In vitro Phase I and Phase II metabolism of the new designer benzodiazepine cloniprazepam using liquid chromatography coupled to quadrupole time-of-flight mass spectrometry.

Designer benzodiazepines have recently emerged as a class of new psychoactive substances. These substances are used in recreational settings and as alternatives to prescription benzodiazepines as self-medication for patients suffering from anxiety or other mental disorders. Due to the limited information available on the metabolic fate of these new substances, it is challenging to reliably detect their usage in bioanalytical (e.g. clinical and forensic) settings. The objective of this study was to investigate the in vitro Phase I and Phase II metabolism of the new designer benzodiazepine cloniprazepam and identify potential biomarkers for its detection in human biological fluids. Cloniprazepam was incubated with human liver microsomes and cytosolic fractions to generate both Phase I and II metabolites. The extracts were analysed using liquid chromatography coupled to quadrupole time-of-flight mass spectrometry. Identification of the metabolites was performed using two complementary workflows, including a suspect screening based on in silico predictions and a non-targeted screening. A total of nine metabolites were identified, eight Phase I metabolites and one Phase II metabolite, of which five were specific for cloniprazepam. Clonazepam was the major metabolite of cloniprazepam. Hydroxy-cloniprazepam, dihydroxy-cloniprazepam, 3-keto-cloniprazepam, 7-amino-cloniprazepam, hydroxy-clonazepam, 7-amino-clonazepam and 3-hydroxy-7-amino-clonazepam were formed through oxidation, hydroxylation, and/or reduction of the nitro-group. Glucuronidated hydroxy-cloniprazepam was the only Phase II metabolite detected. Five metabolites were specific for cloniprazepam. This study provided a set of human in vitro biotransformation products which can assist specific detection of cloniprazepam consumption in future studies.

7-aminoclonazepam is superior to clonazepam for detection of clonazepam use in oral fluid by LC-MS/MS.

BACKGROUND: Clonazepam (CLON) is not only frequently prescribed in addiction management but is also commonly abused. Therefore many addiction clinics perform oral fluid (OF) testing, which unlike urine is not subject to adulteration, to monitor CLON compliance. However, CLON and other benzodiazepines can be challenging to detect in OF due to their weakly acidic nature and their presence in low concentrations. We determined the optimal technical and clinical approach for the detection of CLON use using OF. METHODS: We measured CLON and its primary metabolite 7-aminoclonazepam (7AC) by liquid chromatography-tandem mass spectrometry in OF specimens over a 2 month period. The samples were collected using the Orasure Intercept OF sample collection device. RESULTS: One hundred samples were presumptive-positive for 7AC and/or CLON. 91 (91.0%) confirmed positive for 7AC (median, range: 4.2, 0.5-316.7 ng/ml) using the ion ratio test, while only 44 of the 100 (44.0%) samples confirmed positive for CLON (median, range: 3.7, 0.5-217.2 ng/ml) using the ion ratio test. In OF the levels of 7AC were approximately 2.4-fold higher than CLON. The use of 7AC as an analyte for the detection of both CLON compliance and undisclosed use is also recommended. CONCLUSIONS: 7AC should be the analyte measured in OF for the detection of CLON use.

Biosynthesis of human diazepam and clonazepam metabolites.

A screening of fungal and microbial strains allowed to select the best microorganisms to produce in high yields some of the human metabolites of two benzodiazepine drugs, diazepam and clonazepam, in order to study new pharmacological activities and for chemical standard proposes. Among the microorganisms tested, Cunninghamella echinulata ATCC 9244 and Rhizopus arrhizus ATCC 11145 strains, were the most active producers of the mains metabolites of diazepam which included demethylated, hydroxylated derivatives. Beauveria bassiana ATCC 7159 and Chaetomium indicum LCP 984200 produced the 7 amino-clonazepam metabolite and a product of acid hydrolysis of this benzodiazepine.

Evaluation of brain targeting and mucosal integrity of nasally administrated nanostructured carriers of a CNS active drug, clonazepam.

