Design, synthesis and biological assessment of acridine derivatives containing 1,3,4-thiadiazole moiety as novel selective acetylcholinesterase inhibitors.

A novel series of acridine derivatives containing substituted thiadiazol-2-amine moiety was synthesized via multi-component condensation reaction of dimedone, aromatic aldehyde and 5-aryl-1,3,4-thiadiazol-2-amines in the presence of LaCl3 as a catalyst under solvent-free conditions. Anticholinesterase (AChE and BuChE) activity evaluation of the derivatives showed that all the derivatives are capable of inhibiting both enzymes and are highly selective towards AChE. Among them, the ability of 4i and 4d with respective IC50 values of 0.002 and 0.006 µM to inhibit AChE was higher than the reference compound tacrine (IC50 = 0.016 µM). The kinetics studies demonstrated that 4i and 4d inhibit AChE through a competitive/non-competitive mixed mechanism. The HEPG2 cell viability assay evidenced that 4i and 4d significantly exhibit lower hepatotoxicity compared with tacrine. Blind docking experiments performed on TcAChE (PDB ID: 2ACE) indicated that an unknown site is preferred for binding by all the derivatives over classic binding site of the enzyme, site 1 (CAS/PAS). Identification of the residues by protein structure alignment confirmed that this site is site 2 which was recently recognized as a new allosteric site of hAChE. The binding modes of 4i and 4d were also investigated using local docking studies on site 1 and site 2.

Preclinical safety assessment of pathogen reduced red blood cells treated with amustaline and glutathione.

BACKGROUND: The nucleic acid targeted pathogen reduction (PR) system utilizing amustaline (S-303) and glutathione (GSH) is designed to inactivate blood-borne pathogens and leukocytes in red blood cell concentrates (PR-RBCC). Inactivation is attained after amustaline intercalates and forms covalent nucleic acid adducts preventing replication, transcription, and translation. After pathogen inactivation, amustaline spontaneously hydrolyzes to S-300, the primary negatively charged reaction product; amustaline is below quantifiable levels in PR-RBCC. GSH quenches free unreacted amustaline. STUDY DESIGN AND METHODS: The genotoxic and carcinogenic potential of PR-RBCC, the reaction by-products, and S-300 were assessed in accordance with the International Conference on Harmonization (ICH) guidelines and performed in compliance with the Food and Drug Administration (FDA) good laboratory practice standards, 21 CFR Part 58. in vitro bacterial reverse mutagenicity and chromosomal aberration assays were performed with and without exogenous S9 metabolic activation, and in in vivo clastogenicity and carcinogenic assays using validated murine models. RESULTS: PR-RBCCs were not genotoxic in vitro and in vivo and were non-carcinogenic in p53+/- transgenic mice transfused over 26 weeks. Estimated safety margins for human exposure ranged from >90 to >36 fold for 2 to 5 PR-RBCCs per day, respectively. PR-RBCCs and S-300 did not induce chromosome aberration in the in vivo murine bone marrow micronucleus assay at systemically toxic doses. CONCLUSIONS: PR-RBCCs did not demonstrate genotoxicity in vitro or in vivo and were not carcinogenic in vivo. These studies support the safety of PR-RBCCs and suggest that there is no measurable genotoxic hazard associated with transfusion of PR-RBCCs.

Assessment of DNA Topoisomerase I Unwinding Activity, Radical Scavenging Capacity, and Inhibition of Breast Cancer Cell Viability of N-alkyl-acridones and N,N'-dialkyl-9,9'-biacridylidenes.

The anticancer activity of acridone derivatives has attracted increasing interest, therefore, a variety of substituted analogs belonging to this family have been developed and evaluated for their anti-cancer properties. A series of N-alkyl-acridones 1-6 and N,N'-dialkyl-9,9'-biacridylidenes 7-12 with variable alkyl chains were examined for their topoisomerase I activity at neutral and acidic conditions as well as for their binding capacity to calf thymus and possible radical trapping antioxidant activity. It was found that at a neutral pH, topoisomerase I activity of both classes of compounds was similar, while under acidic conditions, enhanced intercalation was observed. N-alkyl-acridone derivatives 1-6 exhibited stronger, dose-dependent, cytotoxic activity against MCF-7 human breast epithelial cancer cells than N,N'-dialkyl-9,9'-biacridylidenes 7-12, revealing that conjugation of the heteroaromatic system plays a significant role on the effective distribution of the compound in the intracellular environment. Cellular investigation of long alkyl derivatives against cell migration exhibited 40-50% wound healing effects and cytoplasm diffusion, while compounds with shorter alkyl chains were accumulated both in the nucleus and cytoplasm. All N,N'-dialkyl-9,9'-biacridylidenes showed unexpected high scavenging activity towards DPPH or ABTS radicals which may be explained by higher stabilization of radical cations by the extended conjugation of heteroaromatic ring system.

