A single-label fluorescent derivatization method for quantitative determination of neurotoxin in vivo by capillary electrophoresis coupled with laser-induced fluorescence detection.

Neurotoxin (NT), a short-chain α-neurotoxin, is the main neurotoxic protein identified from the venom of Naja naja atra. As an effective drug for the analgesis of advanced cancer patients, NT lasts longer than morphine and does not cause addiction. However, achieving a sensitive and high-resolution measurement of NT is difficult because of the extra-low content of NT in vivo. Therefore, developing a novel method to quantify NT is essential to study its pharmacokinetics in vivo. Although NT contains four primary amine groups that could react with the thiourea in fluorescein isothiocyanate (FITC), we developed a simple and reproducible single-label fluorescent derivatization method for NT which is related to the reaction of N-terminal α-amino of NT alone under optimized derivatization conditions. Furthermore, neurotoxin labelled with fluorescein isothiocyanate (NT-FITC) was prepared by high-performance liquid chromatography (HPLC) with a purity value higher than 99.29% and identified by MALDI-TOF/TOF-MS. Finally, NT-FITC could be detected at 0.8 nmol L(-1) in rat plasma using capillary electrophoresis coupled with laser induced fluorescence detection (CE-LIF). In this paper, the established method robustly and reliably quantified NT labelled with FITC via intravenous and intramuscular administrations in vivo. In addition, this work fully demonstrated the pharmacokinetic characteristics of NT in vivo, which could reduce the risk of drug accumulation, optimize therapies, and provide sufficient evidence for the rational use of NT in clinical and research laboratories.

Pharmacokinetics of Naja sumatrana (equatorial spitting cobra) venom and its major toxins in experimentally envenomed rabbits.

BACKGROUND: The optimization of snakebite management and the use of antivenom depend greatly on the knowledge of the venom's composition as well as its pharmacokinetics. To date, however, pharmacokinetic reports on cobra venoms and their toxins are still relatively limited. In the present study, we investigated the pharmacokinetics of Naja sumatrana (Equatorial spitting cobra) venom and its major toxins (phospholipase A2, neurotoxin and cardiotoxin), following intravenous and intramuscular administration into rabbits. PRINCIPAL FINDINGS: The serum antigen concentration-time profile of the N. sumatrana venom and its major toxins injected intravenously fitted a two-compartment model of pharmacokinetics. The systemic clearance (91.3 ml/h), terminal phase half-life (13.6 h) and systemic bioavailability (41.9%) of N. sumatrana venom injected intramuscularly were similar to those of N. sputatrix venom determined in an earlier study. The venom neurotoxin and cardiotoxin reached their peak concentrations within 30 min following intramuscular injection, relatively faster than the phospholipase A2 and whole venom (Tmax=2 h and 1 h, respectively). Rapid absorption of the neurotoxin and cardiotoxin from the injection site into systemic circulation indicates fast onsets of action of these principal toxins that are responsible for the early systemic manifestation of envenoming. The more prominent role of the neurotoxin in N. sumatrana systemic envenoming is further supported by its significantly higher intramuscular bioavailability (Fi.m.=81.5%) compared to that of the phospholipase A2 (Fi.m.=68.6%) or cardiotoxin (Fi.m.=45.6%). The incomplete absorption of the phospholipase A2 and cardiotoxin may infer the toxins' affinities for tissues at the injection site and their pathological roles in local tissue damages through synergistic interactions. CONCLUSION/SIGNIFICANCE: Our results suggest that the venom neurotoxin is absorbed very rapidly and has the highest bioavailability following intramuscular injection, supporting its role as the principal toxin in systemic envenoming.

Brain pharmacokinetics of neurotoxin-loaded PLA nanoparticles modified with chitosan after intranasal administration in awake rats.

CONTEXT: Neurotoxin (NT), an analgesic peptide which was separated from the venom of Naja naja atra, is endowed an exceptional specificity of action that blocks transmission of the nerve impulse by binding to the acetylcholine receptor in the membrane. However, it has limited permeability across the blood-brain barrier (BBB). OBJECTIVE: The purpose of this study was to encapsulate NT within polylactic acid (PLA) nanoparticles (NPs) modified with chitosan (NT-PLA-cNPs) and to evaluate their brain pharmacokinetic behaviors after intranasal (i.n.) administration using a microdialysis technique in free-moving rats. METHODS: NT-PLA-cNPs (NT labeled with fluorescein isothiocyanate) were prepared and characterized. Then, NT-PLA-cNPs were i.n. administered to rats and the fluorescence intensity in the periaqueductal gray (PAG) was monitored for up to 480 min, with NT-PLA-NPs and NT solution as control groups. RESULTS: The NPs prepared were spherical with a homogenous size distribution. The mean particle size, zeta potential, and entrapment efficiency were 140.5 ± 5.4 nm, +33.71 ± 3.24 mV, and 83.51 ± 2.65%, respectively. The brain transport results showed that Tmax of NT-PLA-cNPs was equal with that of NT-PLA-NPs after i.n. administration (150 min). The Cmax and AUC(0-8 h) of each group followed the following order: NT-PLA-cNPs > NT-PLA-NPs. The corresponding absolute bioavailability (Fabs) of NT-PLA-cNPs was about 151% with NT-PLA-NPs as reference preparations. CONCLUSION: These results suggest that NPs modified with chitosan have better brain targeting efficiency and are a promising approach for i.n. delivery of large hydrophilic peptides and proteins in improving the treatment of central nervous system (CNS) disorders.

