Novel piperidinium and pyridinium agents as water-soluble acetylcholinesterase inhibitors for the reversal of neuromuscular blockade

A series of piperidinium and pyridinium agents containing a common structural fragment of 5,6-dimethoxybenzothiophene have been synthesised as water-soluble acetylcholinesterase inhibitors. Several compounds, for example 42 (AChE IC(50) 0.03 microM) have been found to reverse the neuromuscular blockade induced by vecuronium bromide in vitro and in vivo. Coupled with their high water solubility (up to 30-60 mg/mL), these compounds are potentially useful as intravenous reversal agents of neuromuscular blocking agents in surgical anaesthesia.     

Oxyaniliniums as acetylcholinesterase inhibitors for the reversal of neuromuscular block.

A series of oxyanilinium-based AChE inhibitors have been synthesised and tested for the reversal of vecuronium-induced neuromuscular block. Several compounds, for example 2-hydroxy- and 2-methoxy-N,N-dimethyl-N-allylanilinium bromide (3 and 6) showed comparable reversal potencies to edrophonium and clean in vivo cardiovascular profiles.     

Preferences of Mexican anesthesiologists for vecuronium, rocuronium, or other neuromuscular blocking agents: a survey

Background  

Several neuromuscular blocking (NMB) agents are available for clinical use in anesthesia. The present study was performed in order to identify preferences and behaviors of anesthesiologists for using vecuronium, rocuronium or other NMB agents in their clinical practice.     

Quaternary salts of E2020 analogues as acetylcholinesterase inhibitors for the reversal of neuromuscular block

A series benzylpiperidinium and benzylpyridinium quaternary salts have been synthesised and tested for inhibition of acetylcholinesterase and reversal of neuromuscular block induced by vecuronium. Several potent reversal agents have been identified and their haemodynamic effects measured.     

Monoterpene indole alkaloid azine derivatives as MDR reversal agents

Aiming at generating a library of bioactive indole alkaloid derivatives as multidrug resistance (MDR) reversers, two epimeric indole alkaloids (1 and 2) were submitted to chemical transformations, giving rise to twenty-four derivatives (5-28), bearing new aromatic or aliphatic azine moieties. The structure of the compounds was established by 1D and 2D NMR (COSY, HMBC, HMQC and NOESY) experiments. Two different strategies were employed for assessing their anti-MDR potential, namely through the evaluation of their activity as inhibitors of typical MDR ABC transporters overexpressed by cell transfection, such as ABCB1 (P-gp), ABCC1 (MRP1), and ABCG2 (BCRP), or by evaluating their ability as collateral sensitivity (CS) agents in cells overexpressing MRP1. A considerable MDR reversing activity was observed for compounds bearing the aromatic azine moiety. The strongest and most selective P-gp inhibition was found for the epimeric azines 5 and 6, bearing a para-methylbenzylidene moiety. Instead, compounds 17 and 18 that possess a di-substituted benzylidene portion with methoxy and hydroxyl groups, selectively inhibited MRP1 drug-efflux. None of these compounds inhibited BCRP. Compounds 5, 6 and 18 were further investigated in drug combination experiments, which corroborated their anti-MDR potential. Moreover, it was observed that compound 12, with an aromatic azine moiety, and compounds 23-26, sharing a new aliphatic substituent, displayed a CS activity, selectively killing MRP1-overexpressing cells. Among these last compounds, it could be established that addition of 19, 23 and 25 to MRP1-overexpressing cells led to glutathione depletion triggering cell death through apoptosis.     

Design, synthesis and evaluation of naphthalene-2-carboxamides as reversal agents in MDR cancer

A novel class of molecules with structure N-[3-(4-substituted-1-piperazinyl) propyl]-6-methoxy naphthalene-2-carboxamides were designed by generating a pharmacophore for potent MDR reversal activity, using Elacridar (GF 120918) as a query molecule and using MOE software. They were synthesized by condensing 6-methoxynaphthalene-2-carboxylic acid with N-[3-(4-substituted-1-piperazinyl) propyl] amines in the presence of DCC in DMF. They were evaluated in P388 murine lymphocytic leukemia cell line (P388) in vitro using SRB assay for cytotoxicity and in adriamycin-resistant P388 murine lymphocytic leukemia cell line (P388/ADR) using MTT assay for resistant reversal activity. Test compounds were non-toxic at the doses studied (upto 80 microg/ml). They effectively reversed adriamycin resistance at the doses studied (40 and 80 microg/ml). The percentage enhancement in adriamycin activity was in the range 33.58 -90.67 (at 40 microg/ml) and 8.80-46.04 (at 80 microg/ml) and the corresponding reversal potency values were in the range 1.33-1.90 and 1.08-1.46, respectively. Test compounds 2, 3, and 5 exhibited better activity as compared to the standard resistant reversal agent (Verapamil), at same concentration.     

