Exploratory studies with NX-13: oral toxicity and pharmacokinetics in rodents of an orally active, gut-restricted first-in-class therapeutic for IBD that targets NLRX1.

Nucleotide-binding oligomerization domain, leucine rich repeat containing X1 (NLRX1) is an emerging therapeutic target for a spectrum of human diseases. NX-13 is a small molecule therapeutic designed to target and activate NLRX1 to induce immunometabolic changes resulting in lower inflammation and therapeutic responses in inflammatory bowel disease (IBD). This study investigates the safety of NX-13 in a seven-day, repeat-dose general toxicity study in male and female Sprague Dawley rats at oral doses of 500 and 1000 mg/kg. Weights, clinical signs, functional observational battery, clinical pathology and histopathology were used for evaluation. Daily oral dosing of NX-13 up to 1000 mg/kg did not result in any changes in weight, abnormal clinical signs or behavior. No significant differences were observed between treated and control rats in hematology or blood biochemistry. Histopathological evaluation of 12 tissues demonstrated no differences between controls and treated rats. There were no changes in weights of brain, heart, kidney, liver or spleen. Pharmacokinetic analysis of a single oral dose of NX-13 at 10 mg/kg in Sprague Dawley rats provided a maximum plasma concentration of 57 ng/mL at 0.5 h post-dose. Analysis of colon tissue after oral dosing with 1 and 10 mg/kg indicated high peak concentrations (10 and 100 µg/g, respectively) that scale in a dose-proportional manner. These experiments suggest that NX-13 is safe and well-tolerated in rats given oral doses as high as 1000 mg/kg with a favorable gastrointestinal localized pharmacokinetic profile, confirming NX-13 as a promising therapeutic for Crohn's disease and ulcerative colitis.

Intracellular potassium depletion enhances apoptosis induced by staurosporine in cultured trigeminal satellite glial cells.

PURPOSE: Satellite glial cells (SGC) surrounding neurons in sensory ganglia can buffer extracellular potassium, regulating the excitability of injured neurons and possibly influencing a shift from acute to neuropathic pain. SGC apoptosis may be a key component in this process. This work evaluated induction or enhancement of apoptosis in cultured trigeminal SGC following changes in intracellular potassium [K]ic. MATERIALS AND METHODS: We developed SGC primary cultures from rat trigeminal ganglia (TG). Purity of our cultures was confirmed using immunofluorescence and western blot analysis for the presence of the specific marker of SGC, glutamine synthetase (GS). SGC [K]ic was depleted using hypo-osmotic shock and 4 mM bumetanide plus 10 mM ouabain. [K]ic was measured using the K+ fluorescent indicator potassium benzofuran isophthalate (PBFI-AM). RESULTS: SGC tested positive for GS and hypo-osmotic shock induced a significant decrease in [K]ic at every evaluated time. Cells were then incubated for 5 h with either 2 mM staurosporine (STS) or 20 ng/ml of TNF-α and evaluated for early apoptosis and late apoptosis/necrosis by flow cytometry using annexin V and propidium iodide. A significant increase in early apoptosis, from 16 to 38%, was detected in SGC with depleted [K]ic after incubation with STS. In contrast, TNF-α did not increase early apoptosis in normal or [K]ic depleted SGC. CONCLUSION: Hypo-osmotic shock induced a decrease in intracellular potassium in cultured trigeminal SGC and this enhanced apoptosis induced by STS that is associated with the mitochondrial pathway. These results suggest that K+ dysregulation may underlie apoptosis in trigeminal SGC.

Effect of Adjuvant Release Rate on the Immunogenicity of Nanoparticle-Based Vaccines: A Case Study with a Nanoparticle-Based Nicotine Vaccine.

Adjuvants are a critical component for vaccines, especially for a poorly immunogenic antigen, such as nicotine. However, the impact of adjuvant release rate from a vaccine formulation on its immunogenicity has not been well illustrated. In this study, we fabricated a series of hybrid-nanoparticle-based nicotine vaccines to study the impact of adjuvant release rate on their immunological efficacy. It was found that the nanovaccine with a medium or slow adjuvant release rate induced a significantly higher anti-nicotine antibody titer than that with a fast release rate. Furthermore, the medium and slow adjuvant release rates resulted in a significantly lower brain nicotine concentration than the fast release rate after nicotine challenge. All findings suggest that adjuvant release rate affects the immunological efficacy of nanoparticle-based nicotine vaccines, providing a potential strategy to rationally designing vaccine formulations against psychoactive drugs or even other antigens. The hybrid-nanoparticle-based nicotine vaccine with an optimized adjuvant release rate can be a promising next-generation immunotherapeutic candidate against nicotine.

