anxiety

HG1 is a new 5-HT-moduline antagonist which is itself an endogenous tetrapeptide specifically acting as an antagonist of 5-HT 1B auto- and heteroreceptors. Blockade of endogenous 5-HT-moduline might provoke anxiolysis, so it could be a new therapeutic target in anxiety disorders. The aim of our study was to examine the effects of HG1 in three mouse models of anxiety: the four plates test (FPT), the black and white (B&W) model and the elevated plus maze (EPM). Male Swiss mice were intraperitoneally and acutely administered HG1 at the doses of 8, 16, 32 and 64 mg/kg. In these three tests, HG1 exhibited an anxiolytic profile similar to that of diazepam, the referential benzodiazepine compound, without affecting locomotor activity. In the three models used, HG1 was as efficient as benzodiazepine and may consequently exert its anxiolytic effects via the GABA-ergic system. We cannot exclude that it might also act through 5-HT receptors and rather have the profile of a selective serotonin reuptake inhibitor.

Benzodiazepines

Many benzodiazepine compounds are available; the main differences between them relate to their duration of action. Benzodiazepines are often taken in overdose with CNS depressants or ethanol, potentiating their respiratory depressant effects. This may be particularly hazardous with opioids. However, co-ingestion of benzodiazepines may be beneficial if the other agent causes convulsions. For this reason, routine use of benzodiazepine antagonists as a 'diagnostic test' in poisoning is absolutely contraindicated.

Use of clobazam for the treatment of refractory complex partial seizures

Clobazam (CLB) add-on therapy was attempted in 183 patients with intractable complex partial seizures in whom conventional benzodiazepines had been successfully discontinued before initiation of CLB. Although complete remission was initially achieved in 61, tolerance developed in almost half (49.2%) within the first 3 months, whereas 23 out of 31 patients (74.2%) who remained seizure free for the first 3 months continued to be so over the next 3 months. CLB add-on therapy proved to be significantly more effective when concurrent GTC occurred more often than yearly. In the current series, no frank psychotic episodes were elicited among the 61 patients who achieved complete suppression of long-standing complex partial seizures, which was in agreement with previous studies. From these results, we believe that CLB is an effective, safe, and inexpensive medication for add-on therapy in difficult to treat focal epilepsies, especially without concurrent use of conventional benzodiazepine compounds.

Incorporating measures of variability and uncertainty into the prediction of in vivo hepatic clearance from in vitro data.

The existing procedures for quantitative in vitro-in vivo clearance prediction can be significantly biased either by totally neglecting the existing variability and uncertainty by using mean parameter values or by implementing Monte Carlo simulation with statistical distribution of the parameters reconstructed from very small sets of data. The aim of the present study is to develop a methodology for the prediction of in vivo hepatic clearance in the presence of semiquantitative or qualitative data and accounting for the existing uncertainty and variability. The method consists of two steps: 1) transformation of the information available into fuzzy sets (fuzzification); and 2) computation of the in vivo clearance using arithmetic operations with fuzzy sets. To illustrate the approach, rat hepatocyte and microsomal data for eight benzodiazepine compounds are used. A comparison with a standard Monte Carlo procedure is made. The methodology proposed can be used when Monte Carlo simulation may be biased or cannot be implemented. The obtained fuzzy in vivo clearance can be used subsequently in fuzzy simulations of pharmacokinetic models.

Direct LC analysis of five benzodiazepines in human urine and plasma using an ADS restricted access extraction column.

An alkyl-diol-silica (ADS) precolumn was used for the direct and on-line extraction of several benzodiazepines from serum and urine. The protein component of the biological sample was flushed through the ADS column, while simultaneously extracting the benzodiazepine compounds in the pores of the ADS stationary phase. The role of hydrophobic interactions in the extraction mechanism was confirmed. Column switching was employed to elute the extracted analytes from the ADS column into a high-performance liquid chromatography reverse-phase C18 column for the isocratic separation and UV detection of the benzodiazepines. Sample preconcentration via large volume injections was possible, improving the limits of detection. The calculated clonazepam, oxazepam, temazepam, nordazepam and diazepam detection limits were 38.8, 24.2, 31.7, 31.3, 45.0 ng/ml in serum, respectively, and 48.4, 24.5, 31.7, 33.1, 52.9 ng/ml for urine, respectively. The method was linear over the range of 50-10000 ng/ml in both matrices with an average linear coefficient (R(2)) value of 0.9918. The injection repeatability and intra-assay precision of the method were evaluated over ten injections, resulting in a percent relative standard deviation <5%. The ADS extraction column was robust, providing many direct injections of biological fluids for the extraction and subsequent determination of benzodiazepines.

