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At least 19 recordsLinked to original sources
PubMed1997

Testing human hair for flunitrazepam and 7-amino-flunitrazepam by GC/MS-NCI.

To validate information on flunitrazepam use, we investigated human hair for flunitrazepam and its major metabolite 7-amino-flunitrazepam by gas chromatography coupled to mass spectrometry in negative chemical ionization mode of detection. Samples were twice decontaminated with methylene chloride, pulverized in a ball mill and 50 mg of powdered hair were incubated in Soerensen buffer (pH 7.6) in the presence of diazepam-d5 used as internal standard. After liquid-liquid extraction of the incubation medium with diethylether-chloroform (80:20, by vol.), the organic phase was evaporated and the dry extract was derivatizated with heptafluorobutyric anhydride. Benzodiazepines were separated on a 30 m capillary column and detected using single ion monitoring. Among 40 hair samples tested (obtained from drug addicts deceased by heroin overdose), 14 were positive for both flunitrazepam and 7-amino-flunitrazepam and 12 for 7-amino-flunitrazepam only. Concentrations ranged from 31 to 129 pg/mg (mean: 60 pg/mg) and from 3 to 161 pg/mg (mean: 46 pg/mg) for flunitrazepam (14 cases) and 7-amino-flunitrazepam (26 cases), respectively. This first report described the detection of flunitrazepam and 7-amino-flunitrazepam in hair of chronic abusers. Due to the low concentrations observed, negative chemical ionization appears to be the alternative to test flunitrazepam and other benzodiazepines in hair.

Flunitrazepam↗
PubMed1997

Flunitrazepam has an inverse agonistic effect on recombinant alpha6beta2gamma2-GABAA receptors via a flunitrazepam-binding site.

gamma-Aminobutyric acid type A (GABAA) receptor subtypes containing the alpha6-subunit are generally thought to be insensitive to the action of benzodiazepine agonists. We describe the specific binding of the benzodiazepine agonist flunitrazepam to alpha6beta2gamma2-containing GABAA receptors, which has not been observed before and differs from previous reports. With the whole-cell voltage-clamp technique, we observed a functional discrimination between alpha1beta2gamma2- and alpha6beta2gamma2-receptors. Different benzodiazepines had different effects on GABA-evoked chloride currents. The agonist flunitrazepam had an inverse agonistic effect, whereas the antagonist flumazenil increased GABA-induced chloride currents. The action of flunitrazepam on the channel activity of alpha6beta2gamma2-receptors was opposite to its action on alpha1beta2gamma2-receptors. We conclude that flunitrazepam can act as either an agonist or an inverse agonist, depending on the GABAA receptor configuration.

Affinity Labels↗
PubMed1997

Localization of flunitrazepam in artificial membranes. A spectrophotometric study about the effect the polarity of the medium exerts on flunitrazepam acid-base equilibrium.

In the present paper we tried to test the hypothesis that nonspecific flunitrazepam-membrane interactions are consistent with drug molecules accommodated between lipid molecules, becoming an integral part of the bilayer. We developed a spectrophotometric method to determine FNTZH+ equilibrium dissociation constant and applied it to the study of the acid-base equilibria of this drug in homogeneous media of different polarity. In these conditions, pK decreased with the decrement in the dielectric constant (D) of the media. These results, analyzed under the light of the theory developed by Fernandez and Fromherz (1977; J. Phys. Chem. 81, 1755-1761) let us infer that flunitrazepam is localized a region with D = 60. This D value is lower that Dwater = 78 and higher than D of hydrocarbon chains zone (D = 2-5) and would correspond to D of the region of polar groups. This result is compatible with the hypothesis.

Acid-Base Equilibrium↗
PubMed1997

Abuse liability of flunitrazepam.