Ultrastructural Assessment of 2-(acridin-9-ylmethylene)-N-phenylhydrazinecarbothioamide activity on human breast adenocarcinoma cells.

The aim of the present study was to investigate ultrastructural changes induced by (Z)-2-(acridin-9-ylmethylene)-N-phenylhydrazinecarbothioamide (APHCA) treatment on human breast adenocarcinoma cancer cells MCF-7, besides the evaluation of phosphatidylserine externalization and DNA fragmentation in treated cells. Cell viability analysis demonstrated concentration and time-manner cytotoxicity. Treated MCF-7 cells did not expose phosphatidylserine residues to the external plasma membrane surface and DNA fragmentation was not visualized by electrophoresis. Light microscopy showed compromised cell density and presence of vacuolization after APHCA treatment with 60µM. Scanning and transmission electron microscopies revealed hallmarks of autophagy, namely the presence of membrane bebbling and autophagosomes, besides shrunken cells and cell debris in treated MCF-7 cells. However, more specific tests such as the quantification of mammalian autophagy proteins are necessary to determine the kind of death that is trigged by APHCA.

Application of UV-Vis spectrophotometric process for the assessment of indoloacridines as free radical scavenger.

A conventional approach has been used to synthesis Indole fused acridine, 4a-e. In this paper to achieve the target molecule, 4 the reaction was performed via two steps. In step 1, there was a reaction between Carbazolone, 1 and benzophenone, 2 to get dihydroindoloacridine, 3. In step 2, compound, 3 was treated with 5% Palladium/Carbon in the presence of diphenyl ether for 5h to give a dark brown product, 4. The column chromatography was used to purify final product, 4. All the synthesized compounds such as 3 and 4 were characterized by melting point, FTIR, (1)H NMR, and Mass spectra. Further to check the purity of the compounds it was subjected to CHN analyzer. The target molecules such as 3 and 4 were screened for antimicrobial studies against bacteria such as Bacillus subtilis (B. subtilis), Staphylococcus aureus (S. aureus), Klebsiella pneumonia (K. pneumonia), Salmonella typhi (S. typhi); and fungi like Aspergillus niger (A. niger), Aspergillus fumigatus (A. fumigatus). The obtained results clearly proves that the target molecules shown reasonable activity against K. pneumonia and A. niger. Further the compounds were screened for free radical scavenging activity using 2,2-diphenyl-1-picrylhydrazyl (DPPH). The free radical scavenging property was performed using UV-Visible spectroscopy. The results were compared with the standard BHT (Butylated Hydroxy Toluene). Compounds, 4a and 4e were shown higher percentage of inhibition when compare to the standard. The result confirms that further research on indoloacridine will leads effective drug to the market.

Assessment of cerebral P-glycoprotein expression and function with PET by combined [11C]inhibitor and [11C]substrate scans in rats.

INTRODUCTION: The adenosine triphosphate-binding cassette (ABC) transporter P-glycoprotein (Pgp) protects the brain from accumulation of lipophilic compounds by active efflux transport across the blood-brain barrier. Changes in Pgp function/expression may occur in neurological disorders, such as epilepsy, Alzheimer's or Parkinson's disease. In this work we investigated the suitability of the radiolabeled Pgp inhibitors [(11)C]elacridar and [(11)C]tariquidar to visualize Pgp density in rat brain with PET. METHODS: Rats underwent a first PET scan with [(11)C]elacridar (n = 5) or [(11)C]tariquidar (n = 6) followed by a second scan with the Pgp substrate (R)-[(11)C]verapamil after administration of unlabeled tariquidar at a dose which half-maximally inhibits cerebral Pgp (3 mg/kg). Compartmental modeling using an arterial input function and Logan graphical analysis were used to estimate rate constants and volumes of distribution (VT) of radiotracers in different brain regions. RESULTS: Brain PET signals of [(11)C]elacridar and [(11)C]tariquidar were very low (~0.5 standardized uptake value, SUV). There was a significant negative correlation between VT and K1 (i.e. influx rate constant from plasma into brain) values of [(11)C]elacridar or [(11)C]tariquidar and VT and K1 values of (R)-[(11)C]verapamil in different brain regions which was consistent with binding of [(11)C]inhibitors to Pgp and efflux of (R)-[(11)C]verapamil by Pgp. CONCLUSION: The small Pgp binding signals obtained with [(11)C]elacridar and [(11)C]tariquidar limit the applicability of these tracers to measure cerebral Pgp density. PET tracers with higher (i.e. subnanomolar) binding affinities will be needed to visualize the low density of Pgp in brain.