Alum as an adjuvant for nanoparticle based vaccines: A case study with a hybrid nanoparticle-based nicotine vaccine.

The treatment efficacy of a nicotine vaccine largely relies on its ability to induce high titers of nicotine-specific antibodies. Due to its strong immune-potentiating effects, aluminum salt (Alum) has been commonly used as an adjuvant in various nicotine vaccine formulations. In this study, we attempted to improve the immunological performance of a hybrid nanoparticle-based nicotine vaccine (NanoNicVac) by co-administering it with Alum. It was found that Alum severely restricted the release of NanoNicVac at the site of injection. Moreover, Alum damaged the hybrid structure of the vaccine. In the animal trial, mice immunized with NanoNicVac alone achieved an anti-nicotine IgG titer of 3.5 ±â€¯0.2 × 104 after three injections. Unexpectedly, Alum with quantities of 125, 250, 500, and 1000 µg did not enhance the immunogenicity of NanoNicVac. In addition, Alum did not improve the ability of the vaccine to reduce the entry of nicotine into the brain.

Nonclinical Toxicology and Toxicokinetic Profile of an Oral Lanthionine Synthetase C-Like 2 (LANCL2) Agonist, BT-11.

BT-11 is an orally active, gut-restricted investigational therapeutic targeting the lanthionine synthetase C-like 2 pathway with lead indications in ulcerative colitis (UC) and Crohn disease (CD), 2 manifestations of inflammatory bowel disease (IBD). In 5 mouse models of IBD, BT-11 is effective at oral doses of 8 mg/kg. BT-11 was also efficacious at nanomolar concentrations in primary human samples from patients with UC and CD. BT-11 was tested under Good Laboratory Practice conditions in 90-day repeat-dose general toxicity studies in rats and dogs, toxicokinetics, respiratory, cardiovascular and central nervous system safety pharmacology, and genotoxicity studies. Oral BT-11 did not cause any clinical signs of toxicity, biochemical or hematological changes, or macroscopic or microscopic changes to organs in 90-day repeat-dose toxicity studies in rats and dogs at doses up to 1,000 mg/kg/d. Oral BT-11 resulted in low systemic exposure in both rats (area under the curve exposure from t = 0 to t = 8 hours [AUC0-8] of 216 h × ng/mL) and dogs (650 h × ng/mL) and rapid clearance with an average half-life of 3 hours. BT-11 did not induce changes in respiratory function, electrocardiogram parameters, or behavior with single oral doses of 1,000 mg/kg/d. There was no evidence of mutagenic or genotoxic potential for BT-11 up to tested limit doses using an Ames test, chromosomal aberration assay in human peripheral blood lymphocytes, or micronucleus assay in rats. Therefore, nonclinical studies show BT-11 to be a safe and well-tolerated oral therapeutic with potential as a potent immunometabolic therapy for UC and CD with no-observed adverse effect level >1,000 mg/kg in in vivo studies.

Synthesis and Evaluation of Doxorubicin-Loaded Gold Nanoparticles for Tumor-Targeted Drug Delivery.

Doxorubicin is an effective and widely used cancer chemotherapeutic agent, but its application is greatly compromised by its cumulative dose-dependent side effect of cardiotoxicity. A gold nanoparticle-based drug delivery system has been designed to overcome this limitation. Five novel thiolated doxorubicin analogs were synthesized and their biological activities evaluated. Two of these analogs and PEG stabilizing ligands were then conjugated to gold nanoparticles, and the resulting Au-Dox constructs were evaluated. The results show that release of native drug can be achieved by the action of reducing agents such as glutathione or under acidic conditions, but reductive drug release gave the cleanest drug release. Gold nanoparticles (Au-Dox) were prepared with different loadings of PEG and doxorubicin, and one formulation was evaluated for mammalian stability and toxicity. Plasma levels of doxorubicin in mice treated with Au-Dox were significantly lower than in mice treated with the same amount of doxorubicin, indicating that the construct is stable under physiological conditions. Treatment of mice with Au-Dox gave no histopathologically observable differences from mice treated with saline, while mice treated with an equivalent dose of doxorubicin showed significant histopathologically observable lesions.

Hybrid nanoparticle-based nicotine nanovaccines: Boosting the immunological efficacy by conjugation of potent carrier proteins.

