Synergistic Pressor Effect of Atomoxetine and Pyridostigmine in Patients With Neurogenic Orthostatic Hypotension.

Patients with autonomic failure are characterized by disabling orthostatic hypotension because of impaired sympathetic activity, but even severely affected patients have residual sympathetic tone which can be harnessed for their treatment. For example, norepinephrine transporter blockade with atomoxetine raises blood pressure (BP) in autonomic failure patients by increasing synaptic norepinephrine concentrations; acetylcholinesterase inhibition with pyridostigmine increases BP by facilitating ganglionic cholinergic neurotransmission to increase sympathetic outflow. We tested the hypothesis that pyridostigmine will potentiate the pressor effect of atomoxetine and improve orthostatic tolerance and symptoms in patients with severe autonomic failure. Twelve patients received a single oral dose of either placebo, pyridostigmine 60 mg, atomoxetine 18 mg or the combination on separate days in a single blind, crossover study. BP was assessed seated and standing before and 1-hour postdrug. In these severely affected patients, neither pyridostigmine nor atomoxetine improved BP or orthostatic tolerance compared with placebo. The combination, however, significantly increased seated BP in a synergistic manner (133±9/80±4 versus 107±6/66±4 mm Hg for placebo, 105±5/67±3 mm Hg for atomoxetine, and 99±6/64±4 mm Hg for pyridostigmine; P<0.001); the maximal increase in seated BP with the combination was 33±8/18±3 mm Hg at 60 minutes postdrug. Only the combination showed a significant improvement of orthostatic tolerance and symptoms. In conclusion, the combination pyridostigmine and atomoxetine had a synergistic effect on seated BP which was associated with improvement in orthostatic tolerance and symptoms. This pharmacological approach could be useful in patients with severe autonomic failure but further safety and long-term efficacy studies are needed.

Time Course, Behavioral Safety, and Protective Efficacy of Centrally Active Reversible Acetylcholinesterase Inhibitors in Cynomolgus Macaques.

Galantamine hydrobromide and (-)huperzine A, centrally active reversible acetylcholinesterase inhibitors, are potentially superior to the current standard, pyridostigmine bromide, as a pretreatment for organophosphorus chemical warfare nerve agent intoxication. Galantamine, huperzine, and pyridostigmine were compared for time course of acetylcholinesterase inhibition in 12 cynomolgus macaques. Although both galantamine and huperzine shared a similar time course profile for acetylcholinesterase inhibition, huperzine was 88 times more potent than galantamine. The dose for 50% acetylcholinesterase inhibition (ID50) was 4.1 ug/kg for huperzine, 362 ug/kg for galantamine, and 30.9 ug/kg for pyridostigmine. In a safety assessment, galantamine, huperzine, and pyridostigmine were examined using an operant time-estimation task. Huperzine and pyridostigmine were devoid of behavioral toxicity, whereas galantamine was behaviorally toxic at doses producing peak acetylcholinesterase inhibition of about 50% and higher. Following pretreatment with galantamine, huperzine or pyridostigmine, monkeys were challenged with the median lethal dose of soman at the time of peak acetylcholinesterase inhibition and evaluated for overt signs of soman toxicity (cholinergic crisis, convulsions). Both huperzine and galantamine were equally effective at preventing overt signs of soman toxicity, but neither drug was capable of preventing soman-induced neurobehavioral disruption. In contrast, three of four pyridostigmine-pretreated animals exposed to soman exhibited convulsions and required therapy. Full functional recovery required 3-16 days. The degree of acetylcholinesterase inhibition was lower for pyridostigmine, but rates of recovery of acetylcholinesterase activity following soman challenge were comparable for all drug pretreatments. Huperzine may be the more promising centrally active reversible acetylcholinesterase inhibitor due to its greater potency and superior safety profile.

Growth hormone response to standard provocative stimuli and combined tests in very young children with Prader-Willi syndrome.

