A Benzodiazepine Ligand with Improved GABAA Receptor α5-Subunit Selectivity Driven by Interactions with Loop C.

The family of GABAA receptors is an important drug target group in the treatment of sleep disorders, anxiety, epileptic seizures, and many others. The most frequent GABAA receptor subtype is composed of two α-, two ß-, and one γ2-subunit, whereas the nature of the α-subunit critically determines the properties of the benzodiazepine binding site of those receptors. Nearly all of the clinically relevant drugs target all GABAA receptor subtypes equally. In the past years, however, drug development research has focused on studying α5-containing GABAA receptors. Beyond the central nervous system, α5-containing GABAA receptors in airway smooth muscles are considered as an emerging target for bronchial asthma. Here, we investigated a novel compound derived from the previously described imidazobenzodiazepine SH-053-2'F-R-CH3 (SH53d-ester). Although SH53d-ester is only moderately selective for α5-subunit-containing GABAA receptors, the derivative SH53d-acid shows superior (>40-fold) affinity selectivity and is a positive modulator. Using two-electrode voltage clamp electrophysiology in Xenopus laevis oocytes and radioligand displacement assays with human embryonic kidney 293 cells, we demonstrated that an acid group as substituent on the imidazobenzodiazepine scaffold leads to large improvements of functional and binding selectivity for α5ß3γ2 over other αxß3γ2 GABAA receptors. Atom level structural studies provide hypotheses for the improved affinity to this receptor subtype. Mutational analysis confirmed the hypotheses, indicating that loop C of the GABAA receptor α-subunit is the dominant molecular determinant of drug selectivity. Thus, we characterize a promising novel α5-subunit-selective drug candidate. SIGNIFICANCE STATEMENT: In the current study we present the detailed pharmacological characterization of a novel compound derived from the previously described imidazobenzodiazepine SH-053-2'F-R-CH3. We describe its superior (>40-fold) affinity selectivity for α5-containing GABAA receptors and show atom-level structure predictions to provide hypotheses for the improved affinity to this receptor subtype. Mutational analysis confirmed the hypotheses, indicating that loop C of the GABAA receptor α-subunit is the dominant molecular determinant of drug selectivity.

Facile synthesis of copper(II) oxide nanospheres covered on functionalized multiwalled carbon nanotubes modified electrode as rapid electrochemical sensing platform for super-sensitive detection of antibiotic.

Herein, we report a super-active electrocatalyst of copper(II) oxide nanoparticles (CuO NPs) decorated functionalized multiwalled carbon nanotubes (CuO NPs@f-MWCNTs) by the ultrasonic method. The as-synthesized CuO NPs@f-MWCNTs was characterized through the FESEM, XPS, XRD and electrochemical impedance spectroscopy (EIS). The combination of highly active CuO NPs and highly conductive f-MWCNTs film with rapid detection enables this nanohybrid to display excellent electrochemical performance towards anesthesia drug. Furthermore, the hybrid electrocatalyst modified SPCE was developed for the determination of flunitrazepam (FTM) for the first time. FTM is important anesthesia drug with high adverse effect in human body. Benefiting from the synergistic reaction of CuO NPs and f-MWCNTs, this nanohybrid exhibited high sensitivity and specificity towards FTM electro-reduction. The CuO NPs@f-MWCNTs film modified SPCE exhibits outstanding electrochemical activity including excellent reproducibility, wide linear range from 0.05 to 346.6 µM with nanomolar limit of detection for FTM detection. Further, the as-modified CuO NPs@f-MWCNTs/SPCE has been applied to determination of FTM in biological and drug samples with satisfactory recovery results, thereby showing a notable potential for extensive (bio) sensor applications.

Quantification of 7-aminoflunitrazepam in human urine by polymeric monolith-based capillary liquid chromatography coupled to tandem mass spectrometry.

