Electroporation-delivered transdermal neostigmine in rats: equivalent action to intravenous administration.

PURPOSE: Transdermal electroporation has become one of the most promising noninvasive methods for drug administration, with greatly increased transport of macromolecules through the skin. The cecal-contracting effects of repeated transdermal electroporation delivery and intravenous administration of neostigmine were compared in anesthetized rats. METHODS: The cecal contractions were detected with implantable strain gauge sensors, and the plasma levels of neostigmine were followed by high-performance liquid chromatography. RESULTS: Both intravenously and EP-administered neostigmine (0.2-66.7 µg/kg) increased the cecal contractions in a dose-dependent manner. For both the low doses and the highest dose, the neostigmine plasma concentrations were the same after the two modes of administration, while an insignificantly higher level was observed at a dose of 20 µg/kg after intravenous administration as compared with the electroporation route. The contractile responses did not differ significantly after the two administration routes. CONCLUSION: The results suggest that electroporation-delivered neostigmine elicits action equivalent to that observed after intravenous administration as concerning both time and intensity. Electroporation permits the delivery of even lower doses of water-soluble compounds through the skin, which is very promising for clinical practice.

Development and validation of a rapid and high-sensitivity liquid chromatography-tandem mass spectrometry assay for the determination of neostigmine in small-volume beagle dog plasma and its application to a pharmacokinetic study.

A simple, rapid and high sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of neostigmine in small-volume beagle dog plasma was developed to assess the plasma pharmacokinetics of neostigmine. After protein precipitation in a Sirocco 96-well filtration plate, the filtrate was directly injected into the LC-MS/MS system. The analytes were separated on a Hanbon Hedera CN column (100 × 4.6 mm, 5 µm) with a mobile phase composed of methanol-water (60:40, v/v) and the water containing 0.01% formic acid at a flow rate of 0.6mL/min, with a split ratio of 1:1 flowing 300 µL into the mass spectrometer. The run time was 3 min. Detection was accomplished by electrospray ionization source in multiple reactions monitoring mode with the precursor-to-product ion transitions m/z 223.0 → 72.0 and 306.0 → 140.0 for neostigmine and anisodamine (internal standard), respectively. The method was sensitive with a lower limit of quantitation of 0.1 ng/mL, and good linearity in the range 0.1-100ng/mL for neostigmine (r ≥ 0.998). All the validation data, such as accuracy, intra-run and inter-run precision, were within the required limits. The method was successfully applied to pharmacokinetic study of neostigmine methylsulfate injection in beagle dogs.

Modified carbon paste sensor for the potentiometric determination of neostigmine bromide in pharmaceutical formulations, human plasma and urine.

A novel, simple, rapid, selective and sensitive method for the determination of neostigmine (Ns) ion in its bulk powder, different pharmaceutical dosage forms, and biological fluids (plasma and urine) using four modified carbon paste electrodes was developed. Sensor 1 is based on ion-association Ns-TPB, sensor 2 used Ns-PT, sensor 3 comprises a mixture of (Ns-PT+Ns-TPB) and sensor 4 was constructed using (Ns-PT+ß-CD). Solvent mediator 2-NPPE exhibited a proper behavior including Nernstian slope ranging from 61.5±0.5 to 64.5±0.5 mV per decade over the pH range of 3.8-10 for the four sensors. Linear responses of Ns within the concentration range 1.0×10(-7)-1.0×10(-2) mol/L were obtained. The response time is very short (≤10s) with a detection limit 6.3×10(-8) M. In flow injection analysis (FIA), sensor 3 shows a Nernstian slope value 75.5±0.5 mV per decade within the concentration range of 1×10(-6)-1×10(-2) mol/L and with a detection limit 7.5×10(-7) mol/L. The utility of mixed or additives of ß-CD had a significant influence on increasing the sensitivity of sensors 3 and 4 compared to sensors 1 and 2. The sensors were applied for the determination of neostigmine (Ns) ion in its bulk powder, different pharmaceutical dosage forms, and biological fluids (plasma and urine). The results obtained were satisfactory with excellent percentage recovery comparable with official method for the assay based on non-aqueous titration using perchloric acid as a titrant.