BACKGROUND: In Prader-Willi syndrome (PWS) a reduced growth hormone (GH) response to several stimulators has been documented in many studies, but none have focused on very young children. We evaluated the pattern of GH secretion in very young PWS patients. PATIENTS AND METHODS: Twenty-seven genetically confirmed PWS children (10 females, aged 0.4-5 years, mean: 2.2 ± 1.4 years) were included. All subjects underwent standard provocative tests (clonidine, CLO; and arginine, ARG) and one combined test [growth hormone-releasing hormone (GHRH) plus pyridostigmine (13 patients) or GHRH plus arginine (14 patients)]. Insulin-like growth factor-1 (IGF-1) levels were also measured. RESULTS: While standard tests (CLO and ARG) showed low GH peak in 85.2 and 70.4% of the patients, respectively, the combined test was found to be normal in 85.2%. IGF-1 was low in 66.7% of patients. Out of 27 patients, 3 (11%) showed a normal GH peak with both standard tests (group A), 6 (22%) to one of the standard tests (group B) and 18 (67%) presented a low response to both standard tests (group C). Four subjects showed low response to both the combined and standard tests and reduced IGF-1. CONCLUSION: Our data suggest that very young PWS children seem to have impaired hypothalamic GHRH secretion with a normal GH pituitary reserve.

Nanosized sustained-release pyridostigmine bromide microcapsules: process optimization and evaluation of characteristics.

BACKGROUND: Pyridostigmine bromide (3-[[(dimethylamino)-carbonyl]oxy]-1-methylpyridinium bromide), a reversible inhibitor of cholinesterase, is given orally in tablet form, and a treatment schedule of multiple daily doses is recommended for adult patients. Nanotechnology was used in this study to develop an alternative sustained-release delivery system for pyridostigmine, a synthetic drug with high solubility and poor oral bioavailability, hence a Class III drug according to the Biopharmaceutics Classification System. Novel nanosized pyridostigmine-poly(lactic acid) microcapsules (PPNMCs) were expected to have a longer duration of action than free pyridostigmine and previously reported sustained-release formulations of pyridostigmine. METHODS: The PPNMCs were prepared using a double emulsion-solvent evaporation method to achieve sustained-release characteristics for pyridostigmine. The preparation process for the PPNMCs was optimized by single-factor experiments. The size distribution, zeta potential, and sustained-release behavior were evaluated in different types of release medium. RESULTS: The optimal volume ratio of inner phase to external phase, poly(lactic acid) concentration, polyvinyl alcohol concentration, and amount of pyridostigmine were 1:10, 6%, 3% and 40 mg, respectively. The negatively charged PPNMCs had an average particle size of 937.9 nm. Compared with free pyridostigmine, PPNMCs showed an initial burst release and a subsequent very slow release in vitro. The release profiles for the PPNMCs in four different types of dissolution medium were fitted to the Ritger-Peppas and Weibull models. The similarity between pairs of dissolution profiles for the PPNMCs in different types of medium was statistically significant, and the difference between the release curves for PPNMCs and free pyridostigmine was also statistically significant. CONCLUSION: PPNMCs prepared by the optimized protocol described here were in the nanometer range and had good uniformity, with significantly slower pyridostigmine release than from free pyridostigmine. This novel sustained-release delivery nanosystem for pyridostigmine might alleviate the need to identify new acetylcholinesterase inhibitors.

Novel taste-masked orally disintegrating tablets for a highly soluble drug with an extremely bitter taste: design rationale and evaluation.

The purpose of this study was to evaluate the taste masking potential of novel solid dispersions (SDs) using Eudragit® EPO as the excipient when incorporated into the orally disintegrating tablets (ODTs) for delivering a highly soluble drug with an extremely bitter taste. The pyridostigmine bromides (PB) SDs (PBSDs) were prepared by solvent evaporation-deposition method. The physicochemical properties of PBSDs were investigated by means of differential scanning calorimetry and Fourier transformed infrared spectroscopy. The dissolution test showed that only about 8% of PB was released from PBSDs in the simulated salivary fluid in 30 s. Therefore, PBSDs were considered taste-masked and selected for formulation of PBODTs. A central composite design was employed for process optimization. Multiple linear regression analysis for process optimization revealed that the optimal PBODTs were obtained, when the microcrystalline cellulose and crospovidone were 17.16 and 5.55 (%, w/w), respectively, and the average in vivo disintegration time was 25 s. The bitterness threshold of PB was examined by a sensory test, and the threshold value was set as 3 mg in each tablet. Taste evaluation of PBODTs in 18 volunteers revealed considerable taste masking with bitterness below the threshold value. PBODTs also revealed rapid drug release (around 99%, 2 min) in the simulated gastric fluid. The mean PB plasma concentration-time profiles of PBODTs and that of the commercial tablets were comparable, with closely similar pattern. Bioequivalence assessment results demonstrated that PBODTs and the commercial tablets were bioequivalent. In conclusion, PBODTs are prepared successfully, with taste masking and rapid disintegration in the oral cavity.