Flunitrazepam is among the most frequently prescribed hypnotics in many countries. Although it was never marketed in the United States, flunitrazepam, in recent years, has been smuggled into the country, and reports of abuse--including alleged use of the drug to facilitate "date rape"--have attracted a great deal of scrutiny. It has been suggested that flunitrazepam may have greater liability for abuse than other benzodiazepines; such suggestions are supported by surveys of opioid abusers, many of whom report a distinct preference for flunitrazepam over other benzodiazepines. Experimental studies of animals and normal human subjects indicate that, although flunitrazepam has high efficacy and is very potent, it is pharmacologically similar to most other benzodiazepines. Although the studies are limited in number and scope, the data show no apparent differences between flunitrazepam and other benzodiazepines in ability to produce drug-taking or drug-seeking behavior, in capacity to produce physiologic dependence, nor in the characteristics of withdrawal after administration of an antagonist or discontinuation of treatment. Similar to other benzodiazepines, flunitrazepam produces dose-dependent effects on psychomotor performance and recall. Flunitrazepam does not seem to be involved in medical emergencies more often than other benzodiazepines, and there is no indication that flunitrazepam is more toxic than other benzodiazepines when taken in overdose by drug abusers or other individuals. Survey research among typical patient populations suggests that flunitrazepam is characteristic of benzodiazepines in that it is used appropriately and conservatively, with low liability for abuse. Thus the reported preference for flunitrazepam among opioid abusers seems to be the only way in which flunitrazepam is distinguished from other benzodiazepines; it is unclear what characteristics of the drug may be responsible for this reported preference. The evidence considered in this review indicates that abuse of flunitrazepam in this special population is not associated with any distinctive threats to the health of the general public.

Animals↗
PubMed2002

The flunitrazepam abuse prevention program at a general hospital in Taiwan: a descriptive study.

The Bureau of Controlled Drugs at Ministry of Health, Executive Yuan in Taiwan announced, on 1 April 2000, the schedules of controlled drugs with abuse potential and implemented a policy on 1 October 2000 to control them. Flunitrazepam (FM2), along with other two benzodiazepines (triazolam and brotizolam), is placed on Schedule III. The aim of the present study was to analyze the pattern of flunitrazepam prescriptions across all medical subspecialty departments at Taipei Medical University-Wan Fang Hospital (TMU-WFH), Taiwan. We analyzed 1170 prescriptions over 12 month period from 1 July 2000 to 31 May 2001. All prescription data were divided into three 4 month periods: period I was when the flunitrazepam prescription was not controlled, period II represented the time when flunitrazepam was placed on Schedule III and when physicians were required to use a special duplicated prescription form and period III was when the TMU-WFH started to set a stricter control for the prescription of flunitrazepam. The results indicated that the number of flunitrazepam prescriptions during period III had decreased significantly compared with period I (P < or = 0.05). Eventually, 45.7% of flunitrazepam-medicated patients were followed up monthly with a restriction of their flunitrazepam supply to no more than 14 days, 22.9% of patients were followed up fortnightly at clinics with a 14 day supply of flunitrazepam, 15.7% were followed up fortnightly with a 14 day restriction of flunitrazepam plus a non-flunitrazepan benzodiazepine supplement, 10.7% were referred to clinics within the Department of Psychiatry and 5% were switched from flunitrazepam to other drugs.

Drug Administration Schedule↗
PubMed1990

Photoaffinity labeling of rat liver microsomal morphine UDP-glucuronosyltransferase by [3H]flunitrazepam.

Benzodiazepines have been shown to competitively inhibit morphine glucuronidation in rat and human hepatic microsomes. Flunitrazepam exerted a potent competitive inhibition of rat hepatic morphine UDP-glucuronosyltransferase (UDPGT) activity (Ki = 130 microM). It has no effect on the activity of p-nitrophenol, 17 beta-hydroxysteroid, 3 alpha-hydroxysteroid, or 4-hydroxybiphenyl UDPGTs. Because flunitrazepam is an effective photoaffinity label for benzodiazepine receptors, studied were performed in solubilized rat hepatic microsomes and with partially purified preparations of morphine UDPGT to determine the enhancement of flunitrazepam inhibition and binding to morphine UDPGT promoted by exposure to UV light. Under UV light, flunitrazepam inhibition was markedly enhanced. UV light exposure also led to a marked increase in binding of [3H]flunitrazepam to microsomal protein, which was protected substantially by preincubation with morphine. Testosterone, androsterone, and UDP-glucuronic acid did not protect against UV-enhanced flunitrazepam binding, and morphine did not reverse flunitrazepam binding once binding had occurred. As morphine UDPGT was purified, a good correlation was found between the increases in specific activity of morphine UDPGT and flunitrazepam binding to protein. Chromatofocusing chromatography showed that flunitrazepam bound only to fractions containing active morphine UDPGT, and no binding to 4-hydroxybiphenyl UDPGT was observed. Fluorography of a sodium dodecyl sulfate-polyacrylamide electrophoresis gel of solubilized hepatic microsomes that had been treated with [3H] flunitrazepam under UV light revealed a band with a monomeric molecular weight between 54,000 and 58,000. This monomeric molecular weight compares favorably with the reported monomeric molecular weight of homogeneous morphine UDPGT (56,000). These studies suggest that flunitrazepam binds rather selectively to the morphine binding site of morphine UDPGT and may prove to be a useful probe for this enzyme.