Congenital myasthenia gravis.

Congenital myasthenia gravis is caused by genetic mutations affecting neuromuscular transmission, characterized by muscle weakness usually starting in childhood. A two and a half years old male child presented with bilateral ptosis and hoarseness of voice. The symptoms progressed giving the clinical impression of congenital myasthenia gravis. A series of tests were done including Ice Pack Test, acetylcholine receptor antibody test, trial of steroids and finally neostigmine test which confirmed the diagnosis. This case illustrates the challenges in diagnosing congenital myasthenia gravis and highlights the potential benefits of neostigmine test in its diagnosis.

[Pharmacokinetics and relative bioavailability of neostigmine bromide sustained-release tablets].

OBJECTIVE: To study the pharmacokinetics and relative bioavailability of neostigmine bromide conventional tablets and sustained-release tablets in rabbits. METHODS: Six healthy rabbits were randomly divided into two groups for a cross self-contrast trial. RP-HPLC was used to detect plasma concentrations of neostigmine bromide. The pharmacokinetic parameters were calculated with the aid of DAS 2.0 software. RESULTS: The main pharmacokinetics parameters of the sustained-release tablets and conventional tablets were as follows, respectively: T(max)(3.67 +/- 1.51) hand (1.58 +/- 0.38) h; C(max) (5.04 +/- 1.19) mg/L and (4.56 +/- 1.70) mg/L; AUC(0 --> infinity) (32.82 +/- 9.88) mg/L x h and (29.84 +/- 14.27) mg/L x h. The relative bioavailability of the neostigmine bromide sustained-release tablets was 115.4%. CONCLUSION: The pharmacokinetics of neostigmine bromide accords with two compartments model, showing constant plasma concentration and relatively high bioavailability.

Comparative study of 2-hydroxy propyl beta cyclodextrin and calixarene as ionophores in potentiometric ion-selective electrodes for neostigmine bromide.

Three novel neostigmine bromide (NEO) selective electrodes were investigated with 2-nitrophenyl octyl ether as a plasticiser in a polymeric matrix of polyvinyl chloride (PVC). Sensor 1 was fabricated using tetrakis(4-chlorophenyl)borate (TpClPB) as an anionic exchanger without incorporation of an ionophore. Sensor 2 used 2-hydroxy propyl ß-cyclodextrin as an ionophore while sensor 3 was constructed using 4-sulfocalix-8-arene as an ionophore. Linear responses of NEO within the concentration ranges of 10(-5) to 10(-2), 10(-6) to 10(-2) and 10(-7) to 10(-2) mol L(-1) were obtained using sensors 1, 2 and 3, respectively. Nernstian slopes of 51.6 ± 0.8, 52.9 ± 0.6 and 58.6 ± 0.4 mV/decade over the pH range of 4-9 were observed. The selectivity coefficients of the developed sensors indicated excellent selectivity for NEO. The utility of 2-hydroxy propyl ß-cyclodextrin and 4-sulfocalix[8]arene as ionophores had a significant influence on increasing the membrane sensitivity and selectivity of sensors 2 and 3 compared to sensor 1. The proposed sensors displayed useful analytical characteristics for the determination of NEO in bulk powder, different pharmaceutical formulations, and biological fluids (plasma and cerebrospinal fluid (CSF)) and in the presence of its degradation product (3-hydroxyphenyltrimethyl ammonium bromide) and thus could be used for stability-indicating methods.

LC/MS/MS analysis of quaternary ammonium drugs and herbicides in whole blood.

Quaternary ammonium drugs (atracurium, bretylium, edrophonium, ipratropium, mivacurium, neostigmine, pancuronium and rocuronium) and herbicides (difenzoquat, diquat and paraquat) in human whole blood were analysed by LC/MS/MS with positive electrospray ionisation (ESI), following extraction with Bond Elut LRC-CBA cartridges. Internal standards were benzyldimethylphenylammonium chloride monohydrate and ethyl viologen for drug and herbicide analysis, respectively. Ion-pair chromatography used heptafluorobutyric acid (15 mM)-ammonium formate (20 mM) buffer adjusted to pH 3.30 with formic acid and a linear gradient from 5 to 90% methanol run over 18 min. Recoveries ranged from 79.7 to 105.1%, detection limits were between 3.6 and 20.4 ng/ml and the intra- and inter-day precisions were less than 18.6% at a concentration of 10 ng/ml. The method was applied to a case of accidental paraquat poisoning in which the concentration of paraquat in blood was 0.64 mg/l, which is within the range associated with fatal paraquat poisoning.