Role of a novel pyridostigmine bromide-phospholipid nanocomplex in improving oral bioavailability.

A novel pyridostigmine bromide (PB)-phospholipid nanocomplex (PBPLC) was prepared to increase the bioavailability of PB. A central composite design approach was employed for process optimization. The physicochemical properties of PBPLC were investigated by means of differential scanning calorimetry, ultraviolet spectroscopy, Fourier transformed infrared spectroscopy and the n-octano/water partition coefficient. The intestinal permeability of PBPLC was observed via a single pass intestinal perfusion in rats. After oral administration of PBPLC, the concentrations of PB at predetermined time points were determined by HPLC, and the pharmacokinetic parameters were computed by DAS 2.1.1 software. Multiple linear regression analysis for process optimization revealed that the optimal PBPLC was obtained when the values of X(1), X(2), and X(3) were 8, 40°C, and 4 mg/mL, respectively. The average particle size and zeta potential of PBPLC with the optimized formulation were 204.60 nm and -25.12 mV, respectively. Non-covalent interactions between PB and phospholipids were found in the PBPLC. The n-octanol/water partition coefficient of PBPLC was substantially increased. PBPLC had better intestinal permeability in comparison with free PB. Mean plasma drug concentration-time curves of PBPLC and free PB after oral administration were both in accordance with the two-compartment open model. The values of pharmacokinetic parameters of PBPLC and free PB were the peak time (T(max)) 2 h vs 2 h, the maximum concentration (C(max)) 22.79 µg/mL vs 6.00 µg/mL, and the value of the area under the concentration vs time curve (AUC(0-∞)) 7128.21 µg·min/mL vs 1772.36 µg·min/mL, respectively. In conclusion, compared with free PB, PBPLC remarkably improves the oral bioavailability of PB, which is likely due to its higher lipophilicity and permeability.

[Pharmacokinetics of mestinon-phospholipid complex in rats].

OBJECTIVE: To determine the pharmacokinetics characteristics of mestinon-phospholipid complex (PBPLC) in rats. METHODS: This study adopted a single-dose, randomized, open-label, two-period crossover trial design. Twelve healthy rats were randomly divided into two groups. One group was orally administered with mestinon-phospholipid complex, and the other group was orally administered with reference mestinon solution (1.5 mg/kg of mestinon). The plasma concentrations of the drugs in ophthalmic vein bloods were determined using HPLC. The pharmacokinetic parameters were calculated with the aid of DAS2.1.1 software. RESULTS: Pharmacokinetic parameters of mestinon-phospholipid complex were Tmax 2 h, Cmax 22.79 microg x min/mL and AUC(0-infinity) 7128.21 microg x min/mL, which were different from those of free mestinon--Tmax, 2 h, Cmax 6.00 microg/mL and AUC(0-infinity) 1772.36 microg x min/mL. The relative bioavailability of mestinon-phospholipid complex was 410.98% of free mestinon. CONCLUSION: The oral bioavailability of mestinon increases remarkably when administered as mestinon-phospholipid complex.

[Bioequivalence of pyridostigmine bromide dispersible tablets in rabbits].

OBJECTIVE: To compare the pharmacokinetic parameters of pyridostigmine bromide dispersible tablets and common tablets in rabbits. METHODS: Twelve rabbits were given an oral dose (60 mg) of pyridostigmine bromide dispersible tablets or common tablets in a randomized crossover study. The plasma concentration of pyridostigmine bromide was determined by reversed-phase ion pair chromatography. The pharmacokinetic parameters were calculated using DAS2.1.1 software. RESULTS: The pharmacokinetic parameters showed no significant differences in rabbit plasma between pyridostigmine bromide dispersible tablets and common tablets. The two tablets had a C(max) of 1.83∓0.08 mg·L(-1) and 1.68∓0.03 mg·L(-1), tmax of 2.33∓0.41 h and 2.58∓0.20 h, AUC(0-24) of 15.50∓0.62 mg·h·L(-1) and 15.14∓0.30 mg·h·L(-1), AUC(0-∞) of 15.82∓0.70 mg·h·L(-1) and 15.57∓0.32 mg·h·L(-1), respectively. The relative bioavailability F(0-24) was 102.38% and F(0-∞) was 101.61% for the dispersible tablets. CONCLUSION: The two tablets are bioequivalent in rabbits.