A series of hybrid nanoparticle-based nicotine nanovaccines (NanoNicVac) were engineered in this work by conjugating potent carrier protein candidates (Keyhole limpet hemocyanin (KLH) multimer, KLH subunit, cross-reactive material 197 (CRM197), or tetanus toxoid (TT)) for enhanced immunological efficacy. NanoNicVac with CRM197 or TT were processed by dendritic cells more efficiently than that with KLH multimer or subunit. NanoNicVac carrying CRM197 or TT exhibited a significantly higher immunogenicity against nicotine and a considerably lower immunogenicity against carrier proteins than NanoNicVac carrying KLH multimer or subunit in mice. The in vivo results revealed that NanoNicVac with CRM197 or TT resulted in lower levels of nicotine in the brain of mice after nicotine challenge. All findings suggest that an enhanced immunological efficacy of NanoNicVac can be achieved by using CRM197 or TT instead of KLH or KLH subunit as carrier proteins, making NanoNicVac a promising next-generation immunotherapeutic candidate against nicotine addiction.

Exploratory Studies With BT-11: A Proposed Orally Active Therapeutic for Crohn's Disease.

Lanthionine synthetase cyclase-like receptor 2 (LANCL2) is a novel therapeutic target for Crohn's disease (CD). BT-11 is a small molecule that binds LANCL2, is orally active, and has demonstrated therapeutic efficacy in 3 validated mouse models of colitis at doses as low as 8 mg/kg/d. Exploratory experiments evaluated BT-11 in male Harlan Sprague Dawley rats with a single oral dose of 500 mg/kg and 80 mg/kg/d for 14 days (n = 10 rats dosed/group). Treated and control rats were observed for behavioral detriments, and blood and tissues were collected for clinical pathology and histopathological examination. A functional observational battery demonstrated no differences between treated and control groups over multiple times of observation for quantal, categorical, and continuous end points, including posture, in cage activity, approach, response to touch, weight, grip strength, body temperature, and time on a rotarod. Histopathological examination of the brain, kidney, liver, adrenal gland, testes, stomach, small and large intestines, duodenum, pancreas, heart, lungs, spleen, thymus, and rib found no significant differences between the groups. Plasma enzymes associated with liver function were transiently elevated 2 to 4 days after the 500 mg/kg single dose but returned to normal values by 8 days and were not observed at any time in rats given 80 mg/kg/d for 14 days. One hour after oral administration of a single dose of 80 mg/kg, BT-11 had a maximal concentration of 21 ng/mL; the half-life was 3 hours. These experimental results demonstrated that BT-11 is well tolerated in rats, and, with further testing, may hold promise as an orally active therapeutic for CD.

In vitro controlled release of antigen in dendritic cells using pH-sensitive liposome-polymeric hybrid nanoparticles.

A hybrid nanoparticle (NP) consisting of a pH sensitive lipid shell and a poly(lactic-co-glycolic) acid (PLGA) core was constructed. This hybrid NP has a mean size of 120.1 ± 8.8 nm and positively charged surface (zeta potential of 14.2 ± 1.4 mV). The lipid shell of the hybrid NP was quickly disintegrated in buffer with a pH of 5.5, which resembles the acidic environment of endosomes in dendritic cell (DC). Less than 20% of the antigen enclosed in pH-sensitive hybrid NP was released into human serum at physiological pH within 24 h, but more than 40% of the enclosed antigen was released within 8 h after pH was adjusted to 5.5. Fast uptake of the pH sensitive hybrid NP by DC was also observed. It was found that pH sensitive hybrid NP displayed faster degradation and antigen release compared to regular hybrid NPs after uptake by DC.

Cefazolin concentration in surgically created wounds treated with negative pressure wound therapy compared to surgically created wounds treated with nonadherent wound dressings.