PURPOSE: The aim of the study was to target clonazepam, a CNS active drug, to the brain through the non-invasive intranasal (in) route using of nanocarriers with proven safety METHOD: in clonazepam nanocarriers were prepared by mixing isopropyl myristate, Tween 80, Cremophor EL or lecithin, polyethylene glycol 200, propylene glycol or ethanol in different ratios with water. in-vitro characterization of the nanocarriers was done by various methods including: polarized light microscopy, particle size determination, viscosity measurements and drug release studies. in-vivo study comparing intranasal and intravenous administration was performed. The drug targeting efficiency (DTE %) and direct nose to brain transport percentage (DTP %) were calculated and nasal integrity assessment was carried out. RESULTS: The obtained formulae had particle size below 100 nm favoring rapid direct nose to brain transport and the time for 100% drug release (T100%) depended on systems composition. Plasma Tmax of clonazepam nanostructured carriers varied from 10-30 min., while their brain Tmax did not exceed 10 min, in comparison with 30 min for iv solution. Although there was no significant difference (p>0.05) between the plasma AUC0-∞ of the different tested nanocarriers and intravenous one, the increase in brain AUC 0 -∞ of different nasal formulations in comparison to that of iv administration (3.6 -7.2 fold) confirms direct nose to brain transport via olfactory region. Furthermore, DTE and DTP% confirmed brain targeting of clonazepam following intranasal administration. CONCLUSION: The results confirmed that intranasal nanocarriers were proved to be safe alternative for iv clonazepam delivery with rapid nose to brain transport.

Surface active benzodiazepine-bromo-alkyl conjugate for potential GABAA-receptor purification.

A conjugable analogue of the benzodiazepine 5-(2-hydroxyphenyl)-7-nitro-benzo[e][1,4]diazepin-2(3H)-one containing a bromide C(12)-aliphatic chain (BDC) at nitrogen N1 was synthesized. One-pot preparation of this benzodiazepine derivative was achieved using microwave irradiation giving 49% yield of the desired product. BDC inhibited FNZ binding to GABA(A)-R with an inhibition binding constant K(i) = 0.89 µM and expanded a model membrane packed up to 35 mN m(-1) when penetrating in it from the aqueous phase. BDC exhibited surface activity, with a collapse pressure π = 9.8 mN m(-1) and minimal molecular area A(min) = 52 Å(2)/molecule at the closest molecular packing, resulted fully and non-ideally mixed with a phospholipid in a monolayer up to a molar fraction x≅ 0.1. A geometrical-thermodynamic analysis along the π-A phase diagram predicted that at low x(BDC) (<0.1) and at all π, including the equilibrium surface pressures of bilayers, dpPC-BDC mixtures dispersed in water were compatible with the formation of planar-like structures. These findings suggest that, in a potential surface grafted BDC, this compound could be stabilize though London-type interactions within a phospholipidic coating layer and/or through halogen bonding with an electron-donor surface via its terminal bromine atom while GABA(A)-R might be recognized through the CNZ moiety.

NCLX is an essential component of mitochondrial Na+/Ca2+ exchange.

Mitochondrial Ca(2+) efflux is linked to numerous cellular activities and pathophysiological processes. Although it is established that an Na(+)-dependent mechanism mediates mitochondrial Ca(2+) efflux, the molecular identity of this transporter has remained elusive. Here we show that the Na(+)/Ca(2+) exchanger NCLX is enriched in mitochondria, where it is localized to the cristae. Employing Ca(2+) and Na(+) fluorescent imaging, we demonstrate that mitochondrial Na(+)-dependent Ca(2+) efflux is enhanced upon overexpression of NCLX, is reduced by silencing of NCLX expression by siRNA, and is fully rescued by the concomitant expression of heterologous NCLX. NCLX-mediated mitochondrial Ca(2+) transport was inhibited, moreover, by CGP-37157 and exhibited Li(+) dependence, both hallmarks of mitochondrial Na(+)-dependent Ca(2+) efflux. Finally, NCLX-mediated mitochondrial Ca(2+) exchange is blocked in cells expressing a catalytically inactive NCLX mutant. Taken together, our results converge to the conclusion that NCLX is the long-sought mitochondrial Na(+)/Ca(2+) exchanger.

