Microliquid/Liquid Interfacial Sensors: Biomimetic Investigation of Transmembrane Mechanisms and Real-Time Determinations of Clemastine, Cyproheptadine, Epinastine, Cetirizine, and Desloratadine.

Antihistamines relieve allergic symptoms by inhibiting the action of histamine. Further understanding of antihistamine transmembrane mechanisms and optimizing the selectivity and real-time monitoring capabilities of drug sensors is necessary. In this study, a micrometer liquid/liquid (L/L) interfacial sensor has served as a biomimetic membrane to investigate the mechanism of interfacial transfer of five antihistamines, i.e., clemastine (CLE), cyproheptadine (CYP), epinastine (EPI), desloratadine (DSL), and cetirizine (CET), and realize the real-time determinations. Cyclic voltammetry (CV) and differential pulse voltammetry (DPV) techniques have been used to uncover the electrochemical transfer behavior of the five antihistamines at the L/L interface. Additionally, finite element simulations (FEMs) have been employed to reveal the thermodynamics and kinetics of the process. Visualization of antihistamine partitioning in two phases at different pH values can be realized by ion partition diagrams (IPDs). The IPDs also reveal the transfer mechanism at the L/L interface and provide effective lipophilicity at different pH values. Real-time determinations of these antihistamines have been achieved through potentiostatic chronoamperometry (I-t), exhibiting good selectivity with the addition of nine common organic or inorganic compounds in living organisms and revealing the potential for in vivo pharmacokinetics. Besides providing a satisfactory surrogate for studying the transmembrane mechanism of antihistamines, this work also sheds light on micro- and nano L/L interfacial sensors for in vivo analysis of pharmacokinetics at a single-cell or single-organelle level.

Synthesis and biological evaluation of BU-4664L derivatives as potential anticancer agents.

BU-4664L is a naturally occurring N-farnesylated dibenzodiazepinone with important biological activities. Herein, we report the synthesis and antitumor evaluation of two series of BU-4664L derivatives bearing different substituent patterns on the dibenzodiazepinone core and with diverse side chains. All of the derivatives displayed micromolar activity against the human prostate cancer PC-3 cells, while lower or no activity against the human lung H460 cells. The most active derivatives were 10a and 16c which exerted antiproliferative activity against PC-3 cells with GI50 values of 5.66 and 5.94 µM, respectively, and thus represent promising lead compounds for further development.

Dibenzazepine-linked isoxazoles: New and potent class of α-glucosidase inhibitors.

α-Glucosidase inhibition is a valid approach for controlling hyperglycemia in diabetes. In the current study, new molecules as a hybrid of isoxazole and dibenzazepine scaffolds were designed, based on their literature as antidiabetic agents. For this, a series of dibenzazepine-linked isoxazoles (33-54) was prepared using Nitrile oxide-Alkyne cycloaddition (NOAC) reaction, and evaluated for their α-glucosidase inhibitory activities to explore new hits for treatment of diabetes. Most of the compounds showed potent inhibitory potency against α-glucosidase (EC 3.2.1.20) enzyme (IC50 = 35.62 ± 1.48 to 333.30 ± 1.67 µM) using acarbose as a reference drug (IC50 = 875.75 ± 2.08 µM). Structure-activity relationship, kinetics and molecular docking studies of active isoxazoles were also determined to study enzyme-inhibitor interactions. Compounds 33, 40, 41, 46, 48-50, and 54 showed binding interactions with critical amino acid residues of α-glucosidase enzyme, such as Lys156, Ser157, Asp242, and Gln353.

Characterization of SPP inhibitors suppressing propagation of HCV and protozoa.