A simplified protocol employing elacridar in rodents: a screening model in drug discovery to assess P-gp mediated efflux at the blood brain barrier.

In the present study we have developed a simple, time, and cost effective in vivo rodent protocol to screen the susceptibility of a test compound for P-glycoprotein (P-gp) mediated efflux at the blood brain barrier (BBB) during early drug discovery. We used known P-gp substrates as test compounds (quinidine, digoxin, and talinolol) and elacridar (GF120918) as a chemical inhibitor to establish the model. The studies were carried out in both mice and rats. Elacridar was dosed intravenously at 5 mg/kg, 0.5 h prior to probe substrate administration. Plasma and brain samples were collected and analyzed using UPLC-MS/MS. In the presence of elacridar, the ratio of brain to plasma area under the curve (B/P) in mouse increased 2, 4, and 38-fold, respectively, for talinolol, digoxin, and quinidine; whereas in rat, a 70-fold increase was observed for quinidine. Atenolol, a non P-gp substrate, exhibited poor brain penetration in the presence or absence of elacridar in both species (B/P ratio ~ 0.1). Elacridar had no significant effect on the systemic clearance of digoxin or quinidine; however, a trend towards increasing volume of distribution and half life was observed. Our results support the utility of elacridar in evaluation of the influence of P-gp mediated efflux on drug distribution to the brain. Our protocol employing a single intravenous dose of elacridar and test compound provides a cost effective alternative to expensive P-gp knockout mice models during early drug discovery.

Assessment of P-glycoprotein activity in the Blood-Brain Barrier (BBB) using Near Infrared Fluorescence (NIRF) imaging techniques.

PURPOSE: To examine functional activity of P-glycoprotein (P-gp) in the blood-brain barrier (BBB) using near infrared fluorescence (NIRF) imaging techniques. METHODS: Cellular accumulation and bi-directional permeability of the NIRF probe, rhodamine 800 (R800) was determined in MDCKMDR1 and MDCKwt monolayers under normal conditions and following P-gp inhibition with GF120918. Functional P-gp activity was also assessed in mice following administration of R800 alone and with GF230918. Quantitative analysis of R800 fluorescence in brain tissue and blood was measured ex-vivo using Odyssey Near Infrared imaging. RESULTS: R800 accumulation was reduced in MDCKMDR1 compared to MDCKwt monolayers. Addition of GF120918, resulted in increased R800 accumulation in MDCKMDR1 monolayers. Permeability of R800 in MDCKMDR1 monolayers was significantly enhanced (4-fold) in the basolateral to apical direction under control conditions and was abolished following treatment with GF120918. With the exception of the choriod plexus, there was very little penetration of R800 into the brain under control conditions. Treatment of mice with GF120918 resulted in a nearly 4-fold increase in R800 fluorescence in the brain. In contrast, GF120918 had no effect on brain penetration of a vascular permeability marker. CONCLUSIONS: In vitro studies demonstrate the P-gp transporter properties of the NIRF probe R800. Preliminary in vivo studies confirm the P-gp transporter liabilities of R800 and suggest this probe may be useful as a molecular imaging agent for examining P-gp activity in the BBB.

Dose-response assessment of tariquidar and elacridar and regional quantification of P-glycoprotein inhibition at the rat blood-brain barrier using (R)-[(11)C]verapamil PET.