Characterization of two classes of benzodiazepine binding sites in Schistosoma mansoni.

As we have recently shown that GABA should be considered a putative neurotransmitter in Schistosoma mansoni, the present work aimed to search for GABAA receptors in adult worms using [3H]-flunitrazepam to label the allosteric benzodiazepine binding site which is classically present on GABAA receptor complexes. We detected a large population (Bmax=8.25+/-1.1 pmol x mg protein(-1)) of high affinity (Kd=33.6+/-1.5 nM) binding sites for flunitrazepam. These sites harboured a singular pharmacological modulation that does not fit well with a mammalian central benzodiazepine receptor, mainly due to a very high affinity for Ro5-4864 and a very low affinity for clonazepam. We also detected a second population of benzodiazepine binding sites labelled with high affinity (IC50=85 nM) by [3H]-PK11195, a selective ligand of the mammalian peripheral benzodiazepine receptor. In conclusion, this work describes the pharmacological properties of a large population of central-like benzodiazepine receptors supporting their study as putative new targets for the development of anti-parasitic agents. We also describe, for the first time, the presence of peripheral benzodiazepine receptors in this parasite.

Effect of common NAT2 variant alleles in the acetylation of the major clonazepam metabolite, 7-aminoclonazepam.

We investigated the role of NAT2 on clonazepam acetylation, using transiently expressed human NAT2 alleles. The NAT25*B and the NAT2*6A variant alleles cause a 20 and 22-fold reduction in the Vmax, respectively. We conclude that NAT2 is responsible for 7-aminoclonazepam acetylation and that NAT2 gene polymorphisms impair such metabolic pathway.

Changes in hippocampal GABAA receptor subunit composition in bipolar 1 disorder.

Postmortem CNS studies have suggested an uncoupling of the gamma-aminobutyric acid (GABA) and benzodiazepine binding sites on the hippocampal GABA(A) receptor in schizophrenia. The GABA(A) receptor is an assembly of discrete subunits that form a ligand-gated ion channel, the binding characteristics of which are defined by receptor subunit composition. Thus, a likely explanation for an uncoupling between the GABA and benzodiazepine binding sites on the GABA(A) receptor would be a change in receptor subunit composition. To test this hypothesis we measured the density of GABA ([(3)H]muscimol) and benzodiazepine ([(3)H]flumazenil) binding sites on the GABA(A) receptor in hippocampi, obtained postmortem, from schizophrenic, bipolar I disorder and control subjects. In addition, we measured the amount of [(3)H]flumazenil binding that could be displaced with zolpidem and clonazepam. Levels of both [(3)H]muscimol and [(3)H]flumazenil binding were significantly decreased in part of the CA2 from subjects with schizophrenia; the decrease in [(3)H]flumazenil being due to decreases in both zolpidem-sensitive and -insensitive radioligand binding. There were complex regionally specific changes in [(3)H]muscimol binding in the hippocampus from subjects with bipolar I disorder but there were no significant changes in the overall levels of [(3)H]flumazenil binding. There were significant decreases in zolpidem-sensitive and increases in zolpidem-insensitive [(3)H]flumazenil binding in most regions of the sections of the hippocampal formation studied in bipolar I disorder. Unlike [(3)H]flumazenil, zolpidem does not bind to the alpha5 subunit of the GABA(A) receptor; therefore, we postulate that there is an increase in GABA(A) receptors containing alpha5 subunit in the hippocampus from subjects with bipolar I disorder.

Three-dimensional isobolographic analysis of interactions between lamotrigine and clonazepam in maximal electroshock-induced seizures in mice.