OBJECTIVE: To compare cefazolin concentrations in biopsied tissue samples collected from surgically created wounds treated with negative pressure wound therapy to those collected from surgically created wounds treated with nonadherent dressings. STUDY DESIGN: Prospective, controlled, experimental study. ANIMALS: Adult female spayed Beagles (n = 12). METHODS: Full thickness cutaneous wounds were created on each antebrachium (n = 24). Immediately after surgery, cefazolin (22 mg/kg intravenously [IV]) was administered to each dog and continued every 8 hours during the study. The right wound was randomly assigned to group I or group II whereas the wound on the contralateral antebrachium was assigned to the other group. Group I wounds were treated with negative pressure wound therapy (NPWT) and group II wounds were treated with nonadherent dressings for 3 days. Dressings were changed and tissue biopsies obtained from wound beds at 24 hours intervals for both groups. Cefazolin wound tissue and plasma concentrations were measured by liquid chromatography mass spectrometry (LC-MS/MS). Blood samples for measuring plasma cefazolin concentrations were collected before biopsy sampling. At the time of surgery and at each subsequent bandage change, wound beds were swabbed and submitted for aerobic and anaerobic culture. RESULTS: After initiating cefazolin treatment, wound tissue antibiotic concentrations between treatment groups were not significantly different at any sampling time. Similarly, after initiating cefazolin treatment, plasma cefazolin concentrations were not significantly different at any sampling time for individual dogs. CONCLUSIONS: Using a canine experimental model, NPWT treatment of surgically created wounds does not statistically impact cefazolin tissue concentrations when compared with conventional nonadherent bandage therapy.

Human blood markers of cholinergic neurotoxicity and neuropathy: A useful guide for laboratory applications.

Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are useful markers to assess the effects of exposure to anticholinesterase insecticides (Anti-AChE). In addition, lymphocyte neuropathy target esterase (LNTE) has been used as biomarker of neuropathic organophosphate compounds (OPs). Thus, this study evaluates the main types of circulating biomarkers related to the cholinergic system and to the neuropathy induced by OPs in standardized human samples. To achieve this objective, total protein of human plasma, erythrocytes and lymphocytes were first standardized, and then AChE, BChE and LNTE activities in human blood were evaluated in the presence of inhibitors. The acceptance criteria of the regulatory agency were respected with coefficients of regression of curves of 0.9972 for cholinesterase and 0.9956 for LNTE analyses. The wavelength established to perform cholinesterase assay was 450 nm and the time of incubation of the enzymes with inhibitors was 30 min. Differences were observed among the IC50 values regarding the in vitro inhibition of AChE, BChE and LNTE in the presence of OPs. In conclusion, the procedures demonstrated by the present work were simple, fast, inexpensive, sensitive, easy to be replicated and suitable to make conclusions about the neurotoxicity induced by Anti-AChE and neuropathic OPs.

Transient alterations of the blood-brain barrier tight junction and receptor potential channel gene expression by chlorpyrifos.

The blood-brain barrier (BBB) is formed by specialized endothelial cells lining capillaries in the central nervous system (CNS). We previously demonstrated that exposure to very low concentrations of the organophosphorus insecticide chlorpyrifos (CPF) decreased electrical resistance across the BBB in vitro, indicating a loss of BBB integrity. The present study examined the transient effects of CPF on expression of genes contributing to tight junctions of the BBB. Rat brain endothelial cells (RBE4) were co-cultured with rat astrocytes on membrane inserts to form an in vitro BBB. The RBE4 cells in the BBB were then exposed to CPF for 2, 4 and 12 h. Total RNA was extracted from RBE4 cells and quantitative real-time PCR (qRT-PCR) was used to quantify levels of gene expression of tight junction proteins claudin5, scaffold proteins zona occludens (ZO1) and transient receptor potential (canonical) channels (TRPC4). Gene expression decreased 2 h after exposure to CPF, especially TRPC4, but the effects were reversed 12 h later. CPF exposure for only 15 min caused less effect than longer exposures, with TRPC4 gene expression above the control at 4 h. These results suggest that altering gene expression for claudin5, TRPC4 and ZO1 by CPF may directly contribute to BBB disruption, and that the alteration is reversible upon removal of CPF.

Effect of chlorpyrifos on VEGF gene expression.

Chlorpyrifos (CPF; 0,0-diethyl 0-(3,5,6-trichloro-2-pyridinyl)-phosphorothioate), a cholinesterase inhibitor, compromised the integrity of the blood-brain barrier (BBB) when used at low concentrations during our previous experiments in vitro. To determine if BBB leakage would also occur in vivo, we used FITC-dextrans to evaluate BBB permeability in CPF-dosed mice. Results indicated BBB leakages that were evident at 2 h after treatment with 70 mg/kg CPF ip. Since vascular endothelial growth factor (VEGF), a potent vasopermeability factor, is a signaling protein that promotes the growth of new blood vessels, we investigated the possible involvement of VEGF in BBB disruption by CPF. We found that VEGF serum concentration was significantly increased at 24 h after CPF exposure. To further explore VEGF involving BBB disruption by CPF treatment, the receptor antagonist for VEGF (sFlt-1) was used for pretreatment before CPF exposure. After sFlt-1 pretreatment, gene expressions of the tight junction (TJ) proteins claudin5 and occludin were significantly downregulated at 1, 2, and 3 h, but returned to control levels at 24 h after CPF treatment. These results suggest that VEGF is involved in BBB disruption by CPF through BBB-TJs regulation.