Using a simple liquid-liquid extraction (LLE) procedure for sample pretreatment, 7-Aminoflunitrazepam (7-aminoFM2), a major metabolite of flunitrazepam (FM2), was determined in urine samples by polymeric monolith-based capillary liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The linearity was found in the range of 0.1-50ngmL-1 with a method detection limit (signal-to-noise ratio of 3) estimated at 0.05ngmL-1. Using the proposed method, good precision and recovery were also found in spiked urine samples at the levels of 0.5, 5.0, and 50ngmL-1 (intra-day/inter-day precision: 0.6-1.8% / 0.1-0.8%; post-spiked/pre-spiked recovery: 95.4-102.9% / 96.3-102.5%). In addition, acceptable relative differences (-24.2 - 0.8%) were observed by analyzing clinical urine samples using this monolith-based capillary LC-MS/MS method compared with the results obtained by the routine GC-MC method. Using the monolithic column, no noticeable deterioration of separation efficiency or carry-over was observed for more than 200 injections of urine samples. The applicability of the developed monolith-based capillary LC-MS/MS method was demonstrated by quantifying 7-aminoFM2 in various clinical urine samples. Based on these experimental results, the proposed LLE-monolith-based capillary LC-MS/MS method shows the potential for routine determination of drug metabolites in human urine for clinical and forensic applications.

Long-term storage of authentic postmortem forensic blood samples at -20°C: measured concentrations of benzodiazepines, central stimulants, opioids and certain medicinal drugs before and after storage for 16-18 years.

The long-term stability of benzodiazepines, opioids, central stimulants and medicinal drugs in authentic postmortem blood samples was studied. All together, 73 samples were reanalyzed after storage at -20°C for 16-18 years. At reanalysis samples containing diazepam, nordiazepam and flunitrazepam demonstrated only small changes during long-term storage when mean and median drug concentrations were compared, while clonazepam concentrations tended to decrease. Samples containing amphetamine, morphine, codeine and 'acidic' medicinal drugs as paracetamol and meprobamate also showed small changes over 16-18 years in mean and median drug concentrations at a group level. For many drugs, however, single samples could demonstrate marked concentration changes, both increases and decreases during storage. For 'alkaline' medicinal drugs, concentration losses were observed in most cases.

Rapid determination of flunitrazepam in alcoholic beverages by desorption electrospray ionization-mass spectrometry.

Desorption electrospray ionization-mass spectrometry (DESI-MS), a novel ambient ionization technique, was used in this study for determining flunitrazepam in various alcoholic beverages. Using this technique, no pretreatment of the samples was necessary and identification of the drug was accomplished in individual samples in minutes. In addition, the acquired mass spectra provide the information of the identity of the drink based on the detected characteristic ions from the matrices. This study also demonstrates the capability of DESI-MS to perform quantitative analysis of simulated evidence samples with a limit of quantification of 3µg/mL. Furthermore it has been shown that this method can be used for high-throughput analysis whereby six samples were analyzed in a row within 6 minutes and no observable sample carry-over was noted. DESI-MS shows potential as a rapid, sensitive, and selective technique for forensic analysis of spiked beverages which are typical evidence of drug facilitated sexual assault and robbery cases.

Coupling between GABA(A)-R ligand-binding activity and membrane organization in ß-cyclodextrin-treated synaptosomal membranes from bovine brain cortex: new insights from EPR experiments.

Correlations between GABA(A) receptor (GABA(A)-R) activity and molecular organization of synaptosomal membranes (SM) were studied along the protocol for cholesterol (Cho) extraction with ß-cyclodextrin (ß-CD). The mere pre-incubation (PI) at 37°C accompanying the ß-CD treatment was an underlying source of perturbations increasing [(3)H]-FNZ maximal binding (70%) and K (d) (38%), plus a stiffening of SMs' hydrocarbon core region. The latter was inferred from an increased compressibility modulus (K) of SM-derived Langmuir films, a blue-shifted DPH fluorescence emission spectrum and the hysteresis in DPH fluorescence anisotropy (A (DPH)) in SMs submitted to a heating-cooling cycle (4-37-4°C) with A (DPH,heating) < A (DPH,cooling). Compared with PI samples, the ß-CD treatment reduced B (max) by 5% which correlated with a 45%-decrement in the relative Cho content of SM, a decrease in K and in the order parameter in the EPR spectrum of a lipid spin probe labeled at C5 (5-SASL), and significantly increased A (TMA-DPH). PI, but not ß-CD treatment, could affect the binding affinity. EPR spectra of 5-SASL complexes with ß-CD-, SM-partitioned, and free in solution showed that, contrary to what is usually assumed, ß-CD is not completely eliminated from the system through centrifugation washings. It was concluded that ß-CD treatment involves effects of at least three different types of events affecting membrane organization: (a) effect of PI on membrane annealing, (b) effect of residual ß-CD on SM organization, and (c) Cho depletion. Consequently, molecular stiffness increases within the membrane core and decreases near the polar head groups, leading to a net increase in GABA(A)-R density, relative to untreated samples.