Prenatal and Neonatal Diazepam Administration Synchronizes Testicular Malate Dehydrogenase Circadian Rhythms in Young Rats

Rat or hamster pups exposed to constant light or darkness since birth exhibit many circadian rhythms synchronized with those of the mother. During early development, a number of cues derived from the maternal circadian system synchronize the fetal and neonatal circadian clock. Maternal pineal sympathetic denervation during early pregnancy disrupts maternal synchronization of parotid α-amylase and testicular malate dehydrogenase circadian rhythms in rat pups. Maternal pineal sympathetic denervation was used to study potential agents able to synchronize the fetal or neonatal circadian clock. Melatonin injection to denervated pregnant mothers prevents the pineal sympathetic denervation effect on those circadian rhythms. We now studied the synchronizing effect of a benzodiazepine compound, diazepam. This GABAA agonist synchronized testicular malate dehydrogenase (MDH) activity of pups when it was injected to sympathetic denervated pregnant dams (a daily dose at 07:00 or 19:00 h from the 14 th to the 20 th day of gestation) or orally administered to the pups (a daily dose at 19:00 h from the 10 th to 24 th day of life). Co-injection of diazepam and GABAA antagonist, flumazenil, blocked the synchronizing effect of diazepam. The results demonstrate that diazepam has a synchronizing effect on the development of the circadian clock in rats and suggest that modulation of maternal GABAA could participate in mammalian maternal synchronization.

Analytical methodology for the detection of benzodiazepine consumption in opioid-dependent subjects.

Benzodiazepines are frequently abused by heroin users, but not all compounds have shown the same abuse liability. We developed an analytical method that was able to detect various benzodiazepine compounds in a single run. Enzymatically hydrolyzed urine underwent a liquid-liquid extraction procedure with chloroform/isopropanol (9:1) at pH 8-9 followed by a solid-liquid clean-up (Bond Elut TCA C18) to obtain appropriate extracts for HPLC analysis. Mobile phase composition was optimized by means of the linear solvation energy relationship methodology based on Reichardt's normalized solvatochromic parameter (E(T)N). The method was validated for the simultaneous detection and quantitation of alprazolam, alpha-hydroxyalprazolam, oxazepam, 7-aminoflunitrazepam, flunitrazepam, nordiazepam, lorazepam, and diazepam. The prevalence of benzodiazepine consumption in 229 opioid-dependent subjects on methadone-maintenance treatment was 48%. Oxazepam and nordiazepam were the benzodiazepines most frequently recoved in urine samples. The prevalence of alprazolam use (40%) was higher than that of flunitrazepam (10%).

Intravenous valproate in pediatric epilepsy patients with refractory status epilepticus.

Since its first clinical use in France in 1963, valproic acid (VPA) has rapidly established itself worldwide as one of the major antiepileptic drugs, with a broad efficacy for treatment of both generalized and partial seizures in children and adults.1 In October 1996, an IV VPA formulation was approved in Germany for the short-term supplementation of an established oral VPA treatment in patients with epilepsy, when oral-to-parenteral substitution becomes necessary.2 However, IV VPA also offers the opportunity to treat epileptic emergency situations such as prolonged or serial seizures, or even patients with status epilepticus (SE). We report on 41 children with therapy-resistant SE treated with IV VPA. The clinical details of our patients are listed in the table. All children had SE that was refractory to commonly recommended IV antiepileptic drugs (i.e., benzodiazepine compounds, phenytoin, and barbiturates). Whereas 22 of these children received antiepileptic long-term therapy that started before this SE event, SE was the initial presentation of an …

Pharmacology of recombinant human GABA A receptor subtypes measured using a novel pH-based high-throughput functional efficacy assay