Signal peptide peptidase (SPP) is an intramembrane aspartic protease involved in the maturation of the core protein of hepatitis C virus (HCV). The processing of HCV core protein by SPP has been reported to be critical for the propagation and pathogenesis of HCV. Here we examined the inhibitory activity of inhibitors for γ-secretase, another intramembrane cleaving protease, against SPP, and our findings revealed that the dibenzoazepine-type structure in the γ-secretase inhibitors is critical for the inhibition of SPP. The spatial distribution showed that the γ-secretase inhibitor compound YO-01027 with the dibenzoazepine structure exhibits potent inhibiting activity against SPP in vitro and in vivo through the interaction of Val223 in SPP. Treatment with this SPP inhibitor suppressed the maturation of core proteins of all HCV genotypes without the emergence of drug-resistant viruses, in contrast to the treatment with direct-acting antivirals. YO-01027 also efficiently inhibited the propagation of protozoa such as Plasmodium falciparum and Toxoplasma gondii These data suggest that SPP is an ideal target for the development of therapeutics not only against chronic hepatitis C but also against protozoiasis.

Simultaneous measurement of epinastine and its metabolite, 9,13b-dehydroepinastine, in human plasma by a newly developed ultra-performance liquid chromatography-tandem mass spectrometry and its application to pharmacokinetic studies.

Epinastine is an antiallergic drug with high selectivity for histamine receptors. It has been reported that 9,13b-dehydroepinastine is present as a metabolite in vivo in humans, but there was little information about their pharmacokinetics (PKs) in humans. Although several analytical methods have been reported for epinastine analysis in different matrices, none are available for its metabolite. Therefore, the purpose of this study was to develop an analytical method to simultaneously measure epinastine and its metabolite, 9,13b-dehydroepinastine, in human plasma samples using an ultra-performance liquid chromatography-tandem mass spectrometer. Analytes were separated on a C18 column. Quantification of this analysis was performed on a triple-quadrupole mass spectrometer. Chromatograms showed high sensitivity, selectivity, and resolution with no interference with plasma constituents. Calibration curves for both epinastine and 9,13b-dehydroepinastine in human plasma were 0.1-50 ng/ml and displayed excellent linearity with correlation coefficients (r2 ) >0.99. The developed analytical method satisfied the criteria of international guidance and was validated. The method could be successfully applied to pharmacokinetic studies of epinastine and, for the first time, the metabolite kinetics of epinastine to 9,13b-dehydroepinastine in humans after oral administration of 20 mg epinastine hydrochloride tablets. Our study is expected to be useful in future studies such as dosage settings and clinical pharmacotherapy.

Dibenzazepine-Loaded Nanoparticles Induce Local Browning of White Adipose Tissue to Counteract Obesity.

Inhibition of Notch signaling via systemic drug administration triggers conversion of white adipocytes into beige adipocytes (browning) and reduces adiposity. However, translation of this discovery into clinical practice is challenged by potential off-target side effects and lack of control over the location and temporal extent of beige adipocyte biogenesis. Here, we demonstrate an alternative approach to stimulate browning using nanoparticles (NPs) composed of FDA-approved poly(lactide-co-glycolide) that enable sustained local release of a Notch inhibitor (dibenzazepine, DBZ). These DBZ-loaded NPs support rapid cellular internalization and inhibit Notch signaling in adipocytes. Importantly, focal injection of these NPs into the inguinal white adipose tissue depots of diet-induced obese mice results in localized NP retention and browning of adipocytes, consequently improving the glucose homeostasis and attenuating body-weight gain of the treated mice. These findings offer new avenues to develop a potential therapeutic strategy for clinical treatment of obesity and its associated metabolic syndrome.

Chemistry and biology of the compounds that modulate cell migration.

Cell migration is a fundamental step for embryonic development, wound repair, immune responses, and tumor cell invasion and metastasis. Extensive studies have attempted to reveal the molecular mechanisms behind cell migration; however, they remain largely unclear. Bioactive compounds that modulate cell migration show promise as not only extremely powerful tools for studying the mechanisms behind cell migration but also as drug seeds for chemotherapy against tumor metastasis. Therefore, we have screened cell migration inhibitors and analyzed their mechanisms for the inhibition of cell migration. In this mini-review, we introduce our chemical and biological studies of three cell migration inhibitors: moverastin, UTKO1, and BU-4664L.