Retrospective population pharmacokinetic/pharmacodynamic analysis of pyridostigmine, a cholinesterase inhibitor, in Chinese males.

OBJECTIVES: We have characterised the population pharmacokinetics-pharmacodynamics of pyridostigmine given as pyridostigmine bromide. METHODS: Over three days 50 healthy Chinese male subjects each received seven doses of 30 mg pyridostigmine bromide orally (3 x 10 mg every 8 h). Plasma concentrations of pyridostigmine and red blood cell acetylcholinesterase (AChE) activity were determined at various times within the eight hours after the first and the seventh doses. The resulting pharmacokinetic data were fitted to a single compartment open model with first-order absorption and elimination. The pharmacodynamics were modelled using an inhibitory E(max) model. The potential influence of demographic and biological covariates on the model parameters was investigated. Nonlinear mixed effects modelling was performed using NONMEM. KEY FINDINGS: The apparent clearance and volume of distribution as well as absorption rate constant of plasma pyridostigmine were estimated to be 136 l/h, 130 l and 0.68 1/h, respectively. The maximum red blood cell AChE activity decrease (E(max)) and plasma pyridostigmine concentration producing 50% of this reduction (EC50) were estimated to be 9.32 AChE units per gram haemoglobin and 51.9 ng/ml, respectively. None of the tested covariates were found to be correlated with any of the model parameters. Dosing simulations suggested that 30 mg repeated every six hours might be needed to achieve steady-state trough percentage inhibition above the recommended 10% in healthy Chinese males. CONCLUSIONS: The pharmacokinetics and the effects of pyridostigmine on red blood cell AChE activity were described using a mixed effects model. For Chinese males, the dosing interval may have been shorter than that recommended for the Caucasian population. Additional studies are needed to confirm these findings.

Formulation design of an HPMC-based sustained release tablet for pyridostigmine bromide as a highly hygroscopic model drug and its in vivo/in vitro dissolution properties.

Pyridostigmine bromide (PB), a highly hygroscopic drug was selected as the model drug. A sustained-release (SR) tablet prepared by direct compression of wet-extruded and spheronized core pellets with HPMC excipients and exhibited a zero-order sustained release (SR) profile. The 2(3) full factorial design was utilized to search an optimal SR tablet formulation. This optimal formulation was followed zero-order mechanism and had specific release rate at different time intervals (released % of 1, 6, and 12 hr were 15.84, 58.56, and 93.10%). The results of moisture absorption by Karl Fischer meter showed the optimum SR tablet could improve the hygroscopic defect of the pure drug (PB). In the in vivo study, the results of the bioavailability data showed the T(max) was prolonged (from 0.65 +/- 0.082 hr to 4.83 +/- 1.60 hr) and AUC(0-t) (from 734.88 +/- 230.68 ng/ml.hr to 1153.34 +/- 488.08 ng/ml.hr) and was increased respectively for optimum PB-SR tablets when compared with commercial immediate release (IR) tablets. Furthermore, the percentages of in vitro dissolution and in vivo absorption in the rabbits have good correlation. We believe that PB-SR tablets designed in our study would improve defects of PB, decrease the frequency of administration and enhance the retention period of drug efficacy in vivo for personnel exposed to contamination situations in war or terrorist attacks in the future.

Formulation design of a highly hygroscopic drug (pyridostigmine bromide) for its hygroscopic character improvement and investigation of in vitro/in vivo dissolution properties.