To facilitate the discovery of novel compounds that modulate human GABA A receptor function, we have developed a high throughput functional assay using a fluorescence imaging system. L(tk-) cells expressing combinations of human GABA A receptor subunits were incubated with the pH-sensitive dye 2′,7′bis-(2-carboxyethyl)-5-(and 6)-carboxyfluorescein, then washed and placed in a 96-well real-time fluorescence plate reader. In buffer adjusted to pH 6.9 there was a robust and persisting acidification response to addition of GABA, which was antagonised by the GABA A receptor antagonist bicuculline. The concentration–response relationship for GABA was modulated by allosteric ligands, including benzodiazepine (BZ) site agonists and inverse agonists. The effects of BZ site ligands on the pH response to GABA for receptors containing α1β3γ2, α3β3γ2 or α5β3γ2 subunits were well correlated with results from electrophysiological studies on the same receptor subunit combinations expressed in Xenopus oocytes. Most modulatory compounds tested were found to be relatively unselective across the three subunit combinations tested; however, some showed subtype-dependent efficacy, such as diazepam, which had highest agonist effects on the α3β3γ2 subtype, substantial but lesser agonism on α1β3γ2 and still substantial but the least agonism on α5β3γ2. This indicates that the α subunit within the recombinant receptor expressed in L(tk-) cells can affect the efficacy of the response to some BZ compounds. Inhibitors of Na +/Cl − cotransport, anion/anion exchange and the gastric type of H +/K + ATPase potently inhibited GABA-evoked acidification, indicating that multiple transporters are involved in the GABA-evoked pH change. This novel fluorescence-based high throughput functional assay allows the rapid characterization of allosteric ligands acting on human GABA A receptors.

Use of Diazepam for transportation of Himalayan Black Bear (Selenarctos thibetinus)

A male wild Himalayan Black Bear (Selenarctos thibetinus) was chemically restrained with diazepam, dose rate of 2 mg/kg body weight intramuscularly in divided doses for translocation. Mild tranquillization was achieved without sedation. However, the animal showed moderate analgesia, muscle relaxation and listlessness. No side effects were exhibited. The animal could be translocated safely and it could fully recover after three hours of drug induction. Introduction Diazepam, a benzodiazepine compound has been used as a feed additive in domestic animals for its tranquillizing, antistress and growth stimulating effects. This group of drugs show dose related tranquilizing, sedative and hypnotic properties. This report discusses the various tranquilizing stages of diazepam in a young male wild Himalayan Black Bear (Selenarctos thibetinus) during its translocation. Materials and Methods A young male wild Himalayan Black Bear had to be tranquilized for translocation from the site of temporary rehabilitation and to its new destination site located 68 km away. Diazepam (Calmpose inj.*) was selected as the drug of choice at a dose rate of 1 mg/kg body weight initially, with a total dose of 25 mg., intramuscularly. The animal calmed with no sedation and was boarded on a jeep inside a temporary wooden cage. The dimension of the cage was enough to allow the animal to move, the roof touching its back. The ambient temperature was 34C. The animal initially responded with grunts on starting the vehicle. After about 10 minutes of journey, the animal became increasingly excited and hard to control, gnawing at the thinner planks. Diazepam 10 mg. was again injected intramuscularly. After 10 minutes, the journey was resumed. The bear got excited intermittently, thereafter with heavy panting due to heat, congestion and probably constant movement of the vehicle. After 10 minutes of the administration of the second dose, the animal became increasingly restless and broke one side of the cage with frantic effort to come out. Diazepam 15 mg. was inReceived 22 October 1998; Accepted 6 December 1999 209 * Diazepam 10mg, per 2ml, Ranbaxy India, Bombay jected again intramuscularly to complete a dose regime of 2 mg/ kg body weight. After 15 minutes of administering the third dose the bear calmed down. The animal lay on its belly with relatively moderate muscle relaxation and analgesia but with complete listlessness. Transportation resumed soon after repairing the cage. On arrival, the bear was allowed to creep out of the cage. The animal recovered fully an hour thereafter. Emesis or other complications were not exhibited. Results and Discussion Diazepam is described as a suitable drug for restraining wild animals (Fowler, 1978). The dosage varies from 1 to 3.5 mg/kg (0.5 to 2 mg/lb), depending on the species and the degree of excitement at the time of injection. Onset of induction is reported to be within 1 to 2 minutes when given intravenously. If given intramuscularly, it takes effect within 15 to 30 minutes, depending on the dose. Diazepam is metabolized slowly. Clinical effects usually disappear within 60 to 90 minutes (Fowler, 1978). In the present study, diazepam at the dose rate of 1 mg / kg body weight was not found to be sufficient, but responded well at the dose rate of 2 mg/kg body weight. However, a higher dose rate may be necessary for bigger and more agile and excited bears. The present dose was found to be completely safe with no visible clinical side effects or toxicity. Similar findings have been reported when large doses of diazepam was given to dogs for prolonged periods (Hall & Clarke, 1983). Acknowledgement NN acknowledges the kind help rendered by the D.F.O., Western Assam Wildlife Division, Mangaldai, Assam, India. He is also grateful to all his well-wishers for their help and advice during clinical monitoring and health recovery of the bear at its temporary rehabilitation site.