Affinity Labels↗
PubMed1988

Hypnotic action of flunitrazepam in the rat: does 5-HT mechanism play a role?

This study examined whether pharmacological manipulation of serotonergic (5-HT) systems would affect the hypnotic action of flunitrazepam in rats. Flunitrazepam, a potent hypnotic, was used alone or combined with parachlorophenylalanine (pCPA), an inhibitor of the synthesis of 5-HT, 8-OH-DPAT, a 5-HT1A receptor agonist and fluvoxamine, an inhibitor of the reuptake of 5-HT. Flunitrazepam increased the amount of orthodox sleep, the latency of rapid eye movement (REM) sleep and decreased the amount of REM sleep. The drug pCPA decreased the total sleep time and the amount of orthodox and REM sleep. Administration of flunitrazepam to pCPA-pretreated rats induced orthodox sleep in an identical way to that found in the controls. The drug 8-OH-DPAT increased wakefulness and the latency of REM sleep. The association of flunitrazepam with 8-OH-DPAT abolished the increase in waking seen after 8-OH-DPAT alone. In contrast, the combined treatment with flunitrazepam and 8-OH-DPAT resulted in a lengthening of the latency of REM sleep significantly greater than that observed with the same dose of each drug alone. Fluvoxamine increased the latency a decrease the amount of REM sleep. The association of fluvoxamine with flunitrazepam induced a decrease in REM sleep, equal to the sum of the effects of the two drugs alone. Fluvoxamine did not modify the other effects of flunitrazepam. The present experiments demonstrate that the association of pCPA, 8-OH-DPAT and fluvoxamine, did not alter the hypnogenic effect of flunitrazepam. The possibility of an involvement of 5-HT mechanisms in the effect of flunitrazepam on the phasic events in sleep is questionable.

8-Hydroxy-2-(di-n-propylamino)tetralin↗
PubMed1993

Deaths involving the benzodiazepine flunitrazepam.

A study of eight deaths involving the benzodiazepine flunitrazepam was performed to assess the contribution of this drug to the fatalities. Coronial deaths in Victoria in the 2-year period to mid-1991 were selected in which either flunitrazepam or flunitrazepam and ethanol were the principal toxicological findings. All bodies were subject to a full autopsy by forensic pathologists, and a full toxicological examination. No significant pathology was found at autopsy in any case. Very high concentrations of 7-aminoflunitrazepam, a metabolite of flunitrazepam, were present in all cases. In four cases no other significant drug was detected, whereas in the other four cases there were significant concentrations of ethanol (mean 1.6 g/L). In these two groups of cases the concentrations of 7-aminoflunitrazepam were 0.45 mg/L and 0.16 mg/L, respectively. Only moderate levels of flunitrazepam were detected, suggesting that 7-aminoflunitrazepam is produced postmortem and may be an important marker of flunitrazepam usage. The causes of death in these eight cases were probably either flunitrazepam toxicity or a combination of flunitrazepam and ethanol toxicity. Only one case appeared likely to have been a suicide. Our observations in these cases suggest that flunitrazepam may cause death in the absence of other drugs or significant disease. The presence of ethanol reduces the amount of flunitrazepam needed to cause death.

Adult↗
PubMed1981

[Combined effect of flunitrazepam on activities of anesthetics and analgesics in mice and rats (author's transl)].

Flunitrazepam in a dose range of 0.01 to 1 mg/kg i.v. produced dose dependent sedation and muscle relaxation, while even 1 mg/kg of flunitrazepam had no anesthetic effect (loss of righting reflex). Flunitrazepam in a dose over 0.01 mg/kg prolonged anesthesia induced by pentobarbital, thiopental and ketamine in mice and rats and combinations of non-effective doses of these anesthetics with flunitrazepam induced anesthesia in mice. Furthermore, flunitrazepam enhanced gaseous anesthesia in mice and rats. Flunitrazepam alone was slightly effective in prevention of acetic acid writhing in mice and the drug enhanced the anti-writhing effects of morphine, pentazocine and thalamonal. Flunitrazepam alone had little analgesic effect on the hot plate test in mice, while it did enhance the effect of morphine. The analgesic effects of morphine and pentazocine on the tail pinch test in rats were potentiated with 0.1 and 1 mg/kg of flunitrazepam. Flunitrazepam had no effect on bradykinin-induced nociceptive response in rats, while 1 mg/kg of the drug enhanced morphine analgesia in this test. Combined effect of flunitrazepam on activities of anesthetics and analgesics may contribute to the clinical effect of this drug in cases of preanesthetic medication.