Brain transport of neurotoxin-I with PLA nanoparticles through intranasal administration in rats: a microdialysis study.

The purpose of this study was to encapsulate neurotoxin-I (NT-I) within polylactic acid (PLA) nanoparticles (NPs) and to evaluate their transport into the brain after intranasal administration (i.n.) using a microdialysis sampling technique. NT-I-NPs (NT-I radiolabeled with sodium [(125)I]iodide) were prepared and characterized. Then, NT-I-NPs were administered i.n. or i.v. to rats and the radioactivities in the olfactory bulbs were monitored for up to 240 min. The nanoparticles prepared were spherical with a homogenous size distribution. The mean particle size, zeta potential and entrapment efficiency were -28.6+/-2.3 mV, 65 nm and 35.5+/-2.8%, respectively. The brain transport results showed that the time to reach the peak level (T(max)) of NT-I-NPs (i.n.) was 65 min, shorter than NT-I-NPs (i.v.) (95 min) or NT-I (i.v.) (145 min). The concentration at peak level (C(max)) and the total area under the concentration-time curves from zero to 4 h (AUC(0-4 h)) of each group followed the following order: NT-I-NPs (i.n.)>NT-I-NPs (i.v.)>NT-I (i.v.). The corresponding absolute bioavailabilities (Fabs) of NT-I-NPs (i.n.) were about 160%, 196% with NT-I-NPs (i.v.) and NT-I (i.v.) as reference preparations, respectively. The brain delivery of NT-I could be enhanced with PLA nanoparticles either through i.n. or i.v. administration. Furthermore, the enhancement was more significant for i.n. than for i.v. administration. Nanoparticles as carriers would be a potential way to improve the brain transport for centrally active peptides.

[Brain delivery of neurotoxin-I-loaded nanoparticles through intranasal administration].

The purpose of this paper is to encapsulate neurotoxin-I (NT-I), a kind of analgesic peptide, into polylactic acid (PLA) nanoparticles (NPs) and to evaluate their transport into the brain after intranasal administration (in) by use of microdialysis sampling technique developed in our laboratory recently. NT-I-NPs (NT-Iradiolabeled with sodium 125I-Iodide) were prepared by a double emulsification solvent evaporation method, and were characterized in terms of surface morphology, particle size distribution, zeta potential and entrapment efficiency. Then, NT-I-NPs were administered intranasally or intravenously to rats and the radioactivities in periaqueductal gray (PAG) were monitored up to 240 min utilizing the microdialysis sampling technique. Nanoparticles prepared were spherical with homogenous size distribution. Their mean particle size and zeta potential measured were (65.3 +/- 10.8) nm and (-28.6 +/- 2.3) mV, respectively. The entrapment efficiency of NT-Iencapsulated into nanoparticles was (35.5 +/- 2.8)%. The brain transport results showed that the time to peak level (Tmax) of NT-I-NPs (in) was (65 +/- 10) min approximately, apparently shorter compared with NT-I-NPs [iv, (95 +/- 10) min] or NT-I [iv, (145 +/- 10) min]. The concentration to peak level (Cmax) and the area under the curves from zero to 4 h (AUC0-4h) of each group followed this order: NT-I-NPs (in) > NT-I-NPs (iv) > NT-I (iv). With nanoparticles as carriers and administered intranasally could be a potential way for centrally active peptides to improve their brain transport. Microdialysis is quite a good technique for the study of drug delivery to the brain.

Delivery of 125I-cobrotoxin after intranasal administration to the brain: a microdialysis study in freely moving rats.

In order to determine the contribution of intranasal (i.n.) administration to the uptake of large molecular weight (MW) substances into central nervous system (CNS), concentration in brain of the centrally acting polypeptide cobrotoxin (NT-I) versus time profiles were studied using dual-probe microdialysis in awake free-moving rats. NT-I, radiolabeled with sodium (125)I-Iodide ((125)I-NT-I), was administered at the dose of 105 microg/kg intravenously and intranasally in the same set of rat (n=15). The (125)I-NT-Inasal preparations were formulated with borneol/menthol eutectic mixture (+BMEM) as an absorption enhancer and without (-BMEM). After application, the dialysates sampled simultaneously from olfactory bulb and cerebellar nuclei were measured in a gamma-counter for radioactivity. The real concentrations of NT-I were recalculated by in vivo recoveries of microdialysis probes. The results showed that the area under the curve (AUC) value in cerebellar nuclei (2283.51+/-34.54 min ng/ml) following i.n. administration (+BMEM) was significantly larger than those (AUC(olfactory)=1141.92+/-26.42 min ng/ml; AUC(cerebellar)=1364.62+/-19.35 min ng/ml) after intravenous (i.v.) bolus, respectively. A prolonged time values to peak concentrations after i.n. application (+BMEM) were observed compared with those following i.v. administration. Also, following i.n. application (+BMEM) the measured time value to peak concentration in cerebellar nuclei (85 min) was statistically longer than that in olfactory bulb (75 min), which could be plausibly an indication for NT-I delivery into brain via nose-brain pathway in the presence of absorption enhancer. i.n. administration (-BMEM) had little or no ability of NT-I delivering into brain. In conclusion, i.n. administration (+BMEM) significantly enhanced brain transport of NT-I with uneven distribution in discrete regions of brain compared with i.v. administration. Additionally, multi-probe microdialysis technique should be considerably valuable in brain delivery studies.