SPECT [I-123]iomazenil measurement of the benzodiazepine receptor in panic disorder

Background: Alterations in benzodiazepine receptor function have long been hypothesized to play a role in anxiety. Animal models of anxiety involving exposure to chronic stress have shown a specific decrease in benzodiazepine receptor binding in frontal cortex and hippocampus. The purpose of this study was to examine benzodiazepine receptor binding patients with panic disorder and comparison subjects. Methods: A quantitative measure related to benzodiazepine receptor binding (Distribution Volume (DV)) was obtained with single photon emission computed tomography (SPECT) imaging of [ 123I]iomazenil and measurement of radioligand concentration in plasma in patients with panic disorder and healthy controls. DV image data were analyzed using statistical parametric mapping (spm96). Results: A decrease in measures of benzodiazepine receptor binding (DV) was found in left hippocampus and precuneus in panic disorder patients relative to controls. Panic disorder patients who had a panic attack compared to patients who did not have a panic attack at the time of the scan had a decrease in benzodiazepine receptor binding in prefrontal cortex. Conclusions: Findings of a decrease in left hippocampal and precuneus benzodiazepine receptor binding may be related to alterations in benzodiazepine receptor binding, or other factors including changes in GABAergic transmission or possible endogenous benzodiazepine compounds. Benzodiazepine receptor function in prefrontal cortex appears to be involved in changes in state-related panic anxiety.

The use of benzodiazepines in anxiety and other disorders

Benzodiazepines have come under scrutiny and attack over recent years because of their abuse liability, withdrawal reactions and development of tolerance. Consequently, practitioners worldwide are discouraged from prescribing them. While some of these risks may have been exaggerated, benzodiazepines remain a useful therapeutic tool, alone or in combination, in a number of psychiatric and medical conditions. Withholding such treatment may be unjustified and detrimental to the patients’ health. Further, benzodiazepines have helped researchers in their attempts to elucidate the neurobiological mechanisms underlying anxiety. This, in return, leads to the development of new effective anxiolytic treatments, with fewer problems compared to the traditional benzodiazepine compounds. Such new agents are already available or at the closing stages of clinical trials.

Sex Differences in Long-Term Consequences of Prenatal Diazepam Exposure: Possible Underlying Mechanisms

Prenatal exposure to diazepam, a benzodiazepine (BZD) compound, leads to pronounced effects on responses to stressors in exposed animals when they reach adulthood. Many of the responses are sex specific. The mechanisms mediating the effects of the exposure on the organism have not been elucidated; however, the time course for the appearance of altered function following in utero drug exposure indicates that the exposure interfered with neural organization of mechanisms mediating responses to stressors. The article discusses possible mechanisms that relate to sites of action of the drug in the developing brain: the GABA A receptor, and the mitochondrial BZD receptor. The mechanisms mediating the sex-specific impact of diazepam on the developing brain appear to be complex and interactive.