Analgesics↗
PubMed1996

An autoradiographic study of [3H]flunitrazepam binding sites in the brain of rat made tolerant to and dependent on pentobarbital.

The effects of continuous administration of pentobarbital on the benzodiazepine receptor labeled by [3H]flunitrazepam were investigated. Animals were made tolerant to pentobarbital by intracerebroventricular (i.c.v.) infusion with pentobarbital (300 micrograms/10 microliters/h) for 6 days through pre-implanted canulae connected to osmotic mini-pumps. The dependent rats were assessed 24 h after cessation of pentobarbital infusion. Changes in [3H]flunitrazepam binding were examined in 37 brain regions at a concentration of [3H]flunitrazepam of 1 nM. In subsequent saturation studies, the binding parameters Bmax and KD were also investigated in 17 brain regions, most of which showed significant changes in [3H]flunitrazepam binding in experiments using a fixed concentration of radioligand. The pentobarbital-tolerant rats showed a significant increase in Bmax with an increase in KD for [3H]flunitrazepam in the ventroposterior nucleus of thalamus. In the dependent rats, a significant increase in Bmax for [3H]flunitrazepam binding, without a change in KD, was observed in all layers of the frontal cortex, the caudate-putamen, olfactory tubercle, and some nuclei in thalamus, compared to those in the control. Increased [3H]flunitrazepam binding in the molecular layer of the olfactory bulb, the ventral pallidum, and the cerebellum of the pentobarbital dependent rats at a fixed concentration of [3H]flunitrazepam was also observed. There was no significant change in [3H]flunitrazepam binding in the hippocampus and several nuclei of the brain stem. These findings suggest that benzodiazepine receptors are closely involved in the development of tolerance to and dependence on pentobarbital. Further studies on changes in gamma-aminobutyric acid (GABA)A receptor subunit mRNA or the effects of pentobarbital on GABAA receptor phosphorylation would be necessary for an explanation of the precise mechanisms underlying the development of tolerance to and dependence on pentobarbital.

Animals↗
PubMed1991

A comparison of the physical dependence inducing properties of flunitrazepam and diazepam.

Dogs dosed chronically (4-7 weeks) with oral flunitrazepam (7.6 mg/kg/day) or diazepam (24-36 mg/kg/day) administered in 4 equally divided doses had dose-related flumazenil precipitated benzodiazepine abstinence scale scores (BPAS) of comparable intensities despite the fact that plasma levels of flunitrazepam and its metabolites were much lower than nordiazepam levels in the diazepam-dependent dog. Both groups of dependent dogs had clonic and tonic-clonic seizures after oral and IV flumazenil. Precipitated abstinence signs persisted longer in the diazepam than in the flunitrazepam-dependent dogs. Differences in the pharmacokinetics of the drugs of dependence, their metabolites, and their interactions at receptor sites offer a partial explanation for the high level of dependence seen in the flunitrazepam dog. The finding that the estimated plasma free concentration of flunitrazepam and its metabolites is equal to or greater than that of diazepam and its metabolites together with the fact that flunitrazepam has a higher affinity for the benzodiazepine receptor than either diazepam, nordiazepam or oxazepam can explain why the intensity of the precipitated abstinence syndrome is comparable in flunitrazepam- and diazepam-dependent dogs. Although the flumazenil-induced precipitated abstinence syndromes observed in flunitrazepam- and diazepam-dependent dogs differed qualitatively they did not differ quantitatively. It is therefore concluded from these data that the doses of flunitrazepam and diazepam, chosen for producing comparable degrees of weight loss during dose escalation, did not differ in the degree of physical dependence that they produced in the dog.

Animals↗
PubMed2005

Flunitrazepam: psychomotor impairment, agitation and paradoxical reactions.