Pyridostigmine bromide (PB) sustained-release (SR) pellets were developed by extrusion-spheronization and fluid-bed methods using Taguchi experimental and 2(3) full factorial design. In vitro studies, the 2(3) full factorial design was utilized to search for the optimal SR pellets with specific release rate at different time intervals (release percent of 2, 6, 12, and 24 hr were 6.24, 33.48, 75.18, and 95.26%, respectively) which followed a zero-order mechanism (n=0.93). The results of moisture absorption by Karl Fischer has shown the optimum SR pellets at 25 degrees C/60% RH, 30 degrees C/65% RH, and 40 degrees C/75% RH chambers from 1 hr-4 weeks, attributing that the moisture absorption was not significantly increased. In the in vivo study, the results of the bioavailability data showed the Tmax (from 0.65+/-0.082 hr-4.82+/-2.12 hr) and AUC0-30 hr (from 734.88+/-230.68 ng/mL.hr-1454.86+/-319.28 ng/mL.hr) were prolonged and increased, as well as Cmax (from 251.87+/-27.51 ng/mL-115.08+/-14.87 ng/mL) was decreased for optimum SR-PB pellets when compared with commercial immediate-release (IR) tablets. Furthermore, a good linear regression relationship (r=0.9943) was observed between the fraction dissolution and fraction absorption for the optimum SR pellets. In this study, the formulation design not only improved the hygroscopic character of PB but also achieved the SR effect.

Extrapolating from animal studies to the efficacy in humans of a pretreatment combination against organophosphate poisoning.

The extrapolation from animal data to therapeutic effects in humans, a basic pharmacological issue, is especially critical in studies aimed to estimate the protective efficacy of drugs against nerve agent poisoning. Such efficacy can only be predicted by extrapolation of data from animal studies to humans. In pretreatment therapy against nerve agents, careful dose determination is even more crucial than in antidotal therapy, since excessive doses may lead to adverse effects or performance decrements. The common method of comparing dose per body weight, still used in some studies, may lead to erroneous extrapolation. A different approach is based on the comparison of plasma concentrations at steady state required to obtain a given pharmacodynamic endpoint. In the present study, this approach was applied to predict the prophylactic efficacy of the anticholinergic drug caramiphen in combination with pyridostigmine in man based on animal data. In two species of large animals, dogs and monkeys, similar plasma concentrations of caramiphen (in the range of 60-100 ng/ml) conferred adequate protection against exposure to a lethal-dose of sarin (1.6-1.8 LD(50)). Pharmacokinetic studies at steady state were required to achieve the correlation between caramiphen plasma concentrations and therapeutic effects. Evaluation of total plasma clearance values was instrumental in establishing desirable plasma concentrations and minimizing the number of animals used in the study. Previous data in the literature for plasma levels of caramiphen that do not lead to overt side effects in humans (70-100 ng/ml) enabled extrapolation to expected human protection. The method can be applied to other drugs and other clinical situations, in which human studies are impossible due to ethical considerations. When similar dose response curves are obtained in at least two animal models, the extrapolation to expected therapeutic effects in humans might be considered more reliable.

Pharmacokinetics of pyridostigmine in a child with postural tachycardia syndrome.

Pyridostigmine has been proposed for the treatment of postural orthostatic tachycardia syndrome in adults at a dose of 60 mg twice daily, but no dosing recommendation exists for children. With the approval of our local ethics board, we tested the pharmacokinetics of pyridostigmine in 6 children with myasthenia and a pediatric index patient with severe postural orthostatic tachycardia syndrome whose condition failed all conventional therapy and who had developed significant postural hypertension. Pyridostigmine was quantified by using a validated, semiautomated, and specific high-performance liquid chromatography/tandem mass spectrometry assay in combination with online column-switching extraction and turbo electrospray ionization. The patient with postural orthostatic tachycardia syndrome showed a dose-dependent favorable response to oral pyridostigmine. Pharmacokinetic evaluation revealed a short half-life of 2.29 hours, similar to the 2.0 +/- 0.63 hours in the patients with myasthenia. The patient with postural orthostatic tachycardia syndrome has subsequently been treated at a dose of 45 mg in the morning, 30 mg at lunchtime, and 15 mg at bedtime; after 9 months, there has been persistent positive effect and without additional blood pressure medication. No major adverse effects occurred. Pyridostigmine has been a safe and effective treatment modality for this child with postural orthostatic tachycardia syndrome. The short half-life suggests that dosing 3 times per day is preferable.

Rapid determination of N,N-diethyl-m-toluamide and permethrin in human plasma by gas chromatography-mass spectrometry and pyridostigmine bromide by high-performance liquid chromatography.