The anticonvulsant effects of lamotrigine (LTG) and clonazepam (CZP) and combinations thereof against maximal electroshock (MES)-induced seizures in mice were investigated using three-dimensional (3D) isobolographic analysis. With this method, the doses of fixed-ratio combinations of the drugs (1:3, 1:1 and 3:1) that elicited 16, 50 and 84% of the maximum anticonvulsant effect were determined. Additionally, to evaluate the characteristics of interactions observed with 3D isobolography, the brain concentrations of both drugs were verified pharmacokinetically. The 3D isobolographic analysis showed that LTG and CZP combined at the fixed ratios of 3:1 and 1:1 interacted synergistically in the MES test for all anticonvulsant effects between 16% and 84% of maximum. In contrast, the combination of LTG and CZP at the fixed ratio of 1:3 showed only pure additivity for all estimated effects in 3D isobolography. Moreover, none of the examined antiepileptic drugs altered the brain concentrations of the coadministered drug, so the observed interactions in the MES test are of a pharmacodynamic nature. The 3D isobolographic findings suggest that in epilepsy therapy, increased efficacy of seizure control (synergistic interaction) might be achieved by using LTG and CZP in combination. In this study, some important problems and assumptions related to statistical analysis of data in 3D isobolography are discussed.

Elimination of 7-aminoclonazepam in urine after a single dose of clonazepam.

The objective of this paper was to determine how long after administration of benzodiazepine clonazepam (CLO), its major metabolite 7-aminoclonazepam (7-ACLO) could be detected in urine collected from 10 healthy volunteers who received a single 3-mg dose of Klonopin (clonazepam). Such data would be of great importance to law enforcement agencies trying to determine the best time interval for urine collection from a victim of drug-facilitated sexual assault in order to reveal drug use. A highly sensitive NCI-GC-MS method for the simultaneous quantitation of CLO and its major metabolite 7-ACLO in urine was developed and validated. The following urine samples were collected from each volunteer: one before CLO administration, and 6 h, and 1, 3, 5, 8, 10, 14, 21 and 28 days after. All urine samples (1 mL) were extracted following addition of the internal standard (D(5)-diazepam) and enzymatic hydrolysis ( beta-glucuronidase) using solid-phase extraction columns. Standard curves for CLO (500-4000 pg x mL(-1)) and 7-ACLO (50-2000 pg x mL(-1)) were prepared by spiking aliquots of negative urine. The urine from every subject was still positive for 7-ACLO 14 days after administration of the drug. Eight of the ten volunteers had measurable amounts of the metabolite 21 days after administration. One volunteer was still positive 28 days after administration. Six of the volunteers had urine concentrations of 7-ACLO that peaked at 1 day after administration. One volunteer had the highest concentration of 7-ACLO at 3 days, two volunteers at 5 days, and one at 8 days. The range of concentrations detected was from 73.0 pg x mL(-1) to 183.2 ng x mL(-1). CLO was not detected in any of the samples.

Deposition of 7-aminoclonazepam and clonazepam in hair following a single dose of Klonopin.

The objective of this paper was to determine whether benzodiazepine clonazepam (CLO) and its major metabolite 7-aminoclonazepam (7-ACLO) could be detected in hair collected from healthy volunteers after receiving a single 3-mg dose of Klonopin (clonazepam). Such data would be of great importance to law enforcement agencies trying to determine the best time interval for hair collection from a victim of drug-facilitated sexual assault (DFSA) in order to reveal drug use. Ten healthy volunteers (6 women and 4 men, 23-49 years old) participated in the study. The following hair samples were collected from each volunteer: one before CLO administration, and 1, 3, 5, 14, 21, and 28 days after. All hair samples were pulverized and 50-mg aliquots were sonicated in methanol and digested with 0.1 N HCl at 55 degrees C for 18-24 h. Internal standard, diazepam-d5 (DIAZ-d5) was used. Both extracts were combined and extracted using HCX solid-phase extraction columns. After derivatization with HFBA all extracts were analyzed using highly sensitive negative chemical ionization gas chrometography-mass spectrometry. Standard curves for CLO (20-100 pg/mg) and 7-ACLO (1-20 pg/mg) were prepared by spiking aliquots (50 mg) of negative hair and had correlation coefficients of 0.985 and 0.989, respectively. In addition, two levels of control hair were prepared for CLO and 7-ACLO. All method validation parameters were within acceptable limits. 7-ACLO was detected in hair of 6 out of 10 volunteers. In two cases 7-ACLO appeared in hair three days after CLO intake and remained detectable for the entire 28-day study period (3.6-8.4 pg/mg and 2.7-3.0 pg/mg), and in two subjects it was detectable 21 days later (4.9 and 2.7 pg/mg and 1.2 and 23 pg/mg). In two volunteers 7-ACLO was detected only on day 28 (1.8 and 3.3 pg/mg). CLO was not detected in any of the samples.

