Can Activation of Acetylcholinesterase by ß-Amyloid Peptide Decrease the Effectiveness of Cholinesterase Inhibitors?

A central event in the pathogenesis of Alzheimer's disease (AD) is the accumulation of senile plaques composed of aggregated amyloid-ß (Aß) peptides. The main class of drugs currently used for the treatment of AD are the acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitors. In this study, it has been shown that Aß augmented AChE activity in vitro, maximum activation of 548 ± 5% was achieved following 48 h of incubation with 10 µM of Aß1-40, leading to a 7.7-fold increase in catalytic efficiency. The observed non-competitive type of AChE activation by Aß1-40 was associated with increased Vmax and unchanged Km. Although BChE activity also increased following incubation with Aß1-40, this was less efficiently achieved as compared with AChE. Ex vivo electrophysiological experiments showed that 10 µM of Aß1-40 significantly decreased the effect of the AChE inhibitor huperzine A on the synaptic potential parameters.

Conjugates of nucleobases with triazole-hydroxamic acids for the reactivation of acetylcholinesterase and treatment of delayed neurodegeneration induced by organophosphate poisoning.

A series of new uncharged conjugates of adenine, 3,6-dimetyl-, 1,6-dimethyl- and 6-methyluracil with 1,2,4-triazole-3-hydroxamic and 1,2,3-triazole-4-hydroxamic acid moieties were synthesized and studied as reactivators of organophosphate-inhibited cholinesterase. It is shown that triazole-hydroxamic acids can reactivate acetylcholinesterase (AChE) inhibited by paraoxon (POX) in vitro, offering reactivation constants comparable to those of pralidoxime (2-PAM). However, in contrast to 2-PAM, triazole-hydroxamic acids demonstrated the ability to reactivate AChE in the brain of rats poisoned with POX. At a dose of 200 mg/kg (i.v.), the lead compound 3e reactivated 22.6 ± 7.3% of brain AChE in rats poisoned with POX. In a rat model of POX-induced delayed neurodegeneration, compound 3e reduced the neuronal injury labeled with FJB upon double administration 1 and 3 h after poisoning. Compound 3e was also shown to prevent memory impairment of POX-poisoned rats as tested in a Morris water maze.

Calix[4]arene-pyrazole conjugates as potential cancer therapeutics.

Tumor selectivity is yet a challenge in chemotherapy-based cancer treatment. A series of calixarenes derivatized at the lower rim with 3-phenyl-1H-pyrazole units with variable upper-rim substituent and conformations of macrocyclic core, alkyl chain length between heterocycle and core, as well as phenolic monomer (5-(4-tert-butylphenyloxy)methoxy-3-phenyl-1H-pyrazole) have been synthesized and characterized in a range of therapeutically relevant cellular models (M-HeLa, MCF7, A-549, PC3, Chang liver, and Wi38) from different target organs/systems. Specific cytotoxicity for M-HeLa cells has been observed in tert-butylcalix[4]arene pyrazoles in 1,3-alternate (compound 7b) and partial cone (compound 7c) conformations with low mutagenicity and haemotoxicity and in vivo toxicity in mice. Compounds 7b,c have induced mitochondrial pathway of apoptosis of M-HeLa cells through caspase-9 activation preceded by the cell cycle arrest at G0/G1 phase. A concomitant overexpression of DNA damage markers in pyrazole-treated M-HeLa cells suggests that calixarene pyrazoles target DNA, which was supported by the presence of interactions between calixarenes and ctDNA at the air-water interface.

Differences in bioavailability and cognitive-enhancing activity exerted by different crystal polymorphs of latrepirdine (Dimebon®).

Introduction: Pharmacokinetic characteristics as well as cognitive-enhancing nootropic activity of latrepirdine (Dimebon®) in relationship with its polymorphic forms have been studied in SD and Wistar rats. Methods: The pharmacokinetics of six polymorphs (A, B, C, D, E, F) of latrepirdine were studied in male SD rats after 7 days of oral administration in corn oil at a dose of 10 mg/kg once a day. Blood and brain samples were taken on the 7th day of administration at 15 min, 30 min, 60 min and 120 min after administration and analyzed for latrepirdine content by LC-MS. The cognitive-enhancing nootropic effect was studied in male and female Wistar rats after 9 days of oral administration in corn oil at a dose of 10 mg/kg, after prior administration of scopolamine, an agent that causes memory impairment similar to that in Alzheimer's disease. The animals' cognitive function was studied in the passive avoidance test. Results: When studying the pharmacokinetics, the highest bioavailability both in the blood and in the brain was demonstrated by polymorph E, whose AUC was the highest relative to other polymorphs. In the study of the cognitive-enhancing nootropic effect, polymorph E also showed the highest activity, whose values of the latent period of entering the dark chamber did not differ from control animals, and differed from other polymorphs. Conclusion: Thus, the crystal structure has been shown to play a key role in the bioavailability and efficacy of latrepirdine, and polymorph E has also been shown to be a promising drug for the treatment of neurodegenerative diseases associated with memory impairment, such as Alzheimer's disease.