Synthesis and evaluation of Strychnos alkaloids as MDR reversal agents for cancer cell eradication

Natural products represent the fourth generation of multidrug resistance (MDR) reversal agents that resensitize MDR cancer cells overexpressing P-glycoprotein (Pgp) to cytotoxic agents. We have developed an effective synthetic route to prepare various Strychnos alkaloids and their derivatives. Molecular modeling of these alkaloids docked to a homology model of Pgp was employed to optimize ligand–protein interactions and design analogues with increased affinity to Pgp. Moreover, the compounds were evaluated for their (1) binding affinity to Pgp by fluorescence quenching, and (2) MDR reversal activity using a panel of in vitro and cell-based assays and compared to verapamil, a known inhibitor of Pgp activity. Compound 7 revealed the highest affinity to Pgp of all Strychnos congeners (K_d = 4.4 μM), the strongest inhibition of Pgp ATPase activity, and the strongest MDR reversal effect in two Pgp-expressing cell lines. Altogether, our findings suggest the clinical potential of these synthesized compounds as viable Pgp modulators justifies further investigation.     

New terpenoids from Maytenus apurimacensis as MDR reversal agents in the parasite Leishmania

Two new sesquiterpenes (1-2) and one new lupane triterpene (3) have been isolated from the roots of Maytenus apurimacensis. The novel beta-dihydroagarofurans are the first sesquiterpenes with a basic polyhydroxy skeleton of 15-deoxyalatol and 4,15-dideoxyalatol that show high MDR reversing activity in the protozoan parasite Leishmania tropica.     

Anionic cyclophanes as potential reversal agents of muscle relaxants by chemical chelation

A series of carboxyl-containing cyclophanes have been designed and synthesised as chemical chelators (or host molecules) of cationic muscle relaxant drugs (or guest molecules). Three of these cyclophane derivatives, 1-3, have been shown by NMR to form 1:1 complexes with the muscle relaxants pancuronium, and gallamine, in D(2)O, with association constants up to 10(4) M(-1). When tested in an in vitro chick biventer muscle preparation, the cyclophanes reversed the neuromuscular block induced by pancuronium and gallamine, with having the most effective reversal against pancuronium (EC(50) 40 microM.     

Papyriferic acid derivatives as reversal agents of multidrug resistance in cancer cells

Forty-one derivatives of papyriferic acid were prepared based on our previous finding that methyl papyriferate (3) showed potent reversing effect on cytotoxicity of colchicine against multidrug resistance (MDR) human cancer cells (KB-C2), and evaluated for their cytotoxicity and effect on reversing P-gp-mediated MDR against KB-C2 cells. 3-O-(Morpholino-β-oxopropanoyl)-12β-acetoxy-3α,25-dihydroxy-(20S,24R)-epoxydammarane (37) significantly increased the sensitivity of colchicine against KB-C2 cells by 185-fold at 5 μg/mL (7.4 μM), and the cytotoxicity of colchicine was recovered to nearly that of sensitive (KB) cells. The other several new amide derivatives also exhibited potent reversal activity comparable to or more potent than methyl papyriferate and verapamil.     

Trapping of an open-channel blocker at the frog neuromuscular acetylcholine channel.

At the ganglionic nicotinic acetylcholine channel (Gurney, A. M., and H. P. Rang, 1984, Br. J. Pharmacol., 82:623-642) and on some cholinergic neuromuscular synapses of Crustacea (Lingle, C., 1983a, J. Physiol. (Lond.), 339:395-417; Lingle, C., 1983b, J. Physiol. (Lond.), 339:419-437), some agents that block cholinergic currents by an open-channel block mechanism appear to become trapped within the channel when it subsequently closes. It is unknown whether trapping of some open-channel blockers might also occur at the neuromuscular nicotinic acetylcholine channel. Here we show that the long-lived cholinergic blocking action of chlorisondamine, a ganglionic nicotinic blocker, can in part be most simply explained by an open-channel block mechanism followed by a subsequent trapping of the blocking molecule within the closed ion channel. Unique structural characteristics of the chlorisondamine molecule place several provocative constraints on the mechanism by which trapping may be occurring.     