HDAC-inhibitor (S)-8 disrupts HDAC6-PP1 complex prompting A375 melanoma cell growth arrest and apoptosis.

Histone deacetylase inhibitors (HDACi) are agents capable of inducing growth arrest and apoptosis in different tumour cell types. Previously, we reported a series of novel HDACi obtained by hybridizing SAHA or oxamflatin with 1,4-benzodiazepines. Some of these hybrids proved effective against haematological and solid cancer cells and, above all, compound (S)-8 has emerged for its activities in various biological systems. Here, we describe the effectiveness of (S)-8 against highly metastatic human A375 melanoma cells by using normal PIG1 melanocytes as control. (S)-8 prompted: acetylation of histones H3/H4 and α-tubulin; G0 /G1 and G2 /M cell cycle arrest by rising p21 and hypophos-phorylated RB levels; apoptosis involving the cleavage of PARP and caspase 9, BAD protein augmentation and cytochrome c release; decrease in cell motility, invasiveness and pro-angiogenic potential as shown by results of wound-healing assay, down-regulation of MMP-2 and VEGF-A/VEGF-R2, besides TIMP-1/TIMP-2 up-regulation; and also intracellular accumulation of melanin and neutral lipids. The pan-caspase inhibitor Z-VAD-fmk, but not the antioxidant N-acetyl-cysteine, contrasted these events. Mechanistically, (S)-8 allows the disruption of cytoplasmic HDAC6-protein phosphatase 1 (PP1) complex in A375 cells thus releasing the active PP1 that dephosphorylates AKT and blocks its downstream pro-survival signalling. This view is consistent with results obtained by: inhibiting PP1 with Calyculin A; using PPP1R2-transfected cells with impaired PP1 activity; monitoring drug-induced HDAC6-PP1 complex re-shuffling; and, abrogating HDAC6 expression with specific siRNA. Altogether, (S)-8 proved very effective against melanoma A375 cells, but not normal melanocytes, and safe to normal mice thus offering attractive clinical prospects for treating this aggressive malignancy.

Study of fluorescence interaction and conformational changes of bovine serum albumin with histamine H1 -receptor--drug epinastine hydrochloride by spectroscopic and time-resolved fluorescence methods.

The fluorescence, ultraviolet (UV) absorption, time resolved techniques, circular dichroism (CD), and infrared spectral methods were explored as tools to investigate the interaction between histamine H1 drug, epinastine hydrochloride (EPN), and bovine serum albumin (BSA) under simulated physiological conditions. The experimental results showed that the quenching of the BSA by EPN was static quenching mechanism and also confirmed by lifetime measurements. The value of n close to unity indicated that one molecule of EPN was bound to protein molecule. The binding constants (K) at three different temperatures were calculated (7.1 × 10(4), 5.5 × 10(4), and 3.9 × 10(4) M(-1)). Based on the thermodynamic parameters (ΔH(0), ΔG(0), and ΔS(0)), the nature of binding forces operating between drug and protein was proposed. The site of binding of EPN in the protein was proposed to be Sudlow's site I based on displacement experiments using site markers viz, warfarin, ibuprofen, and digitoxin. Based on the Förster's theory of non-radiation energy transfer, the binding average distance, r between the donor (BSA) and acceptor (EPN) was evaluated and found to be 4.48 nm. The UV-visible, synchronous fluorescence, CD, and three-dimensional fluorescence spectral results revealed the changes in secondary structure of the protein upon its interaction with EPN.

Dibenzazepines and dibenzoxazepines as sodium channel blockers.

We have identified two related series of dibenzazepine and dibenzoxazepine sodium channel blockers, which showed good potency on Nav1.7 in FLIPR-based and electrophysiological functional assays.

