Pre-Steady-State and Steady-State Kinetic Analysis of Butyrylcholinesterase-Catalyzed Hydrolysis of Mirabegron, an Arylacylamide Drug.

The ß-adrenergic drug Mirabegron, a drug initially used for the treatment of an overactive bladder, has new potential indications and is hydrolyzed by butyrylcholinesterase (BChE). This compound is one of the only arylacylamide substrates to be catabolized by BChE. A steady-state kinetic analysis at 25 °C and pH 7.0 showed that the enzyme behavior is Michaelian with this substrate and displays a long pre-steady-state phase characterized by a burst. The induction time, τ, increased with substrate concentration (τ ≈ 18 min at maximum velocity). The kinetic behavior was interpreted in terms of hysteretic behavior, resulting from a slow equilibrium between two enzyme active forms, E and E'. The pre-steady-state phase with the highest activity corresponds to action of the E form, and the steady state corresponds to action of the E' form. The catalytic parameters were determined as kcat = 7.3 min-1 and Km = 23.5 µM for the initial (burst) form E, and kcat = 1.6 min-1 and Km = 3.9 µM for the final form E'. Thus, the higher affinity of E' for Mirabegron triggers the slow enzyme state equilibrium toward a slow steady state. Despite the complexity of the reaction mechanism of Mirabegron with BChE, slow BChE-catalyzed degradation of Mirabegron in blood should have no impact on the pharmacological activities of this drug.

Human Plasma Butyrylcholinesterase Hydrolyzes Atropine: Kinetic and Molecular Modeling Studies.

The participation of butyrylcholinesterase (BChE) in the degradation of atropine has been recurrently addressed for more than 70 years. However, no conclusive answer has been provided for the human enzyme so far. In the present work, a steady-state kinetic analysis performed by spectrophotometry showed that highly purified human plasma BChE tetramer slowly hydrolyzes atropine at pH 7.0 and 25 °C. The affinity of atropine for the enzyme is weak, and the observed kinetic rates versus the atropine concentration was of the first order: the maximum atropine concentration in essays was much less than Km. Thus, the bimolecular rate constant was found to be kcat/Km = 7.7 × 104 M-1 min-1. Rough estimates of catalytic parameters provided slow kcat < 40 min-1 and high Km = 0.3-3.3 mM. Then, using a specific organophosphoryl agent, echothiophate, the time-dependent irreversible inhibition profiles of BChE for hydrolysis of atropine and the standard substrate butyrylthiocholine (BTC) were investigated. This established that both substrates are hydrolyzed at the same site, i.e., S198, as for all substrates of this enzyme. Lastly, molecular docking provided evidence that both atropine isomers bind to the active center of BChE. However, free energy perturbations yielded by the Bennett Acceptance Ratio method suggest that the L-atropine isomer is the most reactive enantiomer. In conclusion, the results provided evidence that plasma BChE slowly hydrolyzes atropine but should have no significant role in its metabolism under current conditions of medical use and even under administration of the highest possible doses of this antimuscarinic drug.

Tuning the Envelope Structure of Enzyme Nanoreactors for In Vivo Detoxification of Organophosphates.

Encapsulated phosphotriesterase nanoreactors show their efficacy in the prophylaxis and post-exposure treatment of poisoning by paraoxon. A new enzyme nanoreactor (E-nRs) containing an evolved multiple mutant (L72C/Y97F/Y99F/W263V/I280T) of Saccharolobus solfataricus phosphotriesterase (PTE) for in vivo detoxification of organophosphorous compounds (OP) was made. A comparison of nanoreactors made of three- and di-block copolymers was carried out. Two types of morphology nanoreactors made of di-block copolymers were prepared and characterized as spherical micelles and polymersomes with sizes of 40 nm and 100 nm, respectively. The polymer concentrations were varied from 0.1 to 0.5% (w/w) and enzyme concentrations were varied from 2.5 to 12.5 µM. In vivo experiments using E-nRs of diameter 106 nm, polydispersity 0.17, zeta-potential -8.3 mV, and loading capacity 15% showed that the detoxification efficacy against paraoxon was improved: the LD50 shift was 23.7xLD50 for prophylaxis and 8xLD50 for post-exposure treatment without behavioral alteration or functional physiological changes up to one month after injection. The pharmacokinetic profiles of i.v.-injected E-nRs made of three- and di-block copolymers were similar to the profiles of the injected free enzyme, suggesting partial enzyme encapsulation. Indeed, ELISA and Western blot analyses showed that animals developed an immune response against the enzyme. However, animals that received several injections did not develop iatrogenic symptoms.