Benzodiazepines are sedatives used for anxiolysis, hypnosis, muscle relaxation and the treatment of epilepsy. Paradoxical reactions including agitation, talkativeness, confusion, disinhibition, aggression, violent behavior and loss of impulse control may, however, occur in some subjects. It has been claimed that high doses of flunitrazepam may cause aggression on a more regular basis in all individuals. The present study makes use of a Norwegian forensic toxicological database containing analytical results from drivers suspected of driving under the influence and suspects of violent crime to analyze the relationship between behavior and blood flunitrazepam concentration. Four-hundred and fifteen cases of drivers suspected of driving under the influence and seven cases of suspects of violent crime were studied. These selected cases had flunitrazepam as the only drug in blood samples and had been evaluated by a clinical test for impairment (CTI) performed by a police physician at the time of blood sampling. The impaired drivers had higher blood flunitrazepam concentrations than the not impaired drivers. Multivariate analysis revealed that both blood flunitrazepam concentration and age of the suspected drivers had independent impact on impairment, indicating tolerance with age. Most of the effects measured were sedative effects of flunitrazepam and these effects were related to flunitrazepam level. Possible paradoxical reactions were observed in a subgroup of 23 individuals (6%), but these reactions did not relate to blood flunitrazepam concentration. The suspects of violent crime showed similar degree impairment and had not more paradoxical reactions than the suspected drugged drivers. The findings were in agreement with other research that claims paradoxical reactions should be viewed as a reaction in certain individuals, and does not support the notion that flunitrazepam in high concentration produces aggression in all individuals taking the drug.

Adult↗
PubMed2001

Flunitrazepam: an evaluation of use, abuse and toxicity.

The benzodiazepine flunitrazepam is extensively prescribed to patients with insomnia in many countries, but has also become popular among alcohol- and drug abusers. Several reports indicate that it is used as a date rape drug and suggest that it may precipitate violent behavior. Furthermore, flunitrazepam is involved in many fatal intoxications in Sweden. This study was designed and conducted to explore the negative consequences of flunitrazepam abuse in Sweden, and to assess the trends in its use and abuse. The occurrence of flunitrazepam in cases referred to the Department of Forensic Chemistry in Linköping, Sweden 1992-1998, was investigated in detail. The detections were studied separately for different groups; medicolegal death investigations, drug abuse cases, driving under influence cases, and other medicolegal cases. These data were further compared with the sales, and seizures by the Swedish Customs and the Swedish Police. During 1992-1998, 641 fatalities occurred, where the cause of death was attributed to intoxication with flunitrazepam solely (130) or in combination with other drugs, or concomitant conditions (511). In 78% of all driving under influence cases, where flunitrazepam was detected, the analyses also disclosed the presence of illicit drugs. A similar association was seen in drug abuse cases. The seizures reported by the Swedish Customs revealed a substantial and increasing illegal trade. Cases, where flunitrazepam seemingly induced violent behavior were identified, and one of these is described in some detail. It is concluded that the abuse pattern and the toxicity of flunitrazepam should be kept in mind by forensic investigators and that this panorama also should be considered when decisions about the registration and classification of flunitrazepam are made in different countries.

Anti-Anxiety Agents↗
PubMed2006

[Changes in the sale and use of flunitrazepam in Norway after 1999].

BACKGROUND: In Norway there has in later years been much discussion of misuse of flunitrazepam. From 1 January 2003 the drug was moved up one level in the schedule of controlled substances. On 1 August 2004 the manufacturer of the Rohypnol brand withdrew it from the Norwegian market. How did these two events influence the sales and use of drugs containing flunitrazepam? MATERIALS AND METHODS: Sales figures for drugs containing flunitrazepam from the statistics database at the Norwegian Institute of Public Health were studied. The Norwegian prescription database was used to describe new (incident) users of flunitrazepam and the two brands of this drug sold in Norway in 2004. RESULTS AND DISCUSSION: Restrictions on the prescription status of flunitrazepam lead to a decrease in sales from 7.2 defined daily doses (DDD) per 1000 inhabitants per day in 2002 to 3.0 DDD per 1000 inhabitants per day in 2003. This decrease was only partly compensated for by an increase in the sales of nitrazepam (from 5.0 to 6.0 DDD per 1000 inhabitants per day). During the years 1999 to 2004 there was a steady increase in the sales of benzodiazepine-related hypnotics (zopiclone and zolpidem). This shift could mean a change from flunitrazepam to zopiclone. The withdrawal of Rohypnol in August 2004 had only minor effects on the total sales of flunitrazepam. The decline in sales of Rohypnol was almost compensated for by the increase in the overall sales of Flunipam. This was reflected in the fact that in the later months of 2004 there were many new (incident) users of Flunipam, but few new users of flunitrazepam-containing drugs in total. It could be concluded that the restrictions on prescription status of flunitrazepam had a much higher impact than the withdrawal of the Rohypnol brand.

Anti-Anxiety Agents↗
PubMed1998

Impairment of memory and plasma flunitrazepam levels.