Reengineered tricyclic anti-cancer agents.

The phenothiazine and dibenzazepine tricyclics are potent neurotropic drugs with a documented but underutilized anti-cancer side effect. Reengineering these agents (TFP, CPZ, CIP) by replacing the basic amine with a neutral polar functional group (e.g., RTC-1, RTC-2) abrogated their CNS effects as demonstrated by in vitro pharmacological assays and in vivo behavioral models. Further optimization generated several phenothiazines and dibenzazepines with improved anti-cancer potency, exemplified by RTC-5. This new lead demonstrated efficacy against a xenograft model of an EGFR driven cancer without the neurotropic effects exhibited by the parent molecules. Its effects were attributed to concomitant negative regulation of PI3K-AKT and RAS-ERK signaling.

A new enantioselective CE method for determination of oxcarbazepine and licarbazepine after fungal biotransformation.

The present work describes, for the first time, the simultaneous separation of oxcarbazepine (OXC) and its active metabolite 10-hydroxy-10,11-dihydrocarbamazepine (licarbazepine, Lic) by chiral CE. The developed method was employed to monitor the enantioselective biotransformation of OXC into its active metabolite by fungi. The electrophoretic separations were performed using 10 mmol/L of a Tris-phosphate buffer solution (pH 2.5) containing 1% w/v of ß-CD phosphate sodium salt (P-ß-CD) as running electrolyte, -20 kV of applied voltage and a 15°C capillary temperature. The method was linear over the concentration range of 1000-30 000 ng/mL for OXC and 75-900 ng/mL for each Lic enantiomer (r ≥ 0.9952). Within-day precision and accuracy evaluated by RSD and relative errors, respectively, were lower than 15% for all analytes. The validated method was used to evaluate the enantioselective biotransformation of OXC, mediated by fungi, into its active metabolite Lic. This study showed that the fungi Glomerella cingulata (VA1) and Beuveria bassiana were able to enantioselectively metabolize the OXC into Lic after 360 h of incubation. Biotransformation by the fungus Beuveria bassiana showed 79% enantiomeric excess for (S)-(+)-Lic, while VA1 gave an enantiomeric excess of 100% for (S)-(+)-Lic. This study opens a new route to the drug (S)-(+)-licarbazepine.

Carbamazepine derivatives with P2X4 receptor-blocking activity.

Antagonists for the P2 receptor subtype P2X4, an ATP-activated cation channel receptor, have potential as novel drugs for the treatment of neuropathic pain and other inflammatory diseases. In the present study, a series of 47 carbamazepine derivatives including 32 novel compounds were designed, synthesized, and evaluated as P2X4 receptor antagonists. Their potency to inhibit ATP-induced calcium influx in 1321N1 astrocytoma cells stably transfected with the human P2X4 receptor was determined. Additionally, species selectivity (human, rat, mouse) and receptor subtype selectivity (P2X4 vs P2X1, 2, 3, 7) were investigated for selected derivatives. The most potent compound of the present series, which exhibited an allosteric mechanism of P2X4 inhibition, was N,N-diisopropyl-5H-dibenz[b,f]azepine-5-carboxamide (34, IC50 of 3.44µM). The present study extends the so far very limited knowledge on structure-activity relationships of P2X4 receptor antagonists.

Synthesis of a human urate transporter-1 inhibitor, an arginine vasopressin antagonist, and a 17ß-hydroxysteroid dehydrogenase type-3 inhibitor, using ring-expansion of cyclic ketoximes with DIBALH.

Synthesis of three clinical candidates for medicines, a human urate transporter-1 inhibitor, an arginine vasopressin antagonist, and a 17ß-hydroxysteroid dehydrogenase type-3 inhibitor, is described. These compounds were synthesized via construction of their 3,4-dihydro-2H-benzo[b][1,4]oxazine, dibenzodiazepine, and dibenzazocine skeletons, respectively, using the reductive ring-expansion reaction of the corresponding bicyclic or tricyclic oximes with diisobutylaluminum hydride.