Determination of neostigmine in human plasma and cerebrospinal fluid by high-performance liquid chromatography with ultraviolet detection.

A high-performance liquid chromatographic assay coupled with ultraviolet detection has been developed for the determination of neostigmine in human plasma and cerebrospinal fluid. A novel solid-phase extraction procedure was first used for this analyte and allowed good recovery (89+/-4.5%) together with ease and speed of execution. The method was sensitive, reproducible (C.V.<4.5%) and accurate (100+/-6.6%) over the range 2.6-167.0 ng/ml neostigmine concentrations in plasma or cerebrospinal fluid, and was applied successfully to study the pharmacokinetics of neostigmine in patients suffering from chronic postoperative abdominal pain.

Bioavailability of intranasal neostigmine: comparison with intravenous route.

The pharmacokinetics of intranasal (i.n.) neostigmine was compared with the intravenous (i.v.) route in 6 healthy volunteers with a mean age of 25.5 years and a mean weight of 65.8 kg in a crossover design. The doses used were 0.5 mg i.v. and 21.6 mg i.n. Neostigmine was determined by HPLC. The plasmatic profiles of the two routes were similar. An early peak of plasmatic concentration after i.n. administration was observed. The absolute bioequivalence of the i.n. route was ten-fold greater than the oral one.

Pharmacokinetic study of neostigmine after intranasal and intravenous administration in the guinea pig.

It is well known that the nasal route may be an effective alternative to the administration of drugs poorly absorbed via oral administration. Thus an investigation of neostigmine bioavailability after nasal administration was undertaken. The neostigmine kinetic profiles after nasal and intravenous administration in the guinea pig have been compared, and results indicate good nasal absorption of neostigmine. At the same dose, no significant differences have been noticed between the two administration routes, as the area under the curve and the bioavailability index is close to 100%. Moreover nasal administration shows a longer plasmatic elimination compared with the i.v. route (t1/2 beta e.n. = 160.04 min; t1/2 beta i.v. = 23.35 min). Nasal absorption is observed to be dose-related. The present results suggest that nasal administration of neostigmine may be an effective clinical means in Myasthenia gravis therapy.

The extraction and analysis of quaternary ammonium compounds in biological material by GC and GC/MS.

An injection port pyrolysis method for the analysis of quaternary ammonium compounds is reported. Identification and confirmation of the analyte was accomplished by gas chromatography/mass spectrometry. With minor modifications to pH used and solvent composition, a common ion-pair method of extraction of quaternary ammonium compounds from biological matrices with an iodide counter ion is described. Although the combined extraction-identification methodology was limited to neostigmine, succinylcholine, and paraquat, it should easily be applicable to any of the other compounds examined in this study.

Clinical pharmacology of pyridostigmine and neostigmine in patients with myasthenia gravis.

Determination of plasma concentration of pyridostigmine in 20 myasthenic patients on maintenance therapy revealed rather small intraindividual variations within a dose interval. The predose concentration varied considerably between different patients and up to seven fold in patients on the same daily dose. No pharmacokinetic interaction between pyridostigmine and neostigmine was found in five patients studied. In six patients the decrement in the deltoid muscle was studied in parallel with determination of the plasma concentrations following administration of pyridostigmine or neostigmine. In these patients the existence of a "bell-shaped" dose response curve is suggested with the maximal effect at a concentration of 30-60 ng/ml for pyridostigmine and 5-15 ng/ml for neostigmine.

The effect of neostigmine on pyridostigmine bioavailability in myasthenic patients after oral administration.