Mechanisms for resistance to anticancer agents and the reversal of the resistance

MDR results from overexpression of P-glycoprotein (Pgp) and multidrug resistance protein (MRP or MRP1) that function as ATP-dependent efflux pumps. Lung resistance related protein (LRP) is also supposed to be involved in MDR. The human canalicular multispecific organic anion transporter (cMOAT) gene that is responsible for the defects in Dubin-Johnson syndrome was isolated. cMOAT is homologous to MRP1 and supposed to be involved in drug resistance. Human cMOAT cDNA transfected LLC-PK1 cells, LLC/cMOAT-1, have increased resistance to vincristine (VCR), 7-ethyl-10-hydroxycamptothecin (SN-38), and cisplatin. The multidrug resistance (MDR)-reversing agents, cyclosporin A (CsA) and PAK-104P, almost completely reversed the resistance to VCR, SN-38 and cisplatin of LLC/cMOAT-1 cells by interacting with the substrate binding site of cMOAT. Treatment of human colorectal carcinoma SW-620 cells with sodium butyrate(NaB) induced LRP in the cells and conferred resistance to Adrianycin(ADM), VCR, VP-16, gramicidin D and taxol. Two LRP-specific ribozymes inhibited the NaB-induced expression of LRP in SW-620 cells and almost completely abolished their acquisition of the MDR phenotype. The accumulation of ADM, VCR and taxol was not decreased in NaB-treated cells, suggesting that ATP-binding cassette transporters are not involved in the MDR of NaB-treated cells. ADM was mainly located in the nuclei of untreated and the cytoplasm of NaB-treated cells. The accumulation level of ADM in the nuclei isolated from untreated cells or those from treated cells in the presence of anti-LRP polyclonal antibody was higher than that from treated cells in the absence of the antibody. Efflux of ADM from nuclei isolated from NaB-treated cells was enhanced compared with those from untreated cells and NaB-treated cells transfected with a LRP-specific ribozyme. The polyclonal antibody against LRP inhibited the enhanced efflux of ADM from nuclei isolated from NaB-treated cells. These findings indicate that LRP is involved in resistance to ADM, VCR, VP-16, taxol and gramicidin D, and has an important role in the transport of ADM from the nucleus to the cytoplasm.     

Discovery of aromatic amides with triazole-core as potent reversal agents against P-glycoprotein-mediated multidrug resistance

P-glycoprotein (P-gp)-mediated multidrug resistance (MDR) is a major impediment for clinical cancer therapy. 19 novel aromatic amides with triazole-core as MDR reversal agents were designed and synthesized via click chemistry to reverse MDR. Among them, compound 42 was identified as the most promising candidate with high potency (EC₅₀ = 78.1 ± 5.4 nM), low cytotoxity (SI > 1282) and persistent duration in reversing doxorubicin (DOX) resistance in K562/A02 cells. 42 also enhanced the potency of other P-gp associated cytotoxic agents with different structures. In further study, remarkably increased intracellular accumulation of Rh123 and DOX in K562/A02 cells was achieved by compound 42, while CYP3A4 activity had no change by compound 42. These results indicate that compound 42 as a relatively safe modulator of P-gp-mediated MDR has good potential for further development.     

Design and synthesis of tetrahydroisoquinoline derivatives as potential multidrug resistance reversal agents in cancer

Exploration for new MDR-modulator utilizing tetrahydroisoquinoline as scaffold disclosed 6,7-dimethoxy-1-(3,4-dimethoxy)benzyl-2-(N-n-octyl-N'-cyano)guanyl-1,2,3,4-tetrahydroisoquinoline (7) as a readily accessible medicinal lead. Compound 7 possessed potent MDR reversal activity in the range of the reference compound verapamil, and had not cardiovascular activity compared to verapamil.     

Reactivating and cholinolytic action of trimedoxime bromide at the neuromuscular junction of warm-blooded animals

The reactivating and cholinolytic action of trimedoxime bromide was evaluated during experiments on rat soleus and diaphragm muscles accoridng to the amplitude and time course of miniature end-plate potentials and currents (MEPP and MEPC respectively). This agent reactivates acetylcholinesterase (AChE) phosphorylation. The effects of trimedoxime bromide at concentrations of 5·10^–6–5·10^–4 M following AChE inhibition on the amplitude and duration of MEPP arises from complex interaction between the reactivating and cholinolytic effects. A separate evaluation of the reactivating effect (once the reactivating agent had been removed) revealed that this action increases throughout the entire range of trimedoxime bromide concentration: complete reactivation of AChE phosphorylation was observed under the action of 2–5·10^–4 M trimedoxime bromide. Examination of the cholinolytic effect in isolation (with voltage-clamping at the muscle and intact AChE) showed that blockade of open end-plate ionic channels underlies this effect. Reduction in MEPC amplitude together with retarded (but still exponential) decay of signals were distinguishing features of this blockade, confirming that trimedoxime bromide acts as a very fast blocker.S. V. Kurashov Medical Institute, Ministry of Public Health of the RSFSR, Kazan'. Translated from Neirofiziologiya, Vol. 20, No. 3, pp. 351–357, May–June, 1988.     