Novel slow-binding reversible acetylcholinesterase inhibitors based on uracil moieties for possible treatment of myasthenia gravis and protection from organophosphate poisoning.

A series of new compounds in which uracil and 3,6-dimethyluracil moieties are bridged with different spacers were prepared and evaluated in vitro for the acetyl- and butyrylcholinesterase (AChE and BChE) inhibitory activities. These bisuracils are shown to be very effective inhibitors of AChE, inhibiting the enzyme at nano- and lower molar concentrations with extremely high selectivity for AChE vs. BChE. Kinetic analysis showed that the lead compound 2h acts as a slow-binding inhibitor of AChE and possess a long drug-target residence time (τ = 1/koff = 18.6 ± 7.5 min). Moreover, compound 2h ameliorated muscle weakness in myasthenia gravis rat model with a lower effective dose and longer lasting effect than pyridostigmine bromide. Besides, it was shown that compound 2h has an effect of increasing efficiency of antidotal therapy as a pretreatment for poisoning by organophosphates.

Therapy of Organophosphate Poisoning via Intranasal Administration of 2-PAM-Loaded Chitosomes.

Chitosan-decorated liposomes were proposed for the first time for the intranasal delivery of acetylcholinesterase (AChE) reactivator pralidoxime chloride (2-PAM) to the brain as a therapy for organophosphorus compounds (OPs) poisoning. Firstly, the chitosome composition based on phospholipids, cholesterol, chitosans (Cs) of different molecular weights, and its arginine derivative was developed and optimized. The use of the polymer modification led to an increase in the encapsulation efficiency toward rhodamine B (RhB; ~85%) and 2-PAM (~60%) by 20% compared to conventional liposomes. The formation of monodispersed and stable nanosized particles with a hydrodynamic diameter of up to 130 nm was shown using dynamic light scattering. The addition of the polymers recharged the liposome surface (from -15 mV to +20 mV), which demonstrates the successful deposition of Cs on the vesicles. In vitro spectrophotometric analysis showed a slow release of substrates (RhB and 2-PAM) from the nanocontainers, while the concentration and Cs type did not significantly affect the chitosome permeability. Flow cytometry and fluorescence microscopy qualitatively and quantitatively demonstrated the penetration of the developed chitosomes into normal Chang liver and M-HeLa cervical cancer cells. At the final stage, the ability of the formulated 2-PAM to reactivate brain AChE was assessed in a model of paraoxon-induced poisoning in an in vivo test. Intranasal administration of 2-PAM-containing chitosomes allows it to reach the degree of enzyme reactivation up to 35 ± 4%.

Novel Uracil-Based Inhibitors of Acetylcholinesterase with Potency for Treating Memory Impairment in an Animal Model of Alzheimer's Disease.

Novel derivatives based on 6-methyluracil and condensed uracil, 2,4-quinazoline-2,4-dione, were synthesized with terminal meta- and para-benzoate moieties in polymethylene chains at the N atoms of the pyrimidine ring. In the synthesized compounds, the polymethylene chains were varied from having tris- to hexamethylene chains and quaternary ammonium groups; varying substituents (ester, salt, acid) at benzene ring were introduced into the chains and benzoate moieties. In vivo biological experiments demonstrated the potency of these compounds in decreasing the number of ß-amyloid plaques and their suitability for the treatment of memory impairment in a transgenic model of Alzheimer's disease.

Oxime Therapy for Brain AChE Reactivation and Neuroprotection after Organophosphate Poisoning.