Pharmacokinetics and tolerability of eslicarbazepine acetate and oxcarbazepine at steady state in healthy volunteers.

PURPOSE: Investigate the pharmacokinetics of once-daily (QD; 900 mg) and twice-daily (BID; 450 mg) regimens of eslicarbazepine acetate (ESL) and BID (450 mg) regimen of oxcarbazepine (OXC) at steady state in healthy volunteers. METHODS: Single-center, open-label, randomized, three-way (n = 12) crossover studies in healthy volunteers. KEY FINDINGS: Mean eslicarbazepine Cmax,ss (in µm) following ESL QD (87.3) was 33.3% higher (p < 0.05) compared to ESL BID (65.5) and 82.1% higher (p < 0.05) compared to OXC BID (48.0). The mean area under the curve (AUC)ss,0-τ (in µmol h/L) following the last dose of an 8-day repeated dosing was 1156.3, 1117.6, and 968.4 for ESL QD, ESL BID, and OXC BID, respectively. The ratio eslicarbazepine plasma exposure (µmol h/L) to ESL daily-dose (µmol) was 0.381 (1156.3:3037.3), 0.368 (1117.6:3037.3), and 0.271 (968.4:3567.6) for ESL-QD, ESL-BID, and OXC-BID, respectively, which translates into a 40.6% increase in the ability of ESL-QD compared to OXC-BID to deliver into the plasma their major active entity eslicarbazepine. The extent of plasma exposure to ESL minor metabolites: (R)-licarbazepine and oxcarbazepine after ESL-QD was 71.5% and 61.1% lower, respectively, than after OXC-BID. Twenty, 24 and 38 treatment emergent adverse events were reported with ESL-QD, ESL-BID, and OXC-BID, respectively. SIGNIFICANCE: ESL-QD resulted in 33.3% higher peak plasma concentration (Cmax,ss ) of eslicarbazepine and similar extent of plasma exposure (AUCss,0-τ ) when compared to ESL-BID, which may contribute to the efficacy profile reported with once-daily ESL. In comparison to OXC-BID, administration of ESL-QD resulted in 40.6% increase in the delivery of eslicarbazepine into the plasma as well as a significantly lower systemic exposure to (R)-licarbazepine and oxcarbazepine.

Convenient syntheses of halo-dibenz[b,f]azepines and carbamazepine analogues via N-arylindoles.

The dibenz[b,f]azepine heterocyclic system and related molecules with a single 10,11-bond are important templates for well-prescribed drug molecules, notably carbamazepine (anticonvulsant), clomipramine and imipramine (antidepressants). We synthesised a range of halogenated carbamazepine analogues, in connection with metabolic and immunological studies, as probes for structure-metabolism and hypersensitive effects and have published on their metabolic behaviour. While a number of synthetic routes to such analogues are possible, we naturally sought short and efficient methods for our target compounds. In the following report we present an effective two-step synthesis of a range of dibenz[b,f]azepines from appropriate indoles via N-arylation, then acid-catalysed rearrangement, with a critical analysis of other approaches. We showed earlier that this route was effective for fluoro analogues and here present a broader review of its scope. The 5-(carboxamido) side chain of carbamazepine may be added in various ways, affording overall a convenient access to drug molecules.

Pharmacokinetics and drug interactions of eslicarbazepine acetate.