Malathion/oxon and lead acetate increase gene expression and protein levels of transient receptor potential canonical channel subunits TRPC1 and TRPC4 in rat endothelial cells of the blood-brain barrier.

This study examined the effects of malathion and lead on transient receptor potential canonical channel TRPC1/TRPC4 channels in rat brain endothelial cells as a mechanism to explain previously noted blood-brain barrier (BBB) permeability induced by these compounds. Lead, malathion, malaoxon and combinations of these were assessed for protein levels and gene expression of TRPC1/C4 at 2, 4, 8, 16, and 24 hours after exposure. Changes in intracellular free calcium dynamics were also assessed. Compounds increased TRPC1 and TRPC4 protein levels as well as gene expression within 4 hours after exposure. Basal levels of intracellular free calcium were also elevated. Increases in gene and protein expression may be associated with an increase in the numbers of TRP channels, and the increases in intracellular calcium may be associated with activation of such channels. Therefore, upregulation and activation of the TRPC1/TRPC4 may be a mechanism by which these neurotoxicants affect BBB permeability.

Effects of chlorpyrifos on transient receptor potential channels.

The well-known toxicity of chlorpyrifos (CPF) occurs via inhibition of cholinesterase (ChE), but in recent years the detrimental effects of low-dose CPF exposure have been attributed to an unknown non-cholinergic mechanism of action. We previously showed that CPF can alter gene expression of transient receptor potential canonical (TRPC) channels in vitro. In this study, we analyzed the gene expression of TRPCs at various time points after CPF treatment in vivo. The results showed that TRPC1 mRNA expression in mouse brain was significantly reduced 2-8 h after CPF treatment, but the TRPC4 mRNA expression was not significantly changed. To investigate the possible involvement of Transforming Growth Factor-beta1 (TGF-ß1) in contributing to TRPCs gene alteration by CPF, we used TGF-beta receptor inhibitor (LY2109761) as a pretreatment prior to CPF treatment. The serum TGF-ß1 concentration was significantly increased 24 h after CPF treatment. After pretreatment with LY2109761, both TRPC1 and TRPC5 mRNAs were significantly downregulated 1 and 2 h after CPF treatment, but were significantly upregulated 3 and 24 h after CPF treatment. TRPC4 mRNA was also significantly downregulated at 1 h. These results suggest that interference with ion channels, a non-cholinergic mechanism of CPF, may contribute to the cellular neurotoxicity of CPF.

Formulation of Nanovaccines toward an Extended Immunity against Nicotine.

Nicotine vaccines have been investigated to assist with smoking cessation. Because smoking cessation is a long process, past nicotine vaccines required multiple injections to achieve long-term efficacy. It would be of great significance if extended efficacy can be achieved with fewer injections. Here, we report the assembly of lipid-polylactic acid (PLA) and lipid-poly(lactic-co-glycolic acid) (PLGA) hybrid nanoparticle (NP) based nicotine vaccines. Mice immunized with the lipid-PLGA vaccine produced higher titers of nicotine-specific antibodies than the lipid-PLA vaccine in short-term. However, the lipid-PLA vaccine was found to induce long-lasting antibodies. Three months after the immunization, only mice that received first two injections of the lipid-PLGA vaccine and a third injection of the lipid-PLA vaccine achieved a significantly lower brain nicotine concentration of 65.13 ± 20.59 ng/mg than 115.88 ± 37.62 ng/mg from the negative controls. The results indicate that not only the stability of the vaccines but also the combination of the vaccines impacted the long-term efficacy of the immunization. Lastly, both the body weight and the histopathology study suggest that the vaccines were safe to mice. These findings suggest that long-term immunity against nicotine can be realized by a rational administration of nanovaccines of different levels of stability.

Effects of polyhydroxyfullerenes on organophosphate-induced toxicity in mice.