A rapid and highly sensitive gas chromatography-mass spectrometry (GC-MS) method for simultaneous determination of N,N-diethyl-m-toluamide (DEET) and permethrin with (2)H(10)-phenanthrene (98 atom %) as an internal standard and a separate external standard high-performance liquid chromatography (HPLC) method for pyridostigmine bromide (PB) determination in human plasma were developed and validated. The GC-MS method for DEET and permethrin quantification utilizes a one-step extraction with tert-butylmethylether. The HPLC method for PB quantification involves a solid-phase extraction and UV detection. The range of the analytical method for DEET and permethrin was 1 ng/mL to 100 ng/mL and for PB was 5 ng/mL to 100 ng/mL. Recovery from plasma proved to be more than 80%. The intraday precision ranged from 1.3% to 8% for DEET, from 2.1% to 11.4% for permethrin, and from 3.0% to 4.8% for PB. The interday precision was 3% for DEET, ranged from 5% to 9% for permethrin, and from 5% to 9% for PB. The accuracy for the limit of quantification was 92% +/- 8% relative standard deviation (RSD) for DEET, 112% +/- 11% RSD for permethrin, and 109% +/- 5% RSD for PB. All 3 compounds were stable in human plasma at -80 degrees C for at least 12 months and after 2 freeze-thaw cycles with RSD values ranging from 7.1% (DEET, 80 ng/mL) to 8.1% (DEET, 8 ng/mL), from 2.3% (permethrin, 80 ng/mL) to 11.6 % (permethrin, 8 ng/mL), and from 0.2% (PB, 80 ng/mL) to 3.6% (PB, 8 ng/mL). Both methods were successfully applied to pharmacokinetic/ pharmacodynamic studies of combined exposure of DEET (skin application), permethrin (treated uniforms), and PB (30 mg orally three times/day for four doses) in healthy volunteers (n = 81).

Oral administration of pyridostigmine bromide and huperzine A protects human whole blood cholinesterases from ex vivo exposure to soman.

Cholinesterases (ChEs) are classified as acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) according to their substrate specificity and sensitivity to selected inhibitors. The activities of AChE in red blood cells (RBC-AChE) and BChE in serum can be used as potential biomarkers of suppressed and/or heightened activity in the central and peripheral nervous systems. Exposure to organophosphate (OP) chemical warfare agents (CWAs), pesticides, anesthetics, and a variety of drugs such as cocaine, as well as some neurodegenerative and liver disease states, selectively reduces AChE or BChE activity. In humans, the toxicity of pesticides is well documented. Therefore, blood cholinesterase activity can be exploited as a tool for confirming exposure to these agents and possible treatments. Current assays for measurement of RBC-AChE and serum BChE require several labor-intensive processing steps, suffer from wide statistical variation, and there is no inter-laboratory conversion between methods. These methods, which determine only the serum BChE or RBC-AChE but not both, include the Ellman, radiometric, and deltapH (modified Michel) methods. In contrast, the Walter Reed Army Institute of Research Whole Blood (WRAIR WB, US Patent #6,746,850) cholinesterase assay rapidly determines the activity of both AChE and BChE in unprocessed (uncentrifuged) whole blood, uses a minimally invasive blood sampling technique (e.g., blood from a finger prick), and is semi-automated for high-throughput using the Biomek 2000 robotic system. To date, the WRAIR whole blood assay was used to measure AChE and BChE activities in human blood from volunteers in FDA clinical trials. In the first FDA study, 24 human subjects were given either 30 mg PB orally (n = 19) or placebo (n = 5). Blood samples were obtained pre-dosing and 2.5, 5, 8, and 24 h post-dosing. The samples were analyzed for AChE and BChE activity using the WRAIR WB robotic system, and for PB concentration by HPLC. We found that maximal inhibition of AChE (26.2%) and concentration of PB (17.1 ng/mL) occurred at 2.5 h post-PB dosing. AChE activity returned to almost 100% of pre-dose values by 6 h. A dose-dependent linear correlation was found between the amount of PB measured in the blood and the inhibition of AChE. Following soman (GD) exposure, recovered AChE activity was similar to levels that were reversibly protected by the PB administration. Therefore, the WRAIR ChE WB data clearly supports the conclusion that PB is an effective pre-treatment drug for nerve agent exposure (GD). In the second FDA human study for the treatment of Alzheimer's disease, the WRAIR ChE WB assay was used to determine the RBC-AChE and serum BChE profile of healthy elderly volunteers receiving Huperzine A. Huperzine A is a plant-derived reversible and selective AChE inhibitor compared to BChE, and is a more potent inhibitor of AChE than PB. Huperzine A is available as a nutraceutical, a natural supplement reported to improve memory, and has a variety of neuroprotective effects. Individuals received an increasing dose regimen of huperzine A (final dose 200 microg after 4 weeks), which produced more than 50% inhibition of RBC-AChE. Huperzine A was well tolerated by these patients at doses that sequestered more RBC-AChE than PB, and thus warrants further study as a prophylaxis for OP poisoning in addition to Alzheimer's therapy. Due to the documented use of OPs by terrorists and in warfare around the globe, Federal, State, and local authorities need a reliable, fast, inexpensive, and standard method for confirming such an assault in order to initiate appropriate containment, decontamination, and treatment measures. This assay is ideal for prescreening military personnel for atypical ChE activities that would preclude their deployment to areas of potential CWA exposure. The WRAIR WB ChE assay will fulfill the requirement for rapid and reliable monitoring of such exposure in military and civilian populations.