Eslicarbazepine acetate (ESL) is a novel once-daily antiepileptic drug (AED) approved in Europe since 2009 that was found to be efficacious and well tolerated in a phase III clinical program in adult patients with partial onset seizures previously not controlled with treatment with one to three AEDs, including carbamazepine (CBZ). ESL shares with CBZ and oxcarbazepine (OXC) the dibenzazepine nucleus bearing the 5-carboxamide substitute, but is structurally different at the 10,11 position. This molecular variation results in differences in metabolism, preventing the formation of toxic epoxide metabolites such as carbamazepine-10,11-epoxide. Unlike OXC, which is metabolized to both eslicarbazepine and (R)-licarbazepine, ESL is extensively converted to eslicarbazepine. The systemic exposure to eslicarbazepine after ESL oral administration is approximately 94% of the parent dose, with minimal exposure to (R)-licarbazepine and OXC. After ESL oral administration, the effective half-life (t(1/2,eff) ) of eslicarbazepine was 20-24 h, which is approximately two times longer than its terminal half-life (t(1/2)). At clinically relevant doses (400-1,600 mg/day) ESL has linear pharmacokinetics (PK) with no effects of gender or moderate liver impairment. However, because eslicarbazepine is eliminated primarily (66%) by renal excretion, dose adjustment is recommended for patients with renal impairment. Eslicarbazepine clearance is induced by phenobarbital, phenytoin, and CBZ and it dose-dependently decreases plasma exposure of oral contraceptive and simvastatin.

Antioxidant and anti-protease activities of diazepinomicin from the sponge-associated Micromonospora strain RV115.

Diazepinomicin is a dibenzodiazepine alkaloid with an unusual structure among the known microbial metabolites discovered so far. Diazepinomicin was isolated from the marine sponge-associated strain Micromonospora sp. RV115 and was identified by spectroscopic analysis and by comparison to literature data. In addition to its interesting preclinical broad-spectrum antitumor potential, we report here new antioxidant and anti-protease activities for this compound. Using the ferric reducing antioxidant power (FRAP) assay, a strong antioxidant potential of diazepinomicin was demonstrated. Moreover, diazepinomicin showed a significant antioxidant and protective capacity from genomic damage induced by the reactive oxygen species hydrogen peroxide in human kidney (HK-2) and human promyelocytic (HL-60) cell lines. Additionally, diazepinomicin inhibited the proteases rhodesain and cathepsin L at an IC50 of 70-90 µM. It also showed antiparasitic activity against trypomastigote forms of Trypanosoma brucei with an IC50 of 13.5 µM. These results showed unprecedented antioxidant and anti-protease activities of diazepinomicin, thus further highlighting its potential as a future drug candidate.

Dibenzazepin hydrochloride as a new spectrophotometric reagent for determination of hydrogen peroxide in plant extracts.

A rapid, simple, accurate, and sensitive visible spectrophotometric method for the determination of trace amounts of hydrogen peroxide in acidic buffer medium is reported. The proposed method is based on the oxidative coupling of Ampyrone with dibenzazepin hydrochloride by hydrogen peroxide in the buffer medium of pH 4.0 which is catalyzed by ferrous iron. The blue-colored product formed with maximum absorption at 620 nm was found to be stable for 2 h. Beer's law is obeyed for hydrogen peroxide concentration in the range of 0.03-0.42 µg ml(-1). The optimum reaction conditions and other important optical parameters are reported. The molar absorptive and Sandell's sensitivity are found to be 5.89 × 10(4) mol(-1) cm(-1) and 0.57 g/cm(2), respectively. The interference due to diverse ions and complexing agents was studied. The method is successfully applied to the determination of hydrogen peroxide in green plants satisfactorily.

Molecular modeling and UV-vis spectroscopic studies on the mechanism of action of reversed chloroquine (RCQ).

Reversed chloroquine (RCQ) is a multiple ligand compound active against chloroquine-sensitive and resistant falciparum malaria. It is composed by a 4-aminoquinoline moiety (like that present in chloroquine (CQ)) joined to imipramine (IMP), a modulating agent that also showed intrinsic antiplasmodial activity against Brazilian Plasmodium falciparum isolates resistant to CQ. Molecular modeling and ultraviolet-visible spectroscopy (UV-vis) studies strongly suggest that the interaction between RCQ and heme is predominant through the quinoline moiety in a mechanism of action similar to that observed for CQ.