Plasma levels of pyridostigmine and/or neostigmine were monitored in 8 myasthenic patients who were stabilised on oral pyridostigmine bromide only (60-540 mg per day), and in 9 patients who were stabilised on both neostigmine bromide (15-480 mg per day) and pyridostigmine bromide (240-1080 mg per day), over a period of 12 hr (8.00 a.m. - 8.00 p.m.). Maximum plasma concentrations of pyridostigmine in the first and second groups of patients ranged from 12.4 to 64.5 ng per ml and 15.3 to 144.0 ng per ml respectively. Despite this general intersubject variation in bioavailability of pyridostigmine, there was a direct relationship between the area under plasma concentration-time curves (AUC) and total daily dose in the first group of myasthenic patients (r = 0.95). However, no such observation was noticed neither in all 17 patients nor in the 9 patients who were treated with both drugs. Neostigmine was detected in only one of the second group of patients. It was suggested that neostigmine might interfere with the bioavailability of pyridostigmine when both drugs are concurrently administered orally.

Reversed-phase, ion-pair liquid chromatography of quaternary ammonium compounds: determination of pyridostigmine, neostigmine and edrophionium in biological fluids.

A reversed-phase, ion-pair liquid chromatographic method for the quantitative determination of quaternary acetylcholinesterase inhibitors is described. The method uses an ion-pair extraction to isolate the drugs from biological material prior to liquid chromatographic separation and online UV detection at 214 nm. Quantiation down to 5 ng/ml and within-day precison with coefficient of variation (C.V.) of 1.5% (n = 10, x = 100 ng/ml) for neostigmine, C.V., 1.7% (n = 10, x = 80 ng/ml) for pyridostigmine and C.V., 1.5% (n = 10, x = 100 ng/ml) for edrophonium have been achieved. The assay was designed for pharmacokinetic studies of these drugs in anesthetized patients.

Kinetics and metabolism of intramuscular neostigmine in myasthenia gravis.

Neostigmine kinetics and metabolism were studied after intracellular administration to 8 patients with myasthenia gravis. The plasma neostigmine level declined monoexponentially from 21 +/- 2 to 9 +/- 1 ng/ml between 30 and 120 min. The data were interpreted in terms of a 1-compartment model. Estimates of plasma half-life (t1/2) ranged from 51.1 to 90.5 min; apparent volume of distribution varied from 32.0 to 60.6 1; and total body clearance from 434 to 549 ml/min. Approximately 80% of the drug was eliminated in urine within 24 hr either as unchanged neostigmine or its metabolites. Approximately 50% of the dose was eliminated as the unchanged drug, 15% as 3-hydroxyphenyltrimethylammonium, and 15% as other unidentified metabolites. The neostigmine t1/2, based on the urinary excretion of the unchanged drug, ranged from 90.2 to 118.7 min. It was concluded that neostigmine was eliminated by renal and extrarenal mechanisms.

The simultaneous monitoring of plasma levels of neostigmine and pyridostigmine in man.

The synthesis of a series of pyridostigmine analogues wa reported. From these analogues N,N-dipropylcarbamoyloxy-1-methylpyridinium bromide was considered the most suitable compound for use as a common internal marker for the simultaneous determination of neostigmine and pyridostigmine in human plasma. The assay involved a preliminary ion-pair extraction of the drugs and the internal marker from plasma using potassium-iodide glycine buffer. The extract was analysed by a GC system (10% OV-17 on chromosorb W-AW, 100-120 mesh) linked to a nitrogensensitive detector. The calibration graphs of neostigmine and pyridostigmine were linear and reproducible over the range 5 ng to 100 ng per ml in 3 ml plasma samples. This assay procedure has been used to monitor simultaneously the plasma levels of neostigmine (4.7 to 33 ng per ml) and pyridostigmine (2.7 to 18.6 ng per ml) of a myasthenic patient over a period of twelve hours with repeated dosing of neostigmine bromide (30 mg) and pyridostigmine bromide (60 mg).

Clearance of neostigmine from the circulation during the antagonism of neuromuscular block.

The plasma concentration of neostigmine was measured in five patients during the antagonism of neuromuscular block. The concentration of the drug decreased rapidly between 2 and 5 min after administration, and then more slowly. Detectable concentrations of neostigmine were present in plasma after 60 min. In the five patients the distribution half-life of neostigmine was less than 1 min; the elimination half-life ranged from 15.4 to 30.1 min.