Richard E. Sweeney

A Common Mechanism for Resistance to Oxime Reactivation of Acetylcholinesterase Inhibited by Organophosphorus Compounds

Administration of oxime therapy is currently the standard approach used to reverse the acute toxicity of organophosphorus (OP) compounds, which is usually attributed to OP inhibition of acetylcholinesterase (AChE). Rate constants for reactivation of OP-inhibited AChE by even the best oximes, such as HI-6 and obidoxime, can vary >100-fold between OP-AChE conjugates that are easily reactivated and those that are difficult to reactivate. To gain a better understanding of this oxime specificity problem for future design of improved reactivators, we conducted a QSAR analysis for oxime reactivation of AChE inhibited by OP agents and their analogues. Our objective was to identify common mechanism(s) among OP-AChE conjugates of phosphates, phosphonates and phosphoramidates that result in resistance to oxime reactivation. Our evaluation of oxime reactivation of AChE inhibited by a sarin analogue, O-methyl isopropylphosphonofluoridate, or a cyclosarin analogue, O-methyl cyclohexylphosphonofluoridate, indicated that AChE inhibited by these analogues was at least 70-fold more difficult to reactivate than AChE inhibited by sarin or cyclosarin. In addition, AChE inhibited by an analogue of tabun (i.e., O-ethyl isopropylphosphonofluoridate) was nearly as resistant to reactivation as tabun-inhibited AChE. QSAR analysis of oxime reactivation of AChE inhibited by these OP compounds and others suggested that the presence of both a large substituent (i.e., ≥ the size of dimethylamine) and an alkoxy substituent in the structure of OP compounds is the common feature that results in resistance to oxime reactivation of OP-AChE conjugates whether the OP is a phosphate, phosphonate or phosphoramidate.

A Combination Drug Treatment Against Ocular Sulfur Mustard Injury

The eye is considered to be one of the most sensitive organs to sulfur mustard [bis(2‐chloroethyl) sulfide (SM)], with injuries ranging from mild conjunctivitis to advanced corneal disease. Even mild ocular involvement from sulfur mustard exposure can result in both physical and psychological incapacitation. In this study we explored the use of Food and Drug Administration (FDA) approved medications (prednisolone acetate ophthalmic suspension, triamcinolone, and cefazolin) as ocular treatments for sulfur mustard injury. Female New Zealand White rabbits were divided into a SM positive control group (n = 8) and a single treatment group (n = 7). At 10, 20, 30, 60, 90, and 120 min after SM exposure, two drops of prednisolone acetate ophthalmic suspension was administered to each treatment group rabbit while the control group received saline drops. At 120 min after SM exposure, each treatment group animal received a single 1.0 mL sub‐Tenons injection containing 20 mg triamcinolone and 50 mg cefazolin. Control group rabbits did not receive an injection. Rabbits were observed for a total of 16 weeks after SM exposure. Corneal thickness, corneal stromal injury, neovascularization (NV), eyelid notching, and chemosis were recorded weekly for 6 consecutive weeks and on week 16 after exposure. The SM treatment group at weeks 2, 3, and 4 had a significantly lower index value for corneal thickness than the SM positive control group. For corneal stromal injury, NV, eyelid notching, and chemosis, significant evidence of a protective effect due to treatment was seen at weeks 4, 5, and 6. In addition, corneal stromal injury was reduced at weeks 2 and 3 and notching at week 2. By week 3, all SM positive control animals developed NV in contrast to 1 of 7 treatment animals. By week 6 all positive control animals still exhibited NV compared to 2 of 7 treatment animals. These data suggest that prednisolone acetate suspension dosed for the first 2 h after SM exposure followed by a single sub‐Tenons injection of a triamcinolone/cefazolin combination is effective in treating the early stages of corneal injury from SM exposure.

Toxicodynamic modeling of highly toxic organophosphorus compounds.

Although the in vitro effect of organophosphorus (OP) compounds on acetylcholine-esterase (AChE) has been studied extensively, the hypothesis that OP inhibition of AChE is the primary mechanism of acute in vivo OP toxicity has been controversial. For example, a recent review (Pope and Liu, 2004) suggested that OP compounds have direct toxic effects on other enzymes, ACh receptors, and receptor/ channel complexes that are independent of AChE inhibition. The purpose of this report is to examine the hypothesis that AChE inhibition is the mechanism of acute toxicity of OP compounds by mathematically modeling the in vivo lethal effects of highly toxic OP compounds and determining the amount of variation in OP toxicity that is explained by AChE inhibition.