One of the main problems in the treatment of poisoning with organophosphorus (OPs) inhibitors of acetylcholinesterase (AChE) is low ability of existing reactivators of AChE that are used as antidotes to cross the blood-brain barrier (BBB). In this work, modified cationic liposomes were developed that can penetrate through the BBB and deliver the reactivator of AChE pralidoxime chloride (2-PAM) into the brain. Liposomes were obtained on the basis of phosphatidylcholine and imidazolium surfactants. To obtain the composition optimized in terms of charge, stability, and toxicity, the molar ratio of surfactant/lipid was varied. For the systems, physicochemical parameters, release profiles of the substrates (rhodamine B, 2-PAM), hemolytic activity and ability to cause hemagglutination were evaluated. Screening of liposome penetration through the BBB, analysis of 2-PAM pharmacokinetics, and in vivo AChE reactivation showed that modified liposomes readily pass into the brain and reactivate brain AChE in rats poisoned with paraoxon (POX) by 25%. For the first time, an assessment was made of the ability of imidazolium liposomes loaded with 2-PAM to reduce the death of neurons in the brains of mice. It was shown that intravenous administration of liposomal 2-PAM can significantly reduce POX-induced neuronal death in the hippocampus.

Enhancement of the Transdermal Delivery of Nonsteroidal Anti-inflammatory Drugs Using Liposomes Containing Cationic Surfactants.

New hybrid liposomes based on cationic amphiphiles with different structures of the head group (cetyltrimethylammonium bromide (CTAB), 3-hexadecyl-1-hydroxyethylimidazolium bromide (IA-16(OH)), 1-(butylcarbamoyl)oxyethyl-3-hexadecylimidazolium bromide (IAC 16(Bu)), and hexadecylmethylpyrrolidinium bromide (PR-16)) were developed for transdermal administration of nonsteroidal anti-inflammatory drugs. The different surfactant/lipid compositions were studied to obtain stable liposomes with high functionality. The hydrodynamic diameter of cationic liposomes was ∼110 nm. An admixture of cationic surfactants and PC liposomes improves the physicochemical properties of vesicles and transdermal diffusion rate and prolongs the release of drugs. Liposomal diclofenac sodium (DS) and ketoprofen (KP) were tested (using Franz cells) for transdermal penetration. Drug diffusion monitoring for 48 h demonstrated that the maximum DS and KP penetration through the synthetic membranes (Strat-M) is characterized by values of 255 ± 2 and 186 ± 3 µg/cm2, respectively. The influence of the surfactant head group on the properties (stability, release profile, permeability) of cationic liposomes was shown for the first time. While the drug specificity is evident for the rate of release, the permeability increases as follows: conventional liposomes < CTAB/PC < PR-16/PC < IAC-16(Bu)/PC < IA-16(OH)/PC for both medicines. The rat paw edema model was used to assess the anti-inflammatory effect of the IA-16(OH)/PC leader formulation in vivo. It was found that liposomal DS and KP are effective for relieving rat paw edema. It should be noted that DS-loaded hybrid liposomes demonstrated the highest therapeutic efficacy compared to conventional vesicles.

Balanced modulation of neuromuscular synaptic transmission via M1 and M2 muscarinic receptors during inhibition of cholinesterases.

Organophosphorus (OP) compounds that inhibit acetylcholinesterase are a common cause of poisoning worldwide, resulting in several hundred thousand deaths each year. The pathways activated during OP compound poisoning via overstimulation of muscarinic acetylcholine receptors (mAChRs) play a decisive role in toxidrome. The antidotal therapy includes atropine, which is a nonspecific blocker of all mAChR subtypes. Atropine is efficient for mitigating depression in respiratory control centers but does not benefit patients with OP-induced skeletal muscle weakness. By using an ex vivo model of OP-induced muscle weakness, we studied the effects of the M1/M4 mAChR antagonist pirenzepine and the M2/M4 mAChR antagonist methoctramine on the force of mouse diaphragm muscle contraction. It was shown that weakness caused by the application of paraoxon can be significantly prevented by methoctramine (1 µM). However, neither pirenzepine (0.1 µM) nor atropine (1 µM) was able to prevent muscle weakness. Moreover, the application of pirenzepine significantly reduced the positive effect of methoctramine. Thus, balanced modulation of neuromuscular synaptic transmission via M1 and M2 mAChRs contributes to paraoxon-induced muscle weakness. It was shown that methoctramine (10 µmol/kg, i.p.) and atropine (50 µmol/kg, i.p.) were equieffective toward increasing the survival of mice poisoned with a 2xLD50 dose of paraoxon.

Microemulsions and nanoemulsions modified with cationic surfactants for improving the solubility and therapeutic efficacy of loaded drug indomethacin.

