HI-6 and 2-PAM in sheep: pharmacokinetics and effects on muscle tissue following intramuscular injection

The pharmacokinetics of 2-PAM, a component of the current nerve agent antidote therapy for U.S. military forces was compared to the pharmacokinetics of another acetylcholinesterase reactivator HI-6. Additionally, the effects of these compounds on muscle tissue following intramuscular injection was examined. Plasma concentrations of the oximes were determined by HPLC. Plasma concentration-time profiles for both oximes fit a one-compartment open model with first-order absorption and elimination. The results demonstrate that the half-time of absorption of HI-6 was significantly higher than that for 2-PAM. Musculoirritancy was assessed on the basis of quantitative histological examinations of the injection sites and by the measurement of serum creatinine phosphokinase. Comparison of the scores from the histological sections demonstrate no difference between the two oximes. Serum creatinine phosphokinase values were elevated following injections of HI-6, but were not consistently elevated following the 2-PAM injections.     

Chemical Stability of the Hagedorn Oximes HGG-12 and HI-6

The chemical stability of the soman antidotes HGG-12 and HI-6 was studied over the pH range 2–9 at various temperatures. Maximum stability for both oximes was found at pH 2. From Arrhenius plots the predicted shelf life (10% decomposition) was 2.6 years at 25° C and 60 years at 4 °C for both oximes. The apparent energy of activation was 113kJ/mol. The decay rate of both oximes apparently was not influenced by oxime concentration, buffer composition, light, and oxygen. By ion-pair HPLC the following degradation products were indentified: pyridine-2-carbaldoxime, pyridine-2-carbonitrile, 2-pyridone, formaldehyde and 3-benzoylpyridine (for HGG-12) or isonicotinamide and isonicotinic acid (for HI-6). Two additional pyridinium compounds have not yet been identified. The pattern of degradation products varied considerably with pH.     

A comparison of cholinergic effects of HI-6 and pralidoxime-2-chloride (2-PAM) in soman poisoning

The effects of HI-6 and pralidoxime chloride (2-PAM) on soman-induced lethality, time to death and several cholinergic parameters in rats were compared to understand the beneficial action of HI-6. Treatment with atropine sulfate (ATS) or HI-6 alone protected against 1.2 and 2.5 LD50s of soman respectively, whereas 2-PAM or methylated atropine (AMN) alone afforded no protection. Addition of ATS, but not AMN, to HI-6-treated rats enhanced the protection from 2.5 to 5.5 LD50s. HI-6 increased the time-to-death, while 2-PAM had no effect; a combination of HI-6 and ATS provided the most significant increase in time-to-death. Cholinesterase (ChE) activity was not altered in any tissue by ATS, HI-6 or 2-PAM treatment individually, but was markedly inhibited in all tissues by 100 micrograms/kg of soman. In soman-poisoned rats, the HI-6, but not the 2-PAM, group had significantly higher levels of ChE in blood and other peripheral tissues than did the group given soman alone. Neither HI-6 nor 2-PAM affected soman-inhibited ChE in the brain. Additional ATS treatment had no effect on ChE activity. HI-6 and 2-PAM neither modified baseline brain acetylcholine (ACh) or choline (Ch) levels nor protected against soman-induced ACh or Ch elevation. 2-PAM exhibited a 4-fold more potent in vitro inhibition of 3H-quinuclidinyl benzilate (3H-QNB) binding and sodium-dependent high-affinity Ch uptake (HACU) than did HI-6 in brain tissues. The findings that 2-PAM is a more potent in vitro inhibitor of muscarinic receptor binding and HACU than HI-6, and yet neither elevates ChE activity in the periphery nor protects rats against soman poisoning, indicate the importance of higher ChE activity in the periphery of HI-6-treated rats. Maintenance by HI-6 of a certain amount of active ChE in the periphery appears to be important for survival after soman exposure.     