Specific ligands of the peripheral benzodiazepine receptor induce apoptosis and cell cycle arrest in human esophageal cancer cells.

Esophageal cancer is the most markedly increasing tumor entity in Western countries. Due to very poor 5-year-survival, new therapeutic approaches are mandatory. Peripheral benzodiazepine receptors (PBR) have been implicated in growth control of various tumor models, but they have not been studied yet in esophageal cancer. We used esophageal cancer cell lines and primary cell cultures of human esophageal cancers and evaluated (i) expression and localization of PBR; (ii) PBR-ligand-induced inhibition of cell growth; (iii) induction of apoptosis; and (iv) alterations in cell cycle. Expression of PBR was detected both in cell lines and in primary cell cultures of human esophageal cancers. PBR was localized in the mitochondria. The PBR-specific ligands FGIN-1-27 and PK 11195, but not the centrally acting benzodiazepine clonazepam or the indolacetamide FGIN-1-52, neither of which displaying any affinity to the PBR, inhibited cell proliferation. FGIN-1-27 and PK 11195, but not clonazepam, potently induced apoptosis. FGIN-1-27 was shown to sequentially decrease the mitochondrial membrane potential, then to activate caspase-3 and finally to cause DNA fragmentation. In addition, PBR-specific ligands induced cell cycle arrest in the G1/G0 phase. Our data qualify PBR-specific ligands as innovative proapoptotic and antiproliferative substances. They might prove suitable for the treatment of esophageal cancer.

Evaluation of native GABA(A) receptors containing an alpha 5 subunit.

The type A receptor for gamma-aminobutyric acid (GABA), or GABA(A) receptor, is a pentamer of highly variable quaternary structure. It includes two alpha subunits, drawn from a pool of six genes, which largely determine benzodiazepine pharmacology of the receptor. In brain sections, both [(3)H]RY-80 (ethyl-8-acetylene-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5a][1,4]benzodiazepine-3-carboxylate) and [(3)H]L-655,708 (ethyl (S)-11,12,13,13a-tetrahydro-7-methoxy-9-oxo-9H-imidazo[1,5-a]pyrrolo[2,1-c][1,4]benzodiazepine-1-carboxylate), which are selective for the benzodiazepine site of alpha 5 subunit-containing receptors, showed high-affinity, specific binding, but to fewer regions than did the nonselective benzodiazepine, [(3)H]flunitrazepam. The pattern mirrored alpha 5 mRNA distribution, and was similar to that previously reported for [(3)H]L-655,708 binding. Displacement of [(3)H]RY-80 bound to hippocampal homogenates, and of [(3)H]flunitrazepam bound to cerebellar and hippocampal homogenates showed comparable displacement by flumazenil (K(i)'s 5--7 nM). However, the K(i)'s for diazepam and for clobazam to displace [(3)H]RY-80 binding in hippocampus were about fourfold higher than for [(3)H]flunitrazepam, and the K(i) for clonazepam was sixfold larger, suggesting that these benzodiazepine receptor agonists bind with relatively lower affinity at hippocampal alpha 5-containing receptors.

Quantitation of clonazepam and its major metabolite 7-aminoclonazepam in hair.