In this work, a noncovalent strategy was successfully used to modify colloidal stability andin vitroandin vivoefficacy of two amphiphilic formulations of the anti-inflammatory drug indomethacin. Namely, nanoemulsions and microemulsions based on oleic acid and nonionic surfactants have been produced and compared. The influence of cationic surfactants cetyltrimethylammonium bromide and its carbamate bearing analogue on the size characteristics, stability and ability to provide prolonged action of loaded drug indomethacin has been evaluated. Adding the positively charged molecules in the surface layer of nanoemulsions and microemulsions has shown the stability increase along with maintaining the size characteristics and homogeneity in time. Moreover, the carbamate modified analogue demonstrated beneficial behavior. Indomethacin loaded in microemulsions and nanoemulsions showed prolonged-release (10%-15% release for 5 h) compared to a free drug (complete release for 5 h). The rate of release of indomethacin from nanoemulsions was slightly higher than from microemulsions and insignificantly decreased with an increase in the concentration of the cationic surfactant. For carbamate surfactant nanocarrier loaded with fluorescence probe Nile Red, the ability to penetrate into the cell was supported by flow cytometry study and visualized by fluorescence microscopy.In vitrotests on anti-inflammatory activity of the systems demonstrated that the blood cell membrane stabilization increased in the case of modified microemulsion. The anti-inflammatory activity of the encapsulated drug was tested in rats using a carrageenan-induced edema model. Nanoemulsions without cationic surfactants appeared more efficient compared to microemulsions. Indomethacin emulsion formulations with carbamate surfactant added showed slower carrageenan-induced edema progression compared to unmodified compositions. Meanwhile, the edema completely disappeared upon treatment with emulsion loaded indomethacin after 4 h in the case of microemulsions versus 5 h in the case of nanoemulsions.

Structure impact on photodynamic therapy and cellular contrasting functions of colloids constructed from dimeric Au(I) complex and hexamolybdenum clusters.

Electrostatically driven self-assembly of [Au2L2]2+ (L is cyclic PNNP ligand) with [{Mo6I8}(L')6]2- (L' = I-, CH3COO-) in aqueous solutions is introduced as facile route for combination of therapeutic and cellular contrasting functions within heterometallic colloids (Mo6-Au2). The nature of L' affects the size and aggregation behavior of crystalline Mo6-Au2 aggregates, which in turn affect the luminescence of the cluster units incorporated into Mo6-Au2 colloids. The spin trap facilitated electron spin resonance spectroscopy technique indicates that the level of ROS generated by Mo6-Au2 colloids is also affected by their size. Both (L' = I-, CH3COO-) Mo6-Au2 colloids undergo cell internalization, which is enhanced by their assembly with poly-DL-lysine (PL) for L' = CH3COO-, but remains unchanged for L' = I-. The colloids PL-Mo6-Au2 (L' = CH3COO-) are visualized as huge crystalline aggregates both outside and inside the cell cytoplasm by confocal microscopy imaging of the incubated cells, while the smaller sized (30-50 nm) PL-Mo6-Au2 (L' = I-) efficiently stain the cell nuclei. Quantitative colocalization analysis of PL-Mo6-Au2 (L' = CH3COO-) in lysosomal compartments points to the fast endo-lysosomal escape of the colloids followed by their intracellular aggregation. The cytotoxicity of PL-Mo6-Au2 differs from that of Mo6 and Au2 blocks, predominantly acting through apoptotic pathway. The photodynamic therapeutic effect of the PL-Mo6-Au2 colloids on the cancer cells correlates with their intracellular trafficking and aggregation.

Mitochondria-targeted mesoporous silica nanoparticles noncovalently modified with triphenylphosphonium cation: Physicochemical characteristics, cytotoxicity and intracellular uptake.

Novel nanocomposite system based on mesoporous silica nanoparticles (MSNs) noncovalently modified with hexadecyltriphenylphosphonium bromide (HTPPB) has been prepared, thoroughly characterized and used for encapsulation of model cargo Rhodamine B (RhB). The high encapsulation efficacy of this dye by HTPPB-modified mesoporous particles was demonstrated by spectrophotometry and thermography techniques. The bioavailability of MSN@HTPPB was testified. Cytotoxicity assay revealed that a marked suppression of M-HeLa cancer cells (epithelioid carcinoma of the cervix) occurs at concentration of 0.06 µg/mL, while the higher viability of Chang liver normal cell line was preserved in the concentration range of 0.98-0.06 µg/mL. Hemolysis assay demonstrated that only 2% of red blood cells are destructed at ~ 30 µg/mL concentration. This allows us to select the most harmless compositions based on MSN@HTPPB with minimal side effects toward normal cells and recommend them for the development of antitumor formulations. Fluorescence microscopy technique testified satisfactory penetration of HTPPB-modified carriers into M-HeLa cells. Importantly, modification of the MSN with HTPPB is shown to promote efficient delivery to mitochondria. To the best of our knowledge, it is one of the first successful examples of noncovalent surface modification of the MSNs with lipophilic phosphonium cation that improves targeted delivery of loads to mitochondria.