Benzodiazepine Compounds as Inhibitors of the Src Protein Tyrosine Kinase: Screening of a Combinatorial Library of 1,4-Benzodiazepines

We screened 1680 spatially separated compounds of a diverse combinatorial library of 1,4-benzodiazepines for their ability to inhibit the kinase activity of protein tyrosine kinases Src, Yes, Abl, Lck, Csk, and fibroblast growth factor receptor. This screening yielded novel ligands for the protein tyrosine kinase Src. In the 1,4-benzodiazepine-2-one scaffold, the preferred substituent at position R1 was 4-hydroxyphenylmethyl or a 3-indolemethyl derived from a tyrosine or tyrptophan used in building the benzodiazepine, while the substituent at R2, introduced by alkylating agents, was preferably aromatic in nature. The preferred ring structure introduced on the bicyclic ring of the scaffold by acid chlorides was a p-hydroxy phenyl group. The lead compound, designated as N-l-Yaa, has a l-4-hydroxyphenylmethyl ring at R1 and a biphenylmethyl substituent at R2. The compound has an IC50 of 73 μM against Src, 2- to 6-fold lower than against other protein tyrosine kinases and >10-fold lower than against other nucleotide-utilizing enzymes. The mechanism of binding of N-l-Yaa to Src is mixed against the peptidic substrate with a Ki of 35 μM and noncompetitive against ATP-Mg with a Ki of 17 μM. Multiple inhibition analysis of the lead compound in the presence of other competitive inhibitors demonstrated that the binding of the lead compound is nonexclusive to the other competitive inhibitor. The inhibitor was found to be nontoxic to the AFB-13-human fibroblasts cells and inhibited the colony formation of HT-29 colon adenocarcinoma cells that are dependent on Src activity.

Characterization of peripheral benzodiazepine receptors in purified large mammal pancreatic islets

In this work, we evaluated the biochemical properties of peripheral benzodiazepine receptors (PBRs) in the porcine endocrine pancreas and their role in insulin release. Binding of [ 3H]1-(2-chlorophenyl-N-methyl-1-methyl-propyl)-3-isoquinolinecarboxamide ([ 3H]PK-11195), a specific ligand of PBRs, to islet membranes was saturable and Scatchard's analysis of saturation curve demonstrated the presence of a single population of binding sites, with a dissociation constant ( K d ) value of 4.75 ± 0.70 nM and a maximum amount of specifically bound ligand ( B max) of 4505 ± 502 fmol/mg of proteins. The pharmacological profile of PBRs was determined as the ability of PK-11195 and several benzodiazepine compounds to displace [ 3H]PK-11195 from these binding sites. The rank order of potency yielded the following affinity results: PK-11195 > 7-chloro-1,3-dihydro-1-methyl-5-(p-chlorophenyl)-2H-1,4-benzodiazepine-2-on (Ro 5-4864) > diazepam ⩾ flunitrazepam ⪢ flumazenil. Secretion studies demonstrated that PK-11195 (1 and 10 μM) and Ro 5-4864 (10 and 50 μM) significantly potentiated insulin secretion from freshly isolated porcine islets at 3.3 mM glucose. This potentiating effect was not observed at 16.7 mM glucose concentration nor by the addition of clonazepam. These results show the presence of PBRs in purified porcine pancreatic islets and suggest an implication of PBRs in the mechanisms of insulin release.

Anxiolytic effect of progesterone is mediated by the neurosteroid allopregnanolone at brain GABAA receptors.

Previous studies from this laboratory have shown that progesterone (PROG) treatment in ovariectomized rats produces an anti-anxiety response similar to that observed after the administration of prototypical anxiolytic benzodiazepine (BDZ) compounds. The PROG-induced anxiolytic response was highly correlated with an increased level of 3 alpha-hydroxy-5 alpha-pregnan-20-one (allopregnanolone) in the blood and brain, and was also associated with a facilitation of GABA-stimulated chloride ion (Cl-) influx in cortical synaptoneurosomes. This correlative evidence suggested that the anxiolytic effect of PROG was a result of its in vivo reduction to the neuroactive steroid, allopregnanolone. In this report, a series of studies was conducted to determine the mechanism(s) by which PROG alters behavior in animal models of anxiety. In the first experiment, ovariectomized rats were injected with PROG (1 mg/0.2 ml, SC) 4 h prior to a test in the elevated plus-maze. Some animals also received an injection of picrotoxin (0.75 mg/kg, IP), a GABAA receptor-gated Cl- channel antagonist, whereas other animals were pretreated with RU 38486 (5 mg/0.2 ml, SC), a progestin receptor antagonist. PROG elicited anxiolytic behavior in the plus-maze, an effect that was blocked by picrotoxin administration. Pretreatment with RU 38486 was not effective in altering PROG-induced anxiolytic behavior in the plus-maze. In a second experiment, the effect of PROG on behavior in the plus-maze was determined in the presence of N,N-diethyl-4-methyl-3-oxo-4-aza-5 alpha-androstane-17 beta-carboxamide (4-MA; 10 mg/0.2 ml, SC), a 5 alpha-reductase inhibitor.(ABSTRACT TRUNCATED AT 250 WORDS)