Clonazepam (CLO) is an anticonvulsant benzodiazepine approved by the Food and Drug Administration for use in the treatment of seizures. It produces pharmacological effects (depression, amnesia) similar to other compounds from the same therapeutic class, and in combination with alcohol, its CNS-depressant action can be significantly potentiated. As with some other benzodiazepines, CLO is a drug possibly used in "date-rape" situations. A method using solid-phase extraction followed by a highly sensitive negative chemical ionization gas chromatography-mass spectrometry for the simultaneous quantitation of CLO and its major metabolite 7-aminoclonazepam (7-ACLO) in hair was developed and validated. The method has potential application to alleged drug-facilitated rape cases. To determine the feasibility of detecting 7-ACLO and CLO in hair, specimens were collected from 10 psychiatric patients treated with CLO, divided into 2-cm segments, and analyzed. Standard curves for 7-ACLO (1-1000 pg/mg) and CLO (10-400 pg/mg) had correlation coefficients of 0.998. All precision and accuracy values were within acceptable limits. 7-ACLO was present in measurable quantities (1.37-1267 pg/mg) in 9 out of 10 patient samples. CLO concentrations in hair were much lower (10.7-180 pg/mg). In 4 out of 10 cases, CLO was not detected in hair. Two patients who had never been treated with CLO before received a single 2-mg dose of the drug. Approximately three weeks later, hair samples were collected, and measurable quantities of 7-ACLO (4.8 pg/mg) were detected in the first segment (proximal) of one of those samples, and traces of the drug were present in the other sample. We concluded that the 7-ACLO is being deposited in hair in much higher quantities than the parent drug and remains there for extended periods of time. Our study also indicates that it is possible to detect 7-ACLO after a single dose of CLO as in the typical date-rape scenarios.

Analysis of clonazepam in a tablet dosage form using smallbore HPLC.

A stability indicating, reversed phase high-performance liquid chromatographic method utilizing a smallbore HPLC column has been developed for the determination of clonazepam in a commercial tablet dosage form. The use of a small bore column results in a substantial solvent savings, as well as a greater mass sensitivity, especially in the identification of degradation peaks in a chromatogram. The method involves ultraviolet detection at 254 nm and utilized a 150 x 3.0 mm i.d. column packed with 3 microm octyldecylsilane particles with a mobile phase of water methanol acetonitrile (40:30:30, v/v/v) at a flow rate of 400 microl min(-1) at ambient temperature, with and without the use of 1,2-dichlorobenzene as the internal standard. The current USP method for the analysis of clonazepam using a 300 x 3.9 mm i.d. conventional octyldecylsilane column was utilized as a comparison to the smallbore method. The retention times for clonazepam and the internal standard on the 3.0 mm i.d. column were 4.0 and 12.5 min, respectively. The intra- and interday RSDs on the 3.0 mm i.d. column were < 0.55% (n =4) using the internal standard, and < 0.19% (n = 4) without the internal standard at the lower limit of the standard curve, 50 microg ml(-1) and had a limit of detection of 24 ng ml(-1). The assay using the 3.0 mm i.d. column was shown to be suitable for measuring clonazepam in a tablet dosage form.

Characterization of binding sites for the omega3 receptor ligands [3H]PK11195 and [3H]RO5-4864 in human brain.