Cationic liposomes mediated transdermal delivery of meloxicam and ketoprofen: Optimization of the composition, in vitro and in vivo assessment of efficiency.

New liposomes modified with pyrrolidinium surfactants containing a hydroxyethyl fragment (CnPB, n = 12, 14, 16) were prepared for transdermal delivery of non-steroidal anti-inflammatory drugs. In order to obtain the optimal composition, the surfactant/lipid molar ratio (0.02/1; 0.029/1; 0.04/1) and the amphiphile hydrocarbon tail length were varied. Rhodamine B was loaded in all formulations, while meloxicam and ketoprofen in selected ones. For liposomes studied the hydrodynamic diameter was in the range of 80-130 nm, the zeta potential ranged from +35 to +50 mV, EE was 75-99%. Liposome modification leads to a prolonged release of the rhodamine B (up to 10-12 h) and faster release of non-steroidal drugs (up to 7-8 h) in vitro. The ability to cross the skin barrier using Franz cells was investigated for liposomal meloxicam and ketoprofen. The total amount of meloxicam and ketoprofen passed through the Strat-M® membranes during 51 h was 51-114 µg/cm2 and 87-105 µg/cm2 respectively. The evaluation of transdermal diffusion ex vivo showed that total amount of liposomal ketoprofen passed through the skin during 51 h was 140-162 µg/cm2. Liposomes modified with C16PB were found as the most effective inflammation reducing formulation in the carrageenan edema model of rat paw.

Cholinesterases in Tripartite Neuromuscular Synapse.

The neuromuscular junction (NMJ) is a tripartite synapse in which not only presynaptic and post-synaptic cells participate in synaptic transmission, but also terminal Schwann cells (TSC). Acetylcholine (ACh) is the neurotransmitter that mediates the signal between the motor neuron and the muscle but also between the motor neuron and TSC. ACh action is terminated by acetylcholinesterase (AChE), anchored by collagen Q (ColQ) in the basal lamina of NMJs. AChE is also anchored by a proline-rich membrane anchor (PRiMA) to the surface of the nerve terminal. Butyrylcholinesterase (BChE), a second cholinesterase, is abundant on TSC and anchored by PRiMA to its plasma membrane. Genetic studies in mice have revealed different regulations of synaptic transmission that depend on ACh spillover. One of the strongest is a depression of ACh release that depends on the activation of α7 nicotinic acetylcholine receptors (nAChR). Partial AChE deficiency has been described in many pathologies or during treatment with cholinesterase inhibitors. In addition to changing the activation of muscle nAChR, AChE deficiency results in an ACh spillover that changes TSC signaling. In this mini-review, we will first briefly outline the organization of the NMJ. This will be followed by a look at the role of TSC in synaptic transmission. Finally, we will review the pathological conditions where there is evidence of decreased AChE activity.

Terbium(III)-thiacalix[4]arene nanosensor for highly sensitive intracellular monitoring of temperature changes within the 303-313 K range.