A 7-day mouse model to assess protection from sulfur mustard (SM) skin injury

The mouse ear vesicant model (MEVM) is a screening tool used to identify protective compounds against acute sulfur mustard (SM)‐induced skin injury. It provides endpoints of edema and histopathology 24 h following a topical SM exposure to assess protection against inflammation and tissue damage. To further evaluate successful compounds, the MEVM was modified for use as a 7‐day model. Dose response studies were conducted with SM to select an optimal challenge dose for the new model. Due to severity of SM‐induced tissue damage by Day 7, edema and histopathology were determined unreliable endpoints. Therefore, a modified Draize scoring system (no damage to extensive necrosis) was incorporated as an endpoint to evaluate tissue damage out to Day 7. To aid in optimal SM dose selection, retro synthetic capsaicin (RSCAP), a protective compound in the MEVM, was evaluated as a treatment 15 min before exposure to 0.06, 0.08, and 0.16 mg SM. The RSCAP compound provided similar significant protection at Day 7 against the 0.06‐ (42% reduction) and 0.08‐mg doses (32% reduction), but was not effective against the severely necrotizing 0.16‐mg SM dose. Based on these results, an optimum SM dose of 0.08 mg was selected. Retro synthetic capsaicin and two pharmacologically inactive analogs were tested as topical treatments 15 min prior to SM challenge. The RSCAP compound significantly reduced injury, whereas the inactive analogs had no protective effect. The RSCAP also significantly reduced SM injury when administered topically 10 min after SM challenge. These data support the use of the 7‐day mouse ear vesicant treatment model (MEVTM) in evaluating candidate antivesicant compounds.

Comparison of hematologic consequences and efficacy of p-aminophenones in mice

Controlled methemoglobin (MHb) formation is one strategy employed to counter cyanide (CN) toxicity. Currently available MHb formers present certain drawbacks and limitations. The purpose of this study was to characterize, in mice, the hematologic effects of the MHb-forming compound p-aminopropiophenone (PAPP), and two structurally-related p-aminophenones, p-aminoheptanoylphenone (PAHP) and p-aminooctanoylphenone (PAOP). Although these three p-aminophenones have been shown previously to be efficacious as pretreatments against CN, a more complete understanding of their hematologic effects is lacking. In addition, because the active form of PAPP has been shown to be its N-hydroxy metabolite, the N-hydroxy metabolites of PAPP, PAHP and PAOP were also tested. Using a hemoximeter, blood samples obtained -2 to +180 min relative to intramuscular (i.m.) or intraperitoneal (i.p.) drug injections were evaluated. Sodium nitrite (NaNO(2)) and the appropriate solvents served as the positive and negative controls, respectively. Dose-, time-, route-, and compound-related effects were observed. MHb and sulfhemoglobin levels increased, whereas levels of those parameters related to oxygen-carrying capacity of the blood, such as, oxygen saturation and oxyhemoglobin decreased. In general, the effects of PAHP and PAOP were longer lasting than those of PAPP and NaNO(2). Furthermore, PAPP and NaNO(2) were equally effective with either route of administration. Conversely, PAHP and PAOP showed larger effects when administered i.p. versus i.m. The animals treated with N-hydroxy metabolites of the p-aminophenones also showed similar changes in the hematological parameters measured. N-hydroxy PAPP was shown to be the most rapidly acting MHb-forming compound examined in this series. It could achieve therapeutic concentrations of MHb within 2 min and thus may be considered as a treatment for CN intoxication. Although additional work is needed, these data provide information that will be useful for the successful development of improved anti-CN MHb formers.

Neutralization effects of interleukin-6 (IL-6) antibodies on sulfur mustard (HD)-induced IL-6 secretion on human epidermal keratinocytes.

The proinflammatory cytokine human interleukin-6 (hIL-6) plays an important role in the early and late courses of inflammation, trauma, and wound healing caused by sulfur mustard (HD). Previously, we demonstrated that hIL-6 might be involved in the early event of structural changes of the signal transducer glycoprotein, which indirectly initiates the cascade of events, such as skin irritation and blister formation observed in the pathophysiology of HD injury. In this present work, we focus on the neutralization effect of IL-6 antibodies with regard to the modulation of hIL-6 secretion. Levels of secreted cytokine hIL-6 in normal human epidermal keratinocytes (NHEK) stimulated with HD (10(-4)M) and incubated for 24h at 37°C were determined by enzyme immunoassay, protein immunocytologic assay and reverse-transcriptase-polymerase chain reaction (RT-PCR). The ratio of HD-treated NHEK to constitutive non-stimulated NHEK controls (S/C) on the induction of hIL-6 is reported. S/C was four-fold higher than non-stimulated NHEK controls as determined by ELISA. By using a more sensitive immunocytologic assay, Luminex(100)™, the increment was verified. hIL-6 levels in NHEK stimulated with HD were 21±11ng/mL as measured by Luminex(100)™. The messenger RNA expression of the cytokine (hIL-6) gene was analyzed semiquantitatively. RT-PCR demonstrated that HD induced an increase in the transcription of hIL-6 gene. Selective immunosuppression, using IL-6 neutralizing antibodies, led to a reduction of such expression of HD-induced transcription of hIL-6 in human keratinocytes. The neutralization by pre-incubating NHEK with monoclonal anti-IL6 antibodies decreased hIL-6 secretion by 76%±1.8 ((*)P<0.05).

Evaluation of the direct actions of HI-6 in reversing soman-induced tetanic fade

The most widely accepted mechanism for the acute toxicity of organophosphorus anticholinesterase agents is irreversible inhibition of acetylcholinesterase (AChE), an enzyme present at all known cholinergic synapses (Taylor, 1990). Inhibition of AChE results in accumulation of acetylcholine (ACh), which then leads to aberrant cholinergic transmission (Katz and Miledi, 1973). The precise nature of the abnormality varies with the synapse, and can include depolarization, desensitization, repetitive firing or sustained activation (Hobbiger, 1976; Adler et al., 1992).