Modification of the photodynamic action of δ-aminolaevulinic acid (ALA) on rat pancreatoma cells by mitochondrial benzodiazepine receptor ligands

We have shown that addition of exogenous delta-aminolaevulinic acid (ALA) to rat pancreatoma AR4-2J cells in culture leads to the increased production of porphobilinogen (PBG) and the accumulation of photoactive protoporphyrin IX (PPix) in these cells. Exposure to light (lambda > 400 nm) at an intensity of 0.2 mW cm-2 for 8 min resulted in an ALA dose-dependent cytolysis of the cells, with an EC50 of 6.6 +/- 0.7 microM. This cytolytic effect was light intensity dependent, with greater cell destruction after exposure to light at an intensity of 0.47 mW cm-2 than at 0.2 mW cm-2; it was also dependent on the duration of illumination, cell survival decreasing with increasing illumination times. The photodestruction of the AR4-2J cells following exposure to ALA can be attributed to the production of endogenous PPix, a photoactive porphyrin that we have shown to generate singlet oxygen upon illumination, whereas ALA itself does not. Further investigation of the molecular mechanisms underlying the photodynamic action of ALA demonstrated the involvement of the mitochondrial (peripheral) benzodiazepine receptor (MBR), a high-affinity recognition site for dicarboxylic porphyrins, and especially PPix. The centrally acting benzodiazepine compounds clonazepam and flumazenil, which have negligible affinities for the MBR, had no effect on ALA-mediated phototoxicity. In contrast, both the isoquinoline carboxamide PK11195 and the benzodiazepine Ro 5-4864 ligands, displaying a high affinity for the MBR, did affect ALA-mediated phototoxicity, each markedly increasing the EC50 for cell photodestruction and thus exerting a photoprotective effect. It is concluded that the MBR may play an important role in the expression of ALA-mediated PPix phototoxicity and that MBR ligands, by diminishing the actions of endogenous PPix, have the potential to rescue cells from porphyrin-induced photolysis.

Clonazepam suppresses GABAB-mediated inhibition in thalamic relay neurons through effects in nucleus reticularis

1. Experiments were carried out using patch-clamp techniques in rat thalamic slices, maintained in vitro, to examine the effects of the benzodiazepine compound, clonazepam (CZP), on intrathalamic inhibition. Bath-applied CZP reduced the gamma-aminobutyric acid-B (GABAB) component of inhibitory postsynaptic potentials and currents (IPSPs and IPSCs, respectively) evoked in rat thalamic somatosensory relay neurons by stimulation of nucleus reticularis thalami (nRt), without consistently affecting the GABAA IPSP. Secondary IPSPs, which occur as a result of intrathalamic oscillations, were dramatically reduced. 2. Voltage-clamp experiments combined with local or bath perfusion of the GABAA antagonist bicuculline methiodide (BMI), demonstrated that nRt is a site of GABAA-mediated postsynaptic inhibition that affects inhibitory output onto relay neurons. BMI enhanced both GABAA and GABAB postsynaptic inhibition in relay neurons when applied to nRt. Focal applications in the ventrobasal relay nucleus near the recording electrode blocked the GABAA-mediated IPSP but had no effects on GABAB inhibitory potentials. 3. Results suggest that CZP acts to facilitate recurrent inhibition in nRt and decrease its inhibitory output onto relay neurons. Intra-nRt GABAA-mediated inhibition thus has an important role in controlling thalamic excitability and in the anti-absence actions of CZP.