N,N'-dicyclohexylsulfamide and N,N'-diphenethylsulfamide are anticonvulsant sulfamides with affinity for the benzodiazepine binding site of the GABA(A) receptor and anxiolytic activity in mice.

A set of sulfamides designed, synthesized and evaluated against maximal electroshock seizure (MES) and pentilenetetrazol (PTZ) tests with promising results, were tested for their affinity for the benzodiazepine binding site of the GABA(A) receptor. The most active compounds, N,N'-dicyclohexylsulfamide (7) and N,N'-diphenethylsulfamide (10), competitively inhibited the binding of [(3)H]-flunitrazepam to the benzodiazepine binding site with K(i)±SEM values of 27.7±4.5µM (n=3) and 6.0±1.2µM (n=3), respectively. The behavioral actions of these sulfamides, i.p. administered in mice, were examined in the plus-maze, hole-board and locomotor activity assays. Compound 7 exhibited anxiolytic-like effects in mice evidenced by a significant increase of the parameters measured in the hole-board test (at 1 and 3mg/kg) and the plus-maze assay (at 1 and 3mg/kg). Compound 10 evidenced anxiolytic activity in the plus-maze and the hole-board tests at 1mg/kg. Locomotor activity of mice was not modified by compound 7 or 10 at the doses tested. Flumazenil, a non selective benzodiazepine binding site antagonist, was able to completely reverse the anxiolytic-like effects of these sulfamides, proving that the GABA(A) receptor is implicated in this action. Anxiety represents a major problem for people with epilepsy. The use of anxiolytic and anticonvulsant sulfamides would be beneficial to individuals who suffer from both disorders.

Autoradiographic analysis of GABAA receptor binding in the neural anxiety network of postpartum and non-postpartum laboratory rats.

Postpartum female rats exhibit a suppression of anxiety-related behaviors when compared to diestrous virgin females, pregnant females, and males. This blunted anxiety promotes optimal maternal care and involves elevated GABA neurotransmission, possibly including greater density of GABA(A) and benzodiazepine receptors in the postpartum brain. We here examined autoradiographic binding of [(3)H]muscimol to measure the total population of GABA(A) receptors and [(3)H]flunitrazepam to assess density of benzodiazepine sites in the medial prefrontal cortex, bed nucleus of the stria terminalis, amygdala, hippocampus, and periaqueductal gray of female rats sacrificed on day 7 postpartum, day 10 of pregnancy, or as diestrous virgins. A group of sexually naïve male rats was also included. We found that [(3)H]muscimol binding did not differ among groups in any site but that diestrous virgin females had greater [(3)H]flunitrazepam binding in the CA1 and dentate gyrus of the hippocampus compared to mid-pregnant females and males. Notably, postpartum and diestrous virgin females did not significantly differ in binding of either ligand in any site examined. This is the first study to evaluate the densities of GABA(A) and benzodiazepine binding sites simultaneously across three female reproductive states and sex with a focus on brain sites influencing anxiety-related behaviors. The results suggest that changes in other GABA(A) receptor characteristics such as subunit composition, or increased presynaptic GABA release during interactions with offspring, must instead play a greater role in the postpartum suppression of anxiety in laboratory rats.

GABA(A) receptor subtype selectivity underlying anxiolytic effect of 6-hydroxyflavone.