Drug and pro-drug substrates and pseudo-substrates of human butyrylcholinesterase.

Butyrylcholinesterase (BChE) is present in plasma and numerous cells and organs. Its physiological function(s) is(are) still unclear. However, this enzyme is of pharmacological and toxicological importance. It displays a broad specificity and is capable of hydrolyzing a wide range of substrates with turnovers differing by several orders of magnitude. Nowaday, these substrates include more than two dozen carboxyl-ester drugs, numerous acetylated prodrugs, and transition state analogues of acetylcholine. In addition, BChE displays a promiscuous hydrolytic activity toward amide bonds of arylacylamides, and slowly hydrolyzes carbamyl- and phosphoryl-esters. Certain pseudo-substrates like carbamates and organophosphates are major drugs of potential medical interest. The existence of a large genetic poly-allelism, affecting the catalytic properties of BChE is at the origin of clinical complications in the use of certain drugs catabolized by BChE. The number of drugs and prodrugs hydrolyzed by BChE is expected to increase in the future. However, very few quantitative data (Km, kcat) are available for most marketed drugs, and except for myorelaxants like succinylcholine and mivacurium, the impact of BChE genetic mutations on catalytic parameters has not been evaluated for most of these drugs.

Terpenes-Modified Lipid Nanosystems for Temozolomide, Improving Cytotoxicity against Glioblastoma Human Cancer Cells In Vitro.

Currently, increasing the efficiency of glioblastoma treatment is still an unsolved problem. In this study, a combination of promising approaches was proposed: (i) an application of nanotechnology approach to create a new terpene-modified lipid system (7% w/w), using soybean L-α-phosphatidylcholine, N-carbonyl-methoxypolyethylene glycol-2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine for delivery of the chemotherapy drug, temozolomide (TMZ, 1 mg/mL); (ii) use of TMZ associated with natural compounds-terpenes (1% w/w) abietic acid and Abies sibirica Ledeb. resin (A. sibirica). Different concentrations and combinations of terpene-lipid systems were employed to treat human cancer cell lines T 98G (glioblastoma), M-Hela (carcinoma of the cervix) and human liver cell lines (Chang liver). The terpene-lipid systems appeared to be unilamellar and of spherical shape under transmission electron microscopy (TEM). The creation of a TMZ-loaded terpene-lipid nanosystem was about 100 nm in diameter with a negative surface charge found by dynamic light scattering. The 74% encapsulation efficiency allowed the release time of TMZ to be prolonged. The modification by terpenes of TMZ-loaded lipid nanoparticles improved by four times the cytotoxicity against human cancer T 98G cells and decreased the cytotoxicity against human normal liver cells. Terpene-modified delivery lipid systems are of potential interest as a combination therapy.

Enzyme Nanoreactor for In Vivo Detoxification of Organophosphates.