Effect of Different Oximes on Rat and Human Cholinesterases Inhibited by Methamidophos: A Comparative In Vitro and In Silico Study

Methamidophos is one of the most toxic organophosphorus (OP) compounds. It acts via phosphorylation of a serine residue in the active site of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), leading to enzyme inactivation. Different oximes have been developed to reverse this inhibition. Thus, our work aimed to test the protective or reactivation capability of pralidoxime and obidoxime, as well as two new oximes synthesised in our laboratory, on human and rat cholinesterases inhibited by methamidophos. In addition, we performed molecular docking studies in non‐aged methamidophos‐inhibited AChE to understand the mechanisms involved. Our results suggested that pralidoxime protected and reactivated methamidophos‐inhibited rat brain AChE. Regarding human erythrocyte AChE, all oximes tested protected and reactivated the enzyme, with the best reactivation index observed at the concentration of 50 μM. Concerning BChE, butane‐2,3‐dionethiosemicarbazone oxime (oxime 1) was able to protect and reactivate the methamidophos‐inhibited BChE by 45% at 50 μM, whereas 2(3‐(phenylhydrazono)butan‐2‐one oxime (oxime 2) reactivated 28% of BChE activity at 100 μM. The two classical oximes failed to reactivate BChE. The molecular docking study demonstrated that pralidoxime appears to be better positioned in the active site to attack the O‐P moiety of the inhibited enzyme, being near the oxyanion hole, whereas our new oximes were stably positioned in the active site in a manner similar to that of obidoxime. In conclusion, our work demonstrated that the newly synthesised oximes were able to reactivate not only human erythrocyte AChE but also human plasma BChE, which could represent an advantage in the treatment of OP compounds poisoning.     

A Comparison of Two Oximes (HI-6 and Obidoxime) for 2-Dimethylaminoethyl-(dimethylamido)-phosphonofluoridate Poisoning

Abstract: The effects of HI-6 (1-[[[(4-aminocarbonyl) pyridinio]methoxy]methyl]-2-(hydroxyimino)-methyl]pyridinium dichloride monohydrate) on the organophosphorus agent 2-dimethylaminoethyl-(dimethylamido)-phosphonofluoridate (GV) inhibited cholinesterases in blood, brain and diaphragm and the stressogenic effect of GV in rats were compared to the effects of obidoxime. In GV-poisoned rats, HI-6 had a significantly higher reactivating effect in blood and brain as well as diaphragm than had obidoxime. HI-6 also practically eliminated the stressogenic effect of the GV compound. On the other hand, hypercorticosteronaemia and increase of liver tyrosine aminotransferase activity during treatment of poisoning with obidoxime were observed. These findings demonstrate the importance of HI-6 for treatment of GV agent poisoning in rats.     

Mechanical Restitution in Atrial Muscle from Human and Rat Hearts: Effects of Agents that Modify Sarcoplasmic Reticulum Function

Abstract: Force of contraction (F_c) of isolated human and rat atrial myocardium shows characteristic patterns of mechanical restitution when single test intervals are interposed in regular stimulation. With several pharmacological agents that modify the function of the sarcoplasmic reticulum we have investigated the role of the sarcoplasmic reticulum in mechanical restitution in these two species. Caffeine, thapsigargin and 2,5-di-(tert-butyl)-l,4-benzohydroquinone (BHQ) were used to reduce Ca²⁺ uptake, ryanodine to open Ca²⁺ release channels, and forskolin to stimulate Ca²⁺ uptake. Under control conditions, F_c recovered rapidly with test intervals shorter than steady-state, and was potentiated with longer than steady-state intervals. In human atrial tissue the maximum potentiation factor was 1.26+0.03 after a mean test interval of 9.70+1.55 s (n=43) as compared to 3.07+0.45 after 30 sec. in rat atria (n=48). Caffeine (3 mM) did not significantly affect steady-state F_c but abolished post-rest potentiation in human and rat preparations. Forskolin (1 μM) enhanced and accentuated the mechanical restitution curve in particular for short test intervals. In the presence of thapsigargin (10 μM), steady-state F_c and mechanical restitution could not be distinguished from time-matched controls exposed to solvent only, indicating that this agent is ineffective in human and rat atrial tissue. In contrast, the putative Ca²⁺ uptake inhibitor BHQ (100 μM) strongly reduced steady-state F_c and decreased potentiation at all intervals in human muscle, but shifted the mechanical restitution curve in parallel to lower values in rat atria. Ryanodine (10 nM) induced post-rest decay in human and depressed both steady-state F_c and post-rest potentiation in rat atrial muscle. From these results it is concluded that human and rat atrial muscle differ in the Ca²⁺ handling by the sarcoplasmic reticulum during mechanical restitution.     