6-Hydroxyflavone (6HF), a naturally occurring flavonoid, was previously reported to bind to type A gamma-aminobutyric acid (GABA(A)) receptors benzodiazepine (BZ) site with moderate binding affinity. In the present study, we showed that 6HF partially potentiated GABA-induced currents in native GABA(A) receptors expressed in cortical neurons via BZ site, as the enhancement was blocked by the antagonist flumazenil. Furthermore, in patch clamp studies, 6HF displayed significant preference for alpha(2)- and alpha(3)-containing subtypes, which were thought to mediate anxiolytic effect, compared to alpha(1)- and alpha(5)-containing subtypes expressed in HEK 293T cells. In mice, 6HF exhibited anxiolytic-like effect in the elevated plus-maze test, unaccompanied at anxiolytic doses by the sedative, cognitive impairing, myorelaxant, motor incoordination and anticonvulsant effects commonly associated with classical BZs when tested in the hole-board, step-through passive avoidance, horizontal wire, rotarod, and pentylenetetrazol (PTZ)-induced seizure tests, respectively. The findings therefore identified 6HF as a promising drug candidate for the treatment of anxiety-like disorders.

Characterization of Escherichia coli nitroreductase NfsB in the metabolism of nitrobenzodiazepines.

Nitrobenzodiazepine (NBDZ) is a sedative-hypnotic drug used in the treatment of anxiety and sleep problems. Overdose of NBDZ may cause severe neurological effects, especially for people in drug abuse or addiction. In the present study, we investigated NBDZ nitroreduction in rat enteric contents and characterized the role of enterobacterial nitroreductase in the reductive pathway. Nitroreduction of flunitrazepam (FZ) was studied in the microsomal membrane fractions of rat liver, jejunum and jejunal microflora using HPLC analysis. In the jejunal microflora, FZ was demonstrated to be significantly reduced to its amino derivative under anaerobic condition. Escherichia coli type I nitroreductase NfsB (EC 1.5.1.34) was found in rat jejunal microflora via PCR technique and Western blotting. The participation of NfsB in FZ nitroreduction was demonstrated from inhibition studies. Kinetic study of the purified recombinant NfsB indicated that nitroreduction of FZ, nitrazepam (NZ) and clonazepam (CZ) are mediated by NfsB, where CZ shows lower k(cat)/K(M) ratio than that of the other two. Finally, two other nitroreductases E. cloacae NR (EC 1.6.99.7) and S. typhimurium Cnr were also found to be responsible for FZ nitroreduction. These results provide that the reduction of NBDZ in normal flora is catalyzed by type I nitroreductase NfsB.

[Comparative study of drug efficacy and drug additives between generic drugs and original drugs].

In the present study, we tested three kinds of sleeping drugs, consisting mainly of triazolam, brotizolam, and flunitrazepam, to compare the drug efficacy of generic drugs with that of original drugs. After these drugs were administered orally to mice, drug efficacy was evaluated in terms of ambulation, onset time of sleep, and duration of sleep in the open field test. For all kinds of sleep-inducing drugs, the drug efficacy of most generic drugs is not necessarily equal to that of the original drug. The main reason for the difference appears to be due to differences in the rate of absorption of the main drug. Any other differences between an original drug and a generic drug are caused by drug additives, the crystal form of the main drug, the formulation, and so on. In this study, the formulation was not the reason for the differences because all of the drugs were pulverized in a mortar and had no special coating. The drug additives for all the drugs are listed and the drug efficacy compared. Unfortunately, the information was not sufficient to shed any light on the differences in drug efficacy. For effective drug therapy, more information on drug additives should be provided.

Exploring ligand recognition and ion flow in comparative models of the human GABA type A receptor.