Flunitrazepam was administered to volunteers in three different oral doses. The effects on psychomotor sedation, attention, working memory and explicit memory were then assessed at various intervals after dosing and compared with levels of the drug in the plasma. Three groups of 12 healthy males with similar levels of education were given placebo or flunitrazepam (1, 2 or 4 mg) in a double-blind, random-sequence study. Volunteers completed a battery of tests at night, 3.5 h after taking the drug and in the morning, 10 h afterward. Blood samples were collected for drug analysis before and after the nocturnal tests and before morning tests. At night, only the highest dose of flunitrazepam (4 mg) induced significant changes in psychomotor sedation, attention, working memory, and prose immediate recall. Doses of 2 and 4 mg flunitrazepam significantly reduced the mean scores of explicit memory (morning tests). Z-scores, calculated from differences between flunitrazepam and placebo, revealed that 2 mg flunitrazepam impaired memory but not alertness or attention. Linear regression analysis of the relationship between plasma levels of flunitrazepam and its effects (Z-scores) indicated that there was a significant positive correlation between peak levels of flunitrazepam at night and impairment of night attention and explicit memory, i.e. delayed recall of prose (r = 0.59, P < 0.01) and trigrams (r = 0.55, P < 0.01). However, memory and attention Z-scores as a function of plasma levels fitted with nonlinear regression analysis to the Emax model had higher correlation coefficients. To produce an effect equal to 50% of the maximum effect for memory impairment, concentrations (EC50) were 6.1 and 6.4 ng/ml for prose and trigrams delayed recall; but for attention they were much higher, at 13.2 ng/ml. The overall results indicate that higher concentrations were needed to impair attention than were required to impair memory.

Adult↗
PubMed1996

Possible involvement of alpha 2-adrenoceptor on the hypnotic action of flunitrazepam.

This study examined whether the pharmacologic manipulation of catecholaminergic systems would affect the hypnotic action of flunitrazepam in rats. Flunitrazepam, a potent hypnotic, was used alone or in combination with alpha-methyl paratyrosine (alpha MPT), an inhibitor of the synthesis of catecholamines, and clonidine (CLN), an alpha 2-adrenoceptor agonist. Flunitrazepam significantly increased the amount of slow-wave sleep and the latency of paradoxical sleep (PS) and decreased the amount of PS. Administration of flunitrazepam to alpha MPT-treated rats significantly increased the number of sleep cycles and PS episodes as compared with flunitrazepam alone. Clonidine decreased total sleep time and significantly decreased PS. The association of flunitrazepam with CLN induced a decrease in PS and waking as compared with flunitrazepam alone. The possible involvement of noradrenergic mechanisms in modulating the effect of flunitrazepam on the rat sleep-waking cycle is proposed.

Adrenergic alpha-2 Receptor Agonists↗
PubMed1990

Flunitrazepam photoaffinity labeling of the GABA(A) receptor reduces inhibition of [3H]Ro15-4513 binding by GABA.

The benzodiazepine drugs modulate gamma-aminobutyric acid (GABA)-mediated synaptic transmission via a high-affinity binding site that is part of the GABA(A) receptor complex, but which is distinct from the GABA binding site. Ro15-4513 is a benzodiazepine negative modulator of GABA action that displays unique anti-ethanol properties both in vivo and in vitro. Ro15-4513 has been reported to photoaffinity label nearly 100% of the benzodiazepine binding sites in rat brain homogenates. In contrast, the benzodiazepine positive modulator flunitrazepam photoaffinity labels only 25% of the sites. Here, we have examined the reversible binding of [3H]Ro15-4513, [3H]flumazenil (Ro15-1788), and [3H]flunitrazepam to embryonic chick brain membranes, and to membranes that have been photoaffinity labeled with nonradioactive flunitrazepam. Photoaffinity labeling with flunitrazepam decreased the subsequent reversible binding of [3H]flunitrazepam and [3H]flumazenil, but increased the binding of [3H]Ro15-4513. The increase in [3H]Ro15-4513 binding after flunitrazepam photoaffinity labeling was due to a decrease in the apparent Kd, with no change in Bmax. Following photoaffinity labeling, negative modulation of [3H]Ro15-4513 binding by GABA was lost, whereas positive modulation of residual [3H]flunitrazepam binding was retained. We conclude that the site photoaffinity labeled by flunitrazepam is distinct from the site responsible for reversible binding of [3H]Ro15-4513.

Affinity Labels↗