The Efficacy of Monopyridinium (2-PAAM, 2-PAEM) and Bispyridinium (Obidoxime,HI-6) Oximes against Mevinphos in Mice

Abstract: The efficacy of two new monopyridinium oximes (2-PAAM, 2-PAEM) and two bispyridinium oximes (obidoxime. HI-6) was tested in combination with atropine sulphate against acute poisoning with the organophosphorus insecticide mevinphos in mice. When mice were treated two min. after mevinphos poisoning, no significant differences in the therapeutic effect of tested oximes were observed. The oximes increased the 24 hr LD₅₀ values of mevinphos about two times in comparison with the 24 hr LD₅₀ values of mevinphos in mice protected with atropine sulphate alone and more than three times in comparison with non-treated intoxicated animals. On the other hand, both monopyridinium oximes were significantly more efficacious than HI-6 and as efficacious as obidoxime when they were administered 30 sec. after mevinphos poisoning. Both monopyridinium oximes and obidoxime increased the 24 hr values of mevinphos almost three times in comparison with the 24 hr values of mevinphos in mice protected with atropine sulphate alone and about twenty-five times in comparison with non-treated intoxicated animals, while the oxime HI-6 less than two times in comparison with the 24 hr values of mevinphos in mice protected with atropine sulphate alone and about fifteen times in comparison with non-treated intoxicated animals. (change-para-here) Use of new monopyridinium oximes seems to be the improvement in the antidotal treatment of poisoning with organophosphorus insecticide mevinphos in comparison with HI-6 but not in comparison with obidoxime when oximes are used in equimolar doses.     

Utility of 2-Pyridine Aldoxime Methyl Chloride (2-PAM) for Acute Organophosphate Poisoning: A Systematic Review and Meta-Analysis

Organophosphates (OP) account for the majority of pesticide-related unintentional or intentional poisonings in lower- and middle-income countries. The therapeutic role of atropine is well-established for patients with acute OP poisoning. The benefit of adding 2-pyridine aldoxime methyl chloride (2-PAM), however, is controversial. We performed a systematic review and meta-analysis of available randomized controlled trials (RCT) to compare 2-PAM plus atropine in comparison to atropine alone for acute OP poisoning. We searched PubMed, EMBASE, and SCOPUS up to March 2017. The Cochrane review handbook was used to assess the risk of bias. Data were abstracted and risk ratios (RR) were calculated for mortality, rate of intubation, duration of intubation, intermediate syndrome, and complications such as hospital-acquired infections, dysrhythmias, and pulmonary edema. We found five studies comprising 586 patients with varying risks of bias. The risk of death (RR = 1.5, 95% CI 0.9–2.5); intubation (RR = 1.3, 95% CI 1.0–1.6); intermediate syndrome (RR = 1.6, 95% CI 1.0–2.6); complications (RR = 1.2, 95% CI 0.8–1.8); and the duration of intubation (mean difference 0.0, 95% CI ? 1.6–1.6) were not significantly different between the atropine plus 2-PAM and atropine alone. Based on our meta-analysis of the available RCTs, 2-PAM was not shown to improve outcomes in patients with acute OP poisoning.     

A Comparison of the Potency of Newly Developed Oximes (K074, K075) and Currently Available Oximes (Obidoxime,HI-6) to Counteract Soman-Induced Neurotoxicity in Rats

The neuroprotective effects of newly developed oximes (K074, K075) and currently available oximes (obidoxime, HI-6) in combination with atropine in rats poisoned with soman were studied. The soman-induced neurotoxicity was monitored using a functional observational battery at 24 h and 7 days after soman challenge. The results indicate that the oxime HI-6 combined with atropine seems to be an effective antidote for a decrease in soman-induced neurotoxicity, whereas the ability of both newly developed oximes (K074, K075) as well as obidoxime to counteract soman-induced acute neurotoxicity is negligible. Due to the absence of their neuroprotective potency, both newly developed oximes are not suitable oximes for antidotal treatment after exposure to soman. The oxime HI-6 is still the best acetylcholinesterase reactivator for the antidotal treatment of acute poisonings with soman.     