Two polyhydroxyfullerenes, which decrease organophosphate (OP)-induced acetylcholinesterase (AChE) inhibition in vitro, were administered by the intraperitoneal (ip) route or applied topically at doses of 0.9-24 mg/kg to protect adult male mice from enzyme-inhibiting and behavioral effects indicative of OP toxicity resulting from exposure to 1.7 - 2 mg/kg diphosphorofluoridate (DFP) ip or 2.3 - 2.7 mg paraoxon topical. Dosing paradigms included OP-fullerene simultaneous administration by the ip route, and 20 min post-OP polyhydroxyfullerene treatment topically. Benefits of OP sequestration by the polyhydroxyfullerene were noted and were dependent on the OP compound as well as timing and route of the polyhydroxyfullerene treatment.

The Safety, Tolerability, and Pharmacokinetics Profile of BT-11, an Oral, Gut-Restricted Lanthionine Synthetase C-Like 2 Agonist Investigational New Drug for Inflammatory Bowel Disease: A Randomized, Double-Blind, Placebo-Controlled Phase I Clinical Trial.

BT-11 is a new oral, gut-restricted, first-in-class investigational drug for Crohn disease (CD) and ulcerative colitis (UC) that targets the lanthionine synthetase C-like 2 (LANCL2) pathway and immunometabolic mechanisms. Oral BT-11 was assessed for safety, tolerability, and pharmacokinetics (PK) in normal healthy volunteers (n = 70) in a randomized, double-blind, placebo-controlled trial. Subjects (n = 70) were randomly assigned to one of five single ascending dose cohorts (up to 100 mg/kg, p.o.) and three multiple ascending dose cohorts [up to 100 mg/kg daily (QD) for seven days, orally]. Safety and tolerability were assessed by adverse event (AE) reporting, vital signs, electrocardiogram, hematology, and clinical chemistry. BT-11 did not increase total or gastrointestinal AE rates, as compared with placebo, and no serious adverse events were observed. Oral BT-11 dosing does not result in any clinically significant findings by biochemistry, coagulation, electrocardiogram, hematology, or urinalysis as compared with placebo. Mean fecal concentrations of BT-11 increased linearly with increasing oral doses, with 2.39 mg/g at 7.7 mg/kg on day 7 of the multiple ascending dose (MAD). Analysis of plasma pharmacokinetics indicates that maximum systemic concentrations are approximately 1/6000th of observed concentrations in feces and the distal gastrointestinal tract. Fecal calprotectin levels were lower in BT-11 treated groups as compared to placebo. BT-11 significantly decreases interferon gamma positive (IFNγ+) and tumor necrosis factor alpha positive (TNFα+) cluster of differentiation 4 positive (CD4+) T cells and increases forkhead box P3 positive (FOXP3+) CD4+ T cells in colonic lamina propria mononuclear cells from patients with CD and patients with UC at concentrations of 0.01 µM when treated ex vivo. BT-11 treatment is well-tolerated with no dose-limiting toxicities up to daily oral doses of 100 mg/kg (16 tablets); whereas the efficacious dose is a single tablet (8 mg/kg). Phase II studies in CD and UC patients are ongoing.

Calcium signaling in neuronal cells exposed to the munitions compound Cyclotrimethylenetrinitramine (RDX).

Cyclotrimethylenetrinitramine (RDX) has been used extensively as an explosive in military munitions. Mechanisms for seizure production, seen in past animal studies, have not been described. Increased calcium levels contribute to excitotoxicity, so in this study neuroblastoma cells are loaded with calcium-indicating dye before application of 1.5 microM to 7.5 mM RDX, with fluorescence recorded for 30 cycles of 11 seconds each. The lowest concentration of RDX increases calcium fluorescence significantly above baseline for cycles 2 to 8; millimolar concentrations increase calcium fluorescence significantly above baseline for cycles 2 to 30. Increases in calcium, like those of 200 nM carbachol, are prevented with 10 mM of calcium chelator ethylene glycol-bis(beta-aminoethyl ether)-N,N,N,N tetra-acetic acid (EGTA, tetrasodium salt). Calcium channel blocker verapamil (20 microM), Ca(2+)-ATPase inhibitor thapsigargin (5 microM), and general membrane stabilizer lidocaine (10 mM) partially attenuate carbachol- and RDX-induced increases in calcium, suggesting that RDX transiently increases intracellular calcium by multiple mechanisms.

Studies Exploring the Interaction of the Organophosphorus Compound Paraoxon with Fullerenes.

In vitro experiments previously published demonstrated the ability of fullerenes to decrease the capability of organophosphorus (OP) compounds to inhibit acetylcholinesterase. Experiments described herein demonstrate molecular level affinity interactions between fullerenes and the OP test compound paraoxon with NMR spectroscopy. The calculated binding constant of 19 M-1 indicates that this binding was not covalent.