We present two comparative models of the GABA(A) receptor. Model 1 is based on the 4-A resolution structure of the nicotinic acetylcholine receptor from Torpedo marmorata and represents the unliganded receptor. Two agonists, GABA and muscimol, two benzodiazepines, flunitrazepam and alprazolam, together with the general anaesthetic halothane, have been docked to this model. The ion flow is also explored in model 1 by evaluating the interaction energy of a chloride ion as it traverses the extracellular, transmembrane and intracellular domains of the protein. Model 2 differs from model 1 only in the extracellular domain and represents the liganded receptor. Comparison between the two models not only allows us to explore commonalities and differences with comparative models of the nicotinic acetylcholine receptor, but also suggests possible protein sub-domain interactions with the GABA(A) receptor not previously addressed.

Effects of flunitrazepam on the Lalpha-H(II) phase transition of phosphatidylethanolamine using merocyanine 540 as a fluorescent indicator.

Previous studies of our group demonstrated that flunitrazepam is a lipophilic drug capable of interacting with membranes through a partition equilibrium phenomenon. Its localization at the phospholipid polar head region could explain the decrease in the size of dipalmitoylphosphatidylcholine (dpPC) vesicles, through a mechanism that involves the increment in the relative volume of this region with a subsequent increase in the vesicle's surface curvature. In the present work, we investigated if flunitrazepam can affect the L(alpha)-H(II) phase transition of phosphatidylethanolamine through a similar mechanism. This study was approached by using merocyanine 540, a dye sensitive to the molecular packing of membrane lipids. A detailed analysis of merocyanine absorption and fluorescence emission and excitation spectra was performed. The results indicated that the fluorescence emitted came mainly from the monomeric form of merocyanine and that it resulted a good indicator of this phase transition, as was previously described. Flunitrazepam did not affect significantly the onset of the phase transition but showed a tendency to diminish the dye fluorescence emission intensity, which could involve a lower partition of merocyanine in the vesicles. Moreover, the results suggest that this drug produced a delay in the completeness of the phase transition and a decrement in the cooperativity of this phenomenon.

Surface activity of thymol: implications for an eventual pharmacological activity.

In the present work, we studied the ability of thymol to affect the organization of model membranes and the activity of an intrinsic membrane protein, the GABA(A) receptor (GABA(A)-R). In this last aspect, we tried to elucidate if the action mechanism of this terpene at the molecular level, involves its binding to the receptor protein, changes in the organization of the receptor molecular environment, or both. The self-aggregation of thymol in water with a critical micellar concentration approximately = 4 microM and its ability to penetrate in monomolecular layers of soybean phosphatidylcholine (sPC) at the air-water interface, even at surface pressures above the equilibrium, lateral pressure of natural bilayers were demonstrated. Thymol affected the self-aggregation of Triton X-100 and the topology of sPC vesicles. It also increased the polarity of the membrane environment sensed by the electrochromic dye merocyanine. A dipolar moment of 1.341 Debye was calculated from its energy-minimized structure. Its effect on the binding of [3H]-flunitrazepam ([3H]-FNZ) to chick brain synaptosomal membranes changed qualitatively from a tendency to the inhibition to a clear activatory regime, up on changing the phase state of the terpene (from a monomeric to a self-aggregated state). Above its CMC, thymol increased the affinity of the binding of [3H]-FNZ (K(d-control)= 2.9, K(d-thymol)= 1.7 nM) without changing the receptor density (B(max-control)= 910, B(max-thymol)= 895 fmol/mg protein). The activatory effect of thymol on the binding of [ [3H]-FNZ was observed even in the presence of the allosteric activator gamma-aminobutyric acid (GABA) at a concentration of maximal activity, and was blocked by the GABA antagonist bicuculline. Changes in the dipolar arrangement and in the molecular packing of GABA(A)-R environment are discussed as possible mediators of the action mechanism of thymol.

Simultaneous determination of flunitrazepam and 7-aminoflunitrazepam in human serum by ion trap gas chromatography-tandem mass spectrometry.