Toxicity of Four Alkylating Agents on in Vitro Rat Embryo Differentiation and Development

The relative developmental toxicity of four direct acting, alkylatingagents was determined in primary cultures of differentiatingrat embryo midbrain (CNS) and limb bud (LB) cells and comparedwith that observed in the rat whole embryo postimplantationculture system. The alkylating agents tested include methylnitrosourea(MNU), ethylnitrosourea (ENU), methyl methanesulfonate (MMS),and ethyl methanesulfonate (EMS). These alkylating agents havebeen shown to produce developmental toxicity following eitherin vitro or in vivo exposure. Viability for both CNS and LBwas assessed by a neutral red dye assay. Differentiation ofCNS cells was assessed by hematoxylin staining of neurons; differentiationof LB cells was assessed by Alcian blue staining of extracellularproteoglycans. Relative potencies of these compounds in thecell culture system were not the same as those observed in theembryo culture system. Whereas rank order of potency in thecell culture system, for viability and differentiation, wasMMS > MNU > ENU > EMS, rank order in the embryo culturesystem, for embryo lethality and malformations, was MNU >ENU > MMS > EMS. Effective concentrations for cell cultureviability and differentiation by MNU and ENU in cell culturewere about three to nine times higher than comparable valuespreviously reported for embryos, while effective concentrationsfor MMS and EMS were two to seven times lower than those observedin the embryos. Differences in potency between the two culturesystems may be related to differences in formation and repairof DNA adducts, as well as differences in culture conditions.     

Effects of Treadmill Exercise on the Recovery of Dopaminergic Neuron Loss and Muscle Atrophy in the 6-OHDA Lesioned Parkinson's Disease Rat Model

This study was to determine the effect of exercise on the recovery of dopaminergic neuron loss and muscle atrophy in 6-OHDA-induced hemi Parkinson''s disease model. Exercise was loaded twice per day for 30 minutes each time, at 5 days after 6-OHDA lesioning and continued for 16 days using a treadmill. Exercise significantly increased the number of tyrosine hydroxylase positive neuron in the lesioned substantia nigra and the expression level of tyrosine hydroxylase in the striatum compared with the control group. To examine which signaling pathways may be involved in the exercise, the phosphorylation of GSK3β and ERK were observed in the striatum. In the control group, basal level of GSK3β phosphorylation was less than in both striatum, but exercise increased it. ERK phosphorylation decreased in the lesioned striatum, but exercise recovered it. These findings suggest that exercise inactivates GSK3β by phosphorylation which may be involved in the neuroprotective effect of exercise on the 6-OHDA-induced cell death. In the exercise group, weight, and Type I and II fiber cross-sectional area of the contralateral soleus significantly recovered and expression of myosin heavy chain and Akt and ERK phosphorylation significantly increased by exercise. These results suggest that exercise recovers Parkinson''s disease induced dopaminergic neuron loss and contralateral soleus muscle atrophy.     

HI-6 in man: efficacy of the oxime in poisoning by organophosphorus insecticides.

The efficacy of the oxime HI-6 was studied as a treatment for organophosphorus poisoning. HI-6 was given four times daily as a single intramuscular injection of 500 mg accompanied by atropine and diazepam therapy. Oxime treatment was started on admission and continued for a minimum of 48 h and a maximum of 7 d. HI-6 rapidly reactivated human blood acetylcholinesterase inhibited by dimethoxy organophosphorus compounds, while the dimethoxy-inhibited enzyme was mainly resistant to the treatment by HI-6. Although both HI-6 and pralidoxime chloride reactivated the red blood cell cholinesterase in quinalphos-poisoned subjects, the return of enzyme activities was more rapid following the use of HI-6. The general improvement of poisoned patients, which was sometimes more rapid than the rise of acetylcholinesterase activity, pointed to direct pharmacological effects of HI-6. No undesirable side-effects were noted in patients when HI-6 plasma concentrations were maintained at levels far above the 'therapeutic' concentration for up to 7 d.     