The kinetics and pharmacology of the isoquinoline and benzodiazepine binding sites of the omega3 or peripheral-type benzodiazepine receptors were studied using the specific ligands [3H] 7-chloro-5-(4-chlorophenyl)-1,3-dihydro-1-methyl-2H-1,4-benzodiazepin -2-one ([3H]PK11195) and [3H]1-(2-Chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarb oxamide ([3H]RO5-4864), respectively. Binding of both ligands was saturable, reversible, displayed nanomolar affinity, and best fit to a single site model. Occipital cortex and cerebellum displayed highest and lowest densities of binding sites respectively; for both ligands. Bmax values of [3H]PK11195 were several-fold higher than that of [3H]RO5-4864 in all regions studied consistent with their binding to distinct subunits of the human peripheral-type benzodiazepine receptor heteromeric complex. However, the isoquinoline and benzodiazepine ligands were found to be mutually competitive at nanomolar concentrations suggesting allosteric interactions between these two sites. Competition binding experiments showed that the binding of both ligands was displaced by diazepam with Ki values in the nM range, and by clonazepam in the microM range. The novel peripheral-type benzodiazepine receptor ligand 2-(4-fluorophenyl)-N,N-di-n-hexyl-1H-indole-3-acetamide (FGIN1-27) displaced only [3H]PK11195 binding with high potency. Heterogeneity of the two sites is observed, manifested by their differential susceptibility towards detergents and alcohols. Histidine residue modification by diethylpyrocarbonate treatment abolished only [3H]PK11195 binding but had no effect on [3H]RO5-4864 binding. These studies demonstrate that the isoquinoline and benzodiazepine sites on the peripheral-type benzodiazepine receptor in human brain manifest many pharmacological characteristics that are distinct from each other and from rodent brain peripheral-type benzodiazepine receptors.

Selective and prolonged MRI enhancement by Mn-TPPS in an experimental rat brain tumour with peripheral benzodiazepine receptors.

Synthesized Mn-TPPS, a paramagnetic metalloporphyrin, is expected to be a tumour specific contrast media for magnetic resonance (MR) imaging. We investigated the enhancing characteristics of Mn-TPPS using a transplanted rat C6 glioma model with peripheral type benzodiazepine (PBD) receptors since porphyrins are thought to possibly be endogenous ligands for PBD receptors. An Mn-TPPS enhancement study was then performed either with or without pretreatment while using peripheral and central type benzodiazepine receptor specific ligands (PK11195 and clonazepam, respectively). A signal intensity analysis disclosed the selective and prolonged enhancement of the brain tumour even at 17 h after the Mn-TPPS injection. This specific enhancement of the tumour, however, was not inhibited nor replaced by benzodiazepines. The tissue concentration of Mn-TPPS was significantly higher in the glioma tissue than the other tissues, while PK11195 pretreatment could not reduce the intratumoural Mn-TPPS concentration. A subcellular distribution study disclosed that Mn-TPPS was readily incorporated into the tumour cells. On the other hand, Mn-TPPS was not specifically distributed in the mitochondrial fraction where PBD receptors exist. The present study therefore indicates that Mn-TPPS could be incorporated into tumour cells and supports the potential use of this agent to improve the diagnostic specificity of MR imaging for brain tumours.

Immunoaffinity purification of gamma-aminobutyric acidA (GABAA) receptors containing gamma 1-subunits. Evidence for the presence of a single type of gamma-subunit in GABAA receptors.

Two polyclonal antibodies were raised against the gamma 1-subunit of gamma-aminobutyric acidA (GABAA) receptors. One of these antibodies could be used for the immunopurification of GABAA receptors containing gamma 1-subunits. These receptors exhibited [3H]muscimol and [3H]flunitrazepam binding, and their benzodiazepine binding properties were dramatically different from those of receptors precipitated by anti-gamma 2- or anti-gamma 3-antibodies. Western blot analysis of the immunopurified GABAA receptors indicated that the gamma 1-subunit exhibits an apparent molecular mass of 45-51 kDa. Furthermore, in addition to gamma 1-subunits, beta 2/3-, alpha 1-, alpha 2-, alpha 3-, and alpha 5-subunits could be detected in immunoaffinity column eluates from total brain. In contrast, gamma 2- or gamma 3-subunits could not be identified in GABAA receptors immunopurified by anti-gamma 1-antibodies. Similarly, gamma 1- and gamma 3-subunits or gamma 1- and gamma 2-subunits could not be identified in GABAA receptors purified by anti-gamma 2- or anti-gamma 3-antibodies, respectively. These data seem to indicate that GABAA receptors contain only a single type of gamma-subunit.