PURPOSE: Overactivity of the multidrug efflux transporter P-glycoprotein (P-gp) at the blood-brain barrier (BBB) is believed to play an important role in resistance to central nervous system drug treatment. (R)-[(11)C]verapamil (VPM) PET can be used to measure the function of P-gp at the BBB, but low brain uptake of VPM hampers the mapping of regional differences in cerebral P-gp function and expression. The aim of this study was to evaluate the dose-response relationship of two potent P-gp inhibitors and to investigate if increased brain uptake of VPM mediated by P-gp inhibition can be used to assess regional differences in P-gp activity. METHODS: Two groups of Sprague-Dawley rats (n = 12) underwent single VPM PET scans at 120 min after administration of different doses of the P-gp inhibitors tariquidar and elacridar. In an additional six rats, paired VPM PET scans were performed before and after administration of 3 mg/kg tariquidar. RESULTS: Inhibitor administration resulted in an up to 11-fold increase in VPM brain distribution volumes (DV) with half-maximum effective dose (ED(50)) values of 3.0 +/- 0.2 and 1.2 +/- 0.1 mg/kg for tariquidar and elacridar, respectively. In paired PET scans, 3 mg/kg tariquidar resulted in regionally different enhancement of brain activity distribution, with lowest DV in cerebellum and highest DV in thalamus. CONCLUSION: Our data show that tariquidar and elacridar are able to increase VPM brain distribution in rat brain up to 11-fold over baseline at maximum effective doses, with elacridar being about three times more potent than tariquidar. Regional differences in tariquidar-induced modulation of VPM brain uptake point to regional differences in cerebral P-gp function and expression in rat brain.

In vitro assessment of novel transcription inhibitors and topoisomerase poisons in rhabdomyosarcoma cell lines.

PURPOSE: Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. Current chemotherapy regimes include the topoisomerase II poison etoposide and the transcription inhibitor actinomycin D. Poor clinical response necessitate identification of new agents to improve patient outcomes. METHODS: We assessed the in vitro cytotoxicity (MTT assay) of DNA intercalating agents in five established human RMS cell lines. These include novel classes of transcription inhibitors and topoisomerase poisons, previously shown to have potential as anti-cancer agents. RESULTS: Amongst the former agents, bisintercalating bis(9-aminoacridine-4-carboxamides) linked through the 9-position, and bis(phenazine-1-carboxamides) linked via their side chains, are compared with established transcription inhibitors. Amongst the latter, monofunctional acridine-4-carboxamides related to N-[2-(dimethylamino)ethyl]acridine-4-carboxamide, DACA, are compared with established topoisomerase poisons. CONCLUSIONS: Our findings specifically highlight the topoisomerase poison 9-amino-DACA, its 5-methylsulphone derivative, AS-DACA, and the bis(phenazine-1-carboxamide) transcription inhibitor MLN944/XR5944, currently in phase I trial, as candidates for further research into new agents for the treatment of RMS.

In vitro p-glycoprotein inhibition assays for assessment of clinical drug interaction potential of new drug candidates: a recommendation for probe substrates.

Because modulation of P-glycoprotein (Pgp) through inhibition or induction can lead to drug-drug interactions by altering intestinal, central nervous system, renal, or biliary efflux, it is anticipated that information regarding the potential interaction of drug candidates with Pgp will be a future regulatory expectation. Therefore, to be able to utilize in vitro Pgp inhibition findings to guide clinical drug interaction studies, the utility of five probe substrates (calcein-AM, colchicine, digoxin, prazosin, and vinblastine) was evaluated by inhibiting their Pgp-mediated transport across multidrug resistance-1-transfected Madin-Darby canine kidney cell type II monolayers with 20 diverse drugs having various degrees of Pgp interaction (e.g., efflux ratio, ATPase, and calcein-AM inhibition). Overall, the rank order of inhibition was generally similar with IC(50) values typically within 3- to 5-fold of each other. However, several notable differences in the IC(50) values were observed. Digoxin and prazosin were the most sensitive probes (e.g., lowest IC(50) values), followed by colchicine, vinblastine, and calcein-AM. Inclusion of other considerations such as a large dynamic range, commercially available radiolabel, and a clinically meaningful probe makes digoxin an attractive probe substrate. Therefore, it is recommended that digoxin be considered as the standard in vitro probe to investigate the inhibition profiles of new drug candidates. Furthermore, this study shows that it may not be necessary to generate IC(50) values with multiple probe substrates for Pgp as is currently done for cytochrome P450 3A4. Finally, a strategy integrating results from in vitro assays (efflux, inhibition, and ATPase) is provided to further guide clinical interaction studies.