Comparison of Cumulative and Non-Cumulative Administration of Vasoactive Agents in Arterial Smooth Muscle Responses in Vitro

Two methods of determining concentration-response curves were compared in isolated endothelium-intact mesenteric arterial rings from Wistar rats: arterial contractile and relaxation responses were elicited by adding compounds cumulatively or introducing a single concentration at a time (non-cumulative method). The contractile responses induced by high concentrations of K⁺ (20-125 mM) were comparable between the two methods, whether or not the responses were elicited in the presence of phentolamine (10 μM) and atenolol (10 μM). Noradrenaline (1 nM - 10 μM) likewise induced similar contractions regardless of method of administration, the only exception being the highest concentration (100 μM) which produced lower contractile force when added directly upon resting tension than after cumulative administration. This difference was abolished by atenolol (10 μM). Arterial smooth muscle relaxations induced by endothelium-dependent (acetylcholine 1 nM - 10 μM) and -independent agents (nitroprusside 1 nM - 1 μM, isoprenaline 10 nM - 100 μM) were similar whether the relaxants were added in a cumulative fashion or in a single concentration introduced upon each precontraction. Thus, cumulative and non-cumulative administration of contractile and relaxing agents give quite comparable results. We conclude that the cumulative method is a reliable and time-saving way of studying vascular smooth muscle responses in vitro.     

A Comparison of the Therapeutic and Reactivating Efficacy of Newly Developed Oximes (K117, K127) and Currently Available Oximes (Obidoxime,Trimedoxime, HI-6) in Tabun-Poisoned Rats and Mice

The potency of newly developed bispyridinium compounds (K117, K127) to reactivate tabun-inhibited acetylcholinesterase and reduce tabun-induced lethal toxic effects was compared with currently available oximes (obidoxime, trimedoxime, oxime HI-6) by using in vivo methods. A study that determined the percentage of reactivation of tabun-inhibited blood and tissue acetylcholinesterase in poisoned rats showed that the reactivating efficacy of newly developed oxime K127 is comparable with obidoxime and trimedoxime in blood but lower than the reactivating potency of trimedoxime and obidoxime in the diaphragm and brain. The potency of another newly developed K117 to reactivate tabun-inhibited acetylcholinesterase is comparable with obidoxime or trimedoxime in the diaphragm, but it is significantly lower than the reactivating potency of trimedoxime and obidoxime in the blood and brain. The oxime, K127, was also found to be relatively effective in reducing lethal toxic effects in tabun-poisoned mice. Its therapeutic efficacy is consistent with the therapeutic potency of obidoxime. On the other hand, the potency of the oxime, K117, to reduce acute toxicity of tabun is significantly lower compared to trimedoxime and obidoxime. The therapeutic efficacy of K117 and K127 corresponds to their potency to reactivate tabun-inhibited acetylcholinesterase, especially in the diaphragm and brain. Contrary to obidoxime and trimedoxime, the oxime, HI-6, is not an effective oxime in the reactivation of tabun-inhibited acetycholinesterase and in reducing the lethal effects of tabun. The reactivating and therapeutic potency of both newly developed oximes does not prevail over the effectiveness of currently available obidoxime and trimedoxime and, therefore, they are not suitable for their replacement of commonly used oximes for the treatment of acute tabun poisoning.     