Facile synthesis and initial PET imaging of novel potential heart acetylcholinesterase imaging agents [11C]pyridostigmine and its analogs.

A series of 11C-labeled analogs of the acetylcholinesterase (AChE) inhibitor pyridostigmine have been synthesized for evaluation as new potential positron emission tomography (PET) imaging agents for heart AChE. The appropriate precursors for radiolabeling were slightly modified from commercial reagents. The new tracers [11C]pyridostigmine (1), [11C]para-pyridostigmine (2) and [11C]ortho-pyridostigmine (3) were prepared by N-[11C]methylation of the precursors using [11C]methyl triflate. Pure target compounds were isolated by a solid-phase extraction (SPE) purification procedure with 60-85% radiochemical yields (decay corrected to end of bombardment), and a synthesis time of 10-15 min. The initial PET dynamic studies of compounds (1-3) in rat heart showed rapid heart uptake and blood pool clearance to give high quality heart images. These results suggest the new tracers delineate the heart very clearly and could be potential heart AChE imaging agents.

Percutaneous absorption of topical N,N-diethyl-m-toluamide (DEET): effects of exposure variables and coadministered toxicants.

Exposure to N,N-diethyl-m-toluamide (DEET) commonly occurs in the general population and has been implicated as a contributory factor to the Gulf War Illness. The focus of the present studies was to determine the effect of coexposure factors, potentially encountered in a military environment, that could modulate transdermal flux of topically applied DEET. Factors investigated were vehicle, dose, coexposure to permethrin, low-level sulfur mustard, occlusion, and simultaneous systemic exposure to pyridostigmine bromide and the nerve agent stimulant diisopropylfluorophosphate (DFP). Studies were conducted using the isolated perfused porcine skin flap (IPPSF), with a few mechanistically oriented studies conducted using in vitro porcine skin and silastic membrane diffusion cells. DEET was quantitated using high-performance liquid chromatography. The vehicle-control transdermal DEET flux in the IPPSF was approximately 2 micrograms/cm2/h for both 7.5 and 75% DEET concentrations, a value similar to that reported in humans. DEET absorption was enhanced by coinfusion of pyridostigmine bromide and DFP, by the presence of sulfur mustard, or by dosing under complete occlusion. The greatest increase in baseline flux was fivefold. In vitro diffusion cell studies indicated that silastic membranes had two orders of magnitude greater permeability than porcine skin, and showed vehicle effects on flux that were not detected in the IPPSF. These results suggest that coexposure to a number of chemicals that potentially could be encountered in a military environment may modulate the percutaneous absorption of topically applied DEET beyond that seen for normal vehicles at typically applied concentrations.

Pyridostigmine bromide modulates topical irritant-induced cytokine release from human epidermal keratinocytes and isolated perfused porcine skin.