Study of the reversal effect of NF449 on neuromuscular blockade induced by d-tubocurarine

AimsThe aim of this study was to investigate the mechanism for the reversal effect of NF449 (a suramin analogue) on the neuromuscular block induced by d-tubocurarine (d-TC).Main methodsNerve-stimulated muscle contractions and end-plate potentials were performed in mouse phrenic nerve-diaphragm preparations. Acetylcholine (ACh)-induced muscle contractions were performed in the chick biventer cervicis preparations. Presynaptic nerve terminal waveform recordings were performed in mouse triangularis sterni preparations.Key findingsAmongst the suramin analogues in this study, only the NF449 and suramin were able to reverse the blockade effect produced by d-TC on nerve-stimulated muscle contractions. Each of these suramin analogues (NF007, NF023, NF279 and NF449) alone has no significant effect on the amplitude of nerve-stimulated muscle contractions. NF449 and suramin also showed the antagonising effects on the inhibition of end-plate potentials induced by d-TC. Furthermore, pre-treatment with NF449 can antagonise the inhibition of d-TC in ACh-induced contractions of chick biventer cervicis muscle. NF449 produced a greater rightward shift of the dose–response inhibition curve for d-TC than did suramin. Because other purinergic 2X (P2X) receptor antagonists, NF023 and NF279, do not have the reverse effects on the neuromuscular blockade of d-TC, the effect of NF449 seems irrelevant to inhibition of P2X receptors.SignificanceThese data suggest that NF449 was able to compete with the binding of d-TC on the nicotinic ACh receptors, and the effect of NF449 was more potent than suramin in reducing the inhibition of d-TC. The structure of NF449 may provide useful information for designing potent antidotes against neuromuscular toxins.     

Melittin as model system for probing interactions between proteins and cyclodextrins

Cylcodextrin sugars are cyclic sugars that have a hydrophilic exterior and a hydrophobic center. This enables cyclodextrins to solubilize hydrophobic molecules in aqueous media. Cyclodextrins may inhibit aggregation by intercalating surface aromatic residues and competing with interprotein aromatic clusters (pi-pi interactions). In order to investigate this concept, the interaction of hydroxypropyl-beta-cyclodextrin (HPBCD) with melittin is studied with steady-state and time-resolved fluorescence, fluorescence polarization, circular dichroism, and IR spectroscopy. HPBCD inhibits the aggregation of melittin. This inhibition and the spectroscopic results are consistent with the lone aromatic tryptophan of the peptide being intercalated within HPBCD.     

Anti-H-2 hybridoma antibodies as immunoselective agents

Using immunoselection with an H-2K^k-specific monoclonal antibody following mutagenesis on an (H-2 ^k/H-2^d) F₁ cell line we have obtained variants that do not react with the selecting monoclonal antibody but continue to react with other monoclonal antibodies directed against the same gene product. The mutants fall into two classes based on their serological profile. This phenotype is suggestive of a structural mutation in the selected gene. If the genetic change involved is a point mutation (as opposed to a deletion), one should be able to obtain revertants. Using the fluorescence-activated cell sorter, we have been able to obtain from one of the monoclonal-antibody-nonseactive mutants cells that do bind the selecting antibody. In order to prove that the presumptive revertant is not a contaminant wild-type cell that inadvertantly got mixed into the resistant mutant, we first introduced an outside marker, resistance to the purine analogue 2-amino-6-mercaptopurine (6-thioguanine), into the monoclonal-antibody-resistant mutant. The revertants obtained using the cell sorter continue to express the nonselective phenotype of resistance to 6-thioguanine, showing that they are not wild-type cells. In addition, their serological characteristics are different from those of either the wild-type cells or the hybrid oma-resistant mutants from which they were derived. Based on the serological analyses, it would seem that we have isolated at least three variant forms of the H-2K^k-gene product.