A method for the determination of flunitrazepam (FNZ) and 7-aminoflunitrazepam (7-AFNZ) in human serum was developed with ion trap gas chromatography (GC)-tandem mass spectrometry. The 7-AFNZ was derivatizated with 50 microl trifluoroacetic anhydride (TFAA), 60 degrees C-20 min. EI mass spectra and tandem mass spectra of FNZ and 7-AFNZ-TFA were m/z 238, 239, 266, 286, 294, 312, 313(M(+)), m/z 350, 351, 360, 378, 379(M(+)), m/z 238, 239, 240 (precursor ion m/z 286, collision energy 1.5 V), and m/z 239, 254, 264, 336 (precursor ion m/z 351, collision energy 1.8 V), respectively. The detection limits of full scan EI mass spectrometry and tandem mass spectrometry for FNZ and 7-AFNZ in human serum were ca. 200 ng/ml, 60 ng/ml, 15 ng/ml and 1 ng/ml, respectively.

Targeted mutation of CCK2 receptor gene modifies the behavioural effects of diazepam in female mice.

RATIONALE: Evidence suggests that GABA and CCK have opposite roles in the regulation of anxiety. OBJECTIVE: The aim of the present work was to study diazepam-induced anxiolytic-like action and impairment of motor co-ordination, and the parameters of benzodiazepine receptors in mice lacking CCK2 receptors. METHODS: The action of diazepam (0.5-3 mg/kg i.p.) was studied in the elevated plus-maze model of anxiety and rotarod test using mice lacking CCK2 receptors. The parameters of benzodiazepine receptors were analysed using [3H]-flunitrazepam binding. RESULTS: In the plus-maze test, the exploratory activity of the homozygous (-/-) mice was significantly higher compared to their wild-type (+/+) littermates. However, the wild-type (+/+) mice displayed higher sensitivity to the anxiolytic-like action of diazepam. Even the lowest dose of diazepam (0.5 mg/kg) induced a significant increase of open arm entries in the wild-type (+/+) mice. A similar effect in the homozygous (-/-) mice was established after the administration of diazepam 1 mg/kg. The highest dose of diazepam (3 mg/kg) caused a prominent anxiolytic-like effect in the wild-type (+/+) mice, whereas in the homozygous (-/-) animals suppression of locomotor activity was evident. The performance of the homozygous (-/-) mice in the rotarod test did not differ from that of the wild-type (+/+) littermates. However, a difference between the wild-type (+/+) and homozygous (-/-) animals became evident after treatment with diazepam. Diazepam (0.5 and 3 mg/kg) induced significantly stronger impairment of motor co-ordination in the homozygous (-/-) mice compared to their wild-type (+/+) littermates. The density of benzodiazepine binding sites was increased in the cerebellum, but not in the cerebral cortex and hippocampus, of the homozygous (-/-) mice. CONCLUSIONS: Female mice lacking CCK2 receptors are less anxious than their wild-type (+/+) littermates. The reduced anxiety in homozygous (-/-) mice probably explains why the administration of a higher dose of diazepam is necessary to induce an anxiolytic-like action in these animals. The highest dose of diazepam (3 mg/kg) induced significantly stronger suppression of locomotor activity and impairment of motor co-ordination in the homozygous (-/-) mice compared to the wild-type (+/+) littermates. The increase in the action of diazepam is probably related to the elevated density of benzodiazepine receptors in the cerebellum of homozygous (-/-) mice. The present study seems to be in favour of increased tone of the GABAergic system in mice without CCK2 receptors.

[Fatal outcome of poisoning with the benzodiazepines flunitrazepam and diazepam]. / Tödlich verlaufene Intoxikation unter Beteiligung der Benzodiazepine Flunitrazepam und Diazepam.

On a wintry day a 29-year-old woman was found dead beside her car showing head injuries and signs of hypothermia. Several empty packets of sedative and hypnotic drugs were lying inside the car. Toxicological analysis revealed the presence of flunitrazepam (heart blood of the left and right chamber 0.033 mg/L each), norflunitrazepam (left heart blood 0.029 mg/L, right heart blood 0.027 mg/L), 7-amino-flunitrazepam (left heart blood 0.090 mg/L, right heart blood 0.104 mg/L), diazepam (left heart blood 0.395 mg/L, right heart blood 0.386 mg/L), nordazepam (left heart blood 0.112 mg/L, right heart blood 0.115 mg/L) and temazepam (left heart blood 0.034 mg/L, right heart blood 0.033 mg/L). Neither alcohol nor other drugs were found. It was concluded that benzodiazepine intake led to a disturbance of consciousness. Whether the woman died in this situation due to the icy temperature as a result of hypothermia or whether she died or would have died solely due to benzodiazepine overdosage could not be clarified.