A comparison of the neuroprotective efficacy of individual oxime (HI-6) and combinations of oximes (HI-6+trimedoxime, HI-6+K203) in soman-poisoned rats

The ability of two combinations of oximes (HI-6+trimedoxime, HI-6+K203) to reduce soman-induced acute neurotoxic signs and symptoms was compared with the neuroprotective efficacy of the oxime HI-6 alone, using a functional observational battery. Soman-induced neurotoxicity and the neuroprotective effects of HI-6 alone and HI-6 combined with trimedoxime or K203 in rats poisoned with soman at a sublethal dose (90 μg/kg intramuscularly, i.m.; 80% of LD?? value) were monitored by the functional observational battery at 24 hours following soman administration. The results indicate that both tested oxime mixtures combined with atropine were able to allow soman-poisoned rats to survive 24 hours following soman challenge, while 4 nontreated soman-poisoned rats and 1 soman-poisoned rat treated with oxime HI-6 alone combined with atropine died within 24 hours following soman poisoning. While the oxime HI-6 alone combined with atropine treatment was able to eliminate a few soman-induced neurotoxic signs and symptoms, both oxime mixtures showed higher neuroprotective efficacy in soman-poisoned rats. Especially, the combination of HI-6 with trimedoxime was able to eliminate most soman-induced neurotoxic signs and symptoms and markedly reduce acute neurotoxicity of soman in rats. Thus, both tested mixtures of oximes combined with atropine were able to increase the neuroprotective effectiveness of antidotal treatment of acute soman poisonings, compared to the individual oxime.     

Simultaneous Delivery of Chemotherapeutic and Thermal-Optical Agents to Cancer Cells by a Polymeric (PLGA) Nanocarrier: An In Vitro Study

Purpose  

To test the effectiveness of a dual–agent-loaded PLGA nanoparticulate drug delivery system containing doxorubicin (DOX) and indocyanine green (ICG) in a DOX-sensitive cell line and two resistant cell lines that have different resistance mechanisms.     

An in Vitro Comparison of Rat and Chicken Brain Neurotoxic Esterase

An in Vitro Comparison of Rat and Chicken Brain Neurotoxic Esterase.NOVAK, R., AND PADLLA, S. (1986). Fundam. Appl. Toxicol. 6,464–471. A systematic comparison was undertaken to characterizeneurotoxic esterase (NTE) from rat and chicken brain in termsof inhibitor sensitivities, pH optima, and molecular weights.Paraoxon titration of phenyl valerate (PV)-hydrolyzing carboxylesterasesshowed that rat esterases were more sensitive than chicken toparaoxon inhibition at concentrations <1 µM and superimposablewith chicken esterases at concentrations of 2.5–1000 µM.Mipafox titration of the paraoxon-resistant esterases at a fixedparaoxon concentration of 100 µM (mipafox concentration:0-1000 µM) resulted in a mipafox 150 of 7.3 µM forchicken brain NTE and 11.6 µM for rat brain NTE. NTE (i.e.,paraoxon-resistant, mipafox-sensitive esterase activity) comprised80% of chicken and 60% of rat brain paraoxon-resistant activitywith the specific activity of chicken brain NTE approximatelytwice that of rat brain NTE. The pH maxima for NTE from bothspecies was similar showing broad, slightly alkaline optimafrom pH 7.9 to 8.6. [³H]Diisopropyl phosphorofluoridate (DFP)-labeledNTE from the brains of both species had an apparent mol wt of160,000 measured by sodium dodecyl sulfate polyacrylamide gelelectrophoresis. In conclusion, NTE from both species was verysimilar, with the mipafox 150 for rat NTE within the range ofreported values for chicken and human NTE, and the inhibitorparameters of the chicken NTE assay were applicable for therat NTE assay.     

GDF11 Treatment Attenuates the Recovery of Skeletal Muscle Function After Injury in Older Rats

Loss of skeletal muscle mass and function results in loss of mobility for elderly patients. Novel therapies that can protect and/or restore muscle function during aging would have profound effects on the quality of life for this population. Growth differentiation factor 11 (GDF11) has been proposed as a “youthful” circulating factor that can restore cardiac, neural, and skeletal muscle functions in aging animals. However, conflicting data has been recently published that casts doubt on these assertions. We used a complex rat model of skeletal muscle injury that physiologically mimics injuries seen in patients; to investigate the ability of GDF11 and to enhance skeletal muscle regeneration after injury in older rats. Our data showed that GDF11 treatment resulted in a significant increase in tissue fibrosis, accompanied by attenuated functional recovery, as compared to animals treated with vehicle alone. GDF11 impaired the recovery of skeletal muscle function in older rats after injury.