The work introduces hydrophilic PSS-[Tb2(TCAn)2] nanoparticles to be applied as highly sensitive intracellular temperature nanosensors. The nanoparticles are synthesized by solvent-induced nanoprecipitation of [Tb2(TCAn)2] complexes (TCAn - thiacalix[4]arenes bearing different upper-rim substituents: unsubstituted TCA1, tert-buthyl-substituted TCA2, di- and tetra-brominated TCA3 and TCA4) with the use of polystyrenesulfonate (PSS) as stabilizer. The temperature responsive luminescence behavior of PSS-[Tb2(TCAn)2] within 293-333 K range in water is modulated by reversible changes derived from the back energy transfer from metal to ligand (M* → T1) correlating with the energy gap between the triplet levels of ligands and resonant 5D4 level of Tb3+ ion. The lowering of the triplet level (T1) energies going from TCA1 and TCA2 to their brominated counterparts TCA3 and TCA4 facilitates the back energy transfer. The highest ever reported temperature sensitivity for intracellular temperature nanosensors is obtained for PSS-[Tb2(TCA4)2] (SI = 5.25% K-1), while PSS-[Tb2(TCA3)2] is characterized by a moderate one (SI = 2.96% K-1). The insignificant release of toxic Tb3+ ions from PSS-[Tb2(TCAn)2] within heating/cooling cycle and the low cytotoxicity of the colloids point to their applicability in intracellular temperature monitoring. The cell internalization of PSS-[Tb2(TCAn)2] (n = 3, 4) marks the cell cytoplasm by green Tb3+-luminescence, which exhibits detectable quenching when the cell samples are heated from 303 to 313 K. The colloids hold unprecedented potential for in vivo intracellular monitoring of temperature changes induced by hyperthermia or pathological processes in narrow range of physiological temperatures.

Slow-binding reversible inhibitor of acetylcholinesterase with long-lasting action for prophylaxis of organophosphate poisoning.

Organophosphorus (OP) compounds represent a serious health hazard worldwide. The dominant mechanism of their action results from covalent inhibition of acetylcholinesterase (AChE). Standard therapy of acute OP poisoning is partially effective. However, prophylactic administration of reversible or pseudo-irreversible AChE inhibitors before OP exposure increases the efficiency of standard therapy. The purpose of the study was to test the duration of the protective effect of a slow-binding reversible AChE inhibitor (C547) in a mouse model against acute exposure to paraoxon (POX). It was shown that the rate of inhibition of AChE by POX in vitro after pre-inhibition with C547 was several times lower than without C547. Ex vivo pre-incubation of mouse diaphragm with C547 significantly prevented the POX-induced muscle weakness. Then it was shown that pre-treatment of mice with C547 at the dose of 0.01 mg/kg significantly increased survival after poisoning by 2xLD50 POX. The duration of the pre-treatment was effective up to 96 h, whereas currently used drug for pre-exposure treatment, pyridostigmine at a dose of 0.15 mg/kg was effective less than 24 h. Thus, long-lasting slow-binding reversible AChE inhibitors can be considered as new potential drugs to increase the duration of pre-exposure treatment of OP poisoning.

Novel Acetylcholinesterase Inhibitors Based on Uracil Moiety for Possible Treatment of Alzheimer Disease.

In this study, novel derivatives based on 6-methyluracil and condensed uracil were synthesized, namely, 2,4-quinazoline-2,4-dione with ω-(ortho-nitrilebenzylethylamino) alkyl chains at the N atoms of the pyrimidine ring. In this series of synthesized compounds, the polymethylene chains were varied from having tetra- to hexamethylene chains, and secondary NH, tertiary ethylamino, and quaternary ammonium groups were introduced into the chains. The molecular modeling of the compounds indicated that they could function as dual binding site acetylcholinesterase inhibitors, binding to both the peripheral anionic site and active site. The data from in vitro experiments show that the most active compounds exhibit affinity toward acetylcholinesterase within a nanomolar range, with selectivity for acetylcholinesterase over butyrylcholinesterase reaching four orders of magnitude. In vivo biological assays demonstrated the potency of these compounds in the treatment of memory impairment using an animal model of Alzheimer disease.

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.

Biomedical potentialities of cationic geminis as modulating agents of liposome in drug delivery across biological barriers and cellular uptake.

Hydroxyethyl bearing gemini surfactants, alkanediyl-α,ω-bis(N-hexadecyl-N-2-hydroxyethyl-N-methylammonium bromide), 16-s-16(OH), were used to augment phosphatidylcholine based liposomes to achieve higher stability and enhanced cellular uptake and penetration. The developed liposomes were loaded with rhodamine B, doxorubicin hydrochloride, pralidoxime chloride to investigate release properties, cytotoxicity in vitro, as well as ability to cross the blood-brain barrier. At molar ratio of 35:1 (lipid:surfactant) the formulation was found to be of low toxicity, stable for two months, and able to deliver rhodamine B beyond the blood-brain barrier in rats. In vivo, pharmacokinetics of free and formulated 2-PAM in plasma and brain were evaluated, liposomal 2-PAM was found to reactivate 27% of brain acetylcholinesterase, which is, to our knowledge, the first example of such high degree of reactivation after intravenous administration of liposomal drug.