A nanoreactor containing an evolved mutant of Saccharolobus solfataricus phosphotriesterase (L72C/Y97F/Y99F/W263V/I280T) as a catalytic bioscavenger was made for detoxification of organophosphates. This nanoreactor intended for treatment of organophosphate poisoning was studied against paraoxon (POX). Nanoreactors were low polydispersity polymersomes containing a high concentration of enzyme (20 µM). The polyethylene glycol-polypropylene sulfide membrane allowed for penetration of POX and exit of hydrolysis products. In vitro simulations under second order conditions showed that 1 µM enzyme inactivates 5 µM POX in less than 10 s. LD50-shift experiments of POX-challenged mice through intraperitoneal (i.p.) and subcutaneous (s.c.) injections showed that intravenous administration of nanoreactors (1.6 nmol enzyme) protected against 7 × LD50 i.p. in prophylaxis and 3.3 × LD50 i.p. in post-exposure treatment. For mice s.c.-challenged, LD50 shifts were more pronounced: 16.6 × LD50 in prophylaxis and 9.8 × LD50 in post-exposure treatment. Rotarod tests showed that transitory impaired neuromuscular functions of challenged mice were restored the day of experiments. No deterioration was observed in the following days and weeks. The high therapeutic index provided by prophylactic administration of enzyme nanoreactors suggests that no other drugs are needed for protection against acute POX toxicity. For post-exposure treatment, co-administration of classical drugs would certainly have beneficial effects against transient incapacitation.

Surface modification of pralidoxime chloride-loaded solid lipid nanoparticles for enhanced brain reactivation of organophosphorus-inhibited AChE: Pharmacokinetics in rat.

The nanotechnological approach is an innovative strategy of high potential to achieve reactivation of organophosphorus-inhibited acetylcholinesterase in central nervous system. It was previously shown that pralidoxime chloride-loaded solid lipid nanoparticles (2-PAM-SLNs) are able to protect the brain against pesticide (paraoxon) central toxicity. In the present work, we increased brain AChE reactivation efficacy by PEGylation of 2-PAM-SLNs using PEG-lipid N-(carbonyl-methoxypolyethylene glycol-2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine, sodium salt) (DSPE-PEG2000) as a surface-modifier of SLNs. To perform pharmacokinetic study, a simple, sensitive (LLOQ 1.0 ng/mL) high-performance liquid chromatography tandem mass spectrometry with atmospheric pressure chemical ionization by multiple reaction monitoring mode (HPLC-APCI-MS) was developed. The method was compared to mass spectrometry with electrospray ionization. The method was validated for linearity, accuracy, precision, extraction recovery, matrix effect and stability. Acetophenone oxime was used as the internal standard for the quantification of 2-PAM in rat plasma and brain tissue after intravenous administration. 2-PAM-DSPE-PEG2000-SLNs of mean size about 80 nm (PDI = 0.26), zeta-potential of -55 mV and of high in vitro stability, prolonged the elimination phase of 2-PAM from the bloodstream more than 3 times compared to free 2-PAM. An increase in reactivation of POX-inhibited human brain acetylcholinesterase up to 36.08 ± 4.3 % after intravenous administration of 2-PAM-DSPE-PEG2000-SLNs (dose of 2-PAM is 5 mg/kg) was achieved. The result is one of the first examples where this level of brain acetylcholinesterase reactivation was achieved. Thus, the implementation of different approaches for targeting and modifying nanoparticles' surface gives hope for improving the antidotal treatment of organophosphorus poisoning by marketed reactivators.

Design and synthesis of amphiphilic 2-hydroxybenzylphosphonium salts with antimicrobial and antitumor dual action.

Here we report the synthesis and biological evaluation of a series of new 2-hydroxybenzylphosphonium salts (QPS) with antimicrobial and antitumor dual action. The most active compounds exhibit antimicrobial activity at a micromolar level against Gram-positive bacteria Sa (ATCC 209p and clinical isolates), Bc (1-2 µM) and fungi Tm and Ca, and induced no notable hemolysis at MIC. The change in nature of substituents of the same length led to a drastic change of biological activity. Self-assembly behavior of the octadecyl and oleyl derivatives was studied. QPS demonstrated self-assembly within the micromolar range with the formation of nanosized aggregates capable of the solubilizing hydrophobic probe. The synthesized phosphonium salts were tested for cytotoxicity. The most potent salt was active against on M-Hela cell line with IC50 on the level of doxorubicin and good selectivity. According to the cytofluorimetry analysis, the salts induced mitochondria-dependent apoptosis.