Flunitrazepam-membrane non-specific binding and unbinding: two pathways with different energy barriers.

The effect of molecular packing on flunitrazepam's ability to interact with bio-membranes was studied using dipalmitoylphosphatidylcholine monomolecular layers at the air-water interface as a model membrane. Flunitrazepam penetrated from the subphase into monolayers at lateral pressures below 44.8 mN/m and induced their concentration-dependent expansion. As inferred from the values of compressibility modulus, the elasticity of the liquid-condensed phase decreased in the presence of flunitrazepam. Although this drug hardly penetrated into high-packed monolayers, it was easily incorporated in the low-packed ones at an extent sufficient to reach the partition equilibrium. Below a molecular area of 75 A(2), contrary to what would be expected, the drug surface concentration increased as a function of surface pressure, suggesting that after its penetration in disordered phases, it became energetically or physically trapped in newly-formed liquid condensed clusters. The phenomenon of flunitrazepam penetration and release would have different energy barriers depending on the membrane phase-state.

3H-nicotine, 3H-flunitrazepam, and 3H-cocaine incorporation into melanin: a model for the examination of drug-melanin interactions.

To explore drug-melanin interactions, we examined the in vitro tyrosinase-mediated formation of melanin from tyrosine in the presence of the 3H-cocaine (3H-COC), 3H-flunitrazepam (3H-FLU), and 3H-nicotine (3H-NIC) at 10-100,000 ng/mL. Polymerization in the presence of 10 or 100 ng/mL of each drug resulted in almost complete drug incorporation into the melanin pellet. Only 12% (3H-NIC) to 28% (3H-FLU) of the pellet-associated radioactivity could be released upon treatment with 6 M HCl. At 1000-100,000 ng/mL, between 20 and 50% of label became melanin-associated. In each case a significant percentage of melanin-associated radioactivity was resistant to treatment with 6 M HCl. Nicotine-associated radioactivity in the polymer was subject to much greater quenching than was 3H-COC or 3H-FLU, suggesting a much tighter association with the melanin. The subsequent demonstration of a covalent adduct of a melanin intermediate and nicotine has demonstrated the utility of this polymerization system as a model for further chemical characterization of drug-melanin interactions.

Flunitrazepam, a 7-nitro-1,4-benzodiazepine that is unable to bind to the indole-benzodiazepine site of human serum albumin.

Benzodiazepine (BDZ) is generally thought to bind to site II of human serum albumin (HSA), also known as the indole-BDZ site, which is located at subdomain III A of the molecule. However, differences in the binding characteristics of BDZ drugs with HSA have been reported. The photolabeling profiles of HSA with [(3)H]flunitrazepam (FNZP) in the presence and absence of diazepam (DZP) were shown to be identical, suggesting that each drug primarily binds to different regions. The results of fluorescent probe displacement experiments showed that FNZP failed to decrease the fluorescence of dansylsarcosine to an extent similar to that of DZP. In the photoinhibition experiment, site I and site II ligands failed to inhibit the photoincorporation of [(3)H]FNZP to HSA. In order to evaluate the photolabeling specificity of FNZP, an attempt was made to photolabel alpha(1)-acid glycoprotein (AGP) which also binds BDZ with similar affinity as HSA. The effect of myristate (MYR) and DZP on the FNZP photolabeling of these two major drug binding plasma proteins was examined. Photoincorporation was inhibited when HSA was photolabeled with [(3)H]FNZP in the presence of MYR but not in the presence of DZP. Conversely, DZP inhibited the photolabeling of [(3)H]FNZP to AGP. These results suggest that FNZP interacts with HSA at regions which are not located in the preformed binding pocket of subdomain III A.