Gulf War personnel were given pyridostigmine bromide (PB) as a prophylactic treatment against organophosphate nerve agent exposure, and were exposed to the insecticide permethrin and the insect repellent N,N-diethyl-m-toluamide (DEET). The purpose of this study was to assess the effects of PB to modulate release of inflammatory biomarkers after topical chemical exposure to chemical mixtures containing permethrin and DEET applied in ethanol or water vehicles. Treatments were topically applied to isolated perfused porcine skin flaps (IPPSFs). Concentrations of interleukin-8 (IL-8), tumor necrosis factor-alpha (TNF-alpha) and prostaglandin E(2) (PGE(2)) were assayed in perfusate to probe for potential inflammatory effects after complex mixture application. IPPSFs (n=4/treatment) were topically dosed with mixtures of permethrin, DEET, and permethrin/DEET, in ethanol. Each treatment was repeated with perfusate spiked with 50 ng/ml of PB. Perfusate was also spiked with 30 ng/ml diisopropylfluorophosphate to simulate low level organophosphate nerve agent exposure. Timed IPPSF venous effluent samples (0.5,1,2,4, and 8 h) were assayed by ELISA for IL-8 and TNF-alpha and by EIA for PGE(2). Overall, PB infusion caused a decrease or IL-8 and PGE(2) release. Effects on TNF-alpha were vehicle dependent. To probe the potential mechanism of this PB effect, human epidermal keratinocyte HEK cell cultures were exposed to permethrin DEET permethrin/DEET, with and without PB in DMSO. IL-8 was assayed at 1, 2, 4, 8, 12 and 24 h. PB suppressed IL-8 in permethrin and ethanol treatment from 4 to 24 h confirming the IPPSF results. In conclusion, these studies suggest that systemic exposure to PB suppressed IL-8 release at multiple time points in two skin model systems. This interaction merits further study.

Protein binding of isofluorophate in vivo after coexposure to multiple chemicals.

Full toxicologic profiles of chemical mixtures, including dose-response extrapolations to realistic exposures, is a prohibitive analytical problem, even for a restricted class of chemicals. We present an approach to probing in vivo interactions of pesticide mixtures at relevant low doses using a monitor compound to report the response of biochemical pathways shared by mixture components. We use accelerator mass spectrometry (AMS) to quantify [14C]-diisopropylfluorophosphate as a tracer at attomole levels with 1-5% precision after coexposures to parathion (PTN), permethrin (PER), and pyridostigmine bromide separately and in conjunction. Pyridostigmine shows an overall protective effect against tracer binding in plasma, red blood cells, muscle, and brain that is not explained as competitive protein binding. PTN and PER induce a significant 25-30% increase in the amount of tracer reaching the brain with or without pyridostigmine. The sensitivity of AMS for isotope-labeled tracer compounds can be used to probe the physiologic responses of specific biochemical pathways to multiple compound exposures.

Acute effects of an insect repellent, N,N-diethyl-m-toluamide, on cholinesterase inhibition induced by pyridostigmine bromide in rats.

Acute lethal interactions have been previously described between a cholinesterase (ChE) inhibitor, pyridostigmine bromide (PB), and the insect repellent, N,N-diethyl-m-toluamide (DEET). The mechanism of toxic interaction between these agents is unknown. Alterations in membrane permeability caused by DEET could facilitate or enhance absorption, or alter the distribution of peripherally restricted PB, causing increased inhibition of ChE at a given dose. Studies were conducted to investigate PB-induced ChE inhibition in the presence of DEET. Rats received ip injections of PB (1, 2, or 3 mg/kg), DEET (200 mg/kg), or PB + DEET at doses that potentiated acute lethality. ChE activity was measured in heart, diaphragm, blood, whole brain, or specific brain areas using a modified spectrophotometric assay. DEET did not alter PB-induced inhibition of ChE activity in rat diaphragm, heart, or blood. Administration of DEET alone had no effect on ChE activity. PB alone did not inhibit ChE in whole brain, but PB (3 mg/kg) + DEET (200 mg/kg) caused significant inhibition of whole brain ChE activity to approximately 60% of controls. In specific brain areas, (cortex, cerebellum, medulla, hypothalamus, hippocampus, midbrain, and striatum) PB alone did not inhibit ChE activity. PB (3 mg/kg) + DEET (200 mg/kg) reduced ChE activity to approximately 65-75% of controls in each brain area, but those results were not statistically significant. In conclusion, DEET did not alter PB-induced inhibition of ChE activity in the periphery. While DEET may have facilitated the access of PB into the CNS at high doses, it is doubtful that the resulting minor reduction in ChE activity would have resulted in death. It is unlikely that the lethal interaction between PB and DEET is mediated through a cholinergic effect resulting from increased inhibition of ChE.