Genome-free Viral Capsids as Carriers for Positron Emission Tomography Radiolabels

PURPOSE: We have developed a modular synthetic strategy to append imaging agents to a viral capsid. PROCEDURES: The hollow protein shell of bacteriophage MS2 (mtMS2) was labeled on its inside surface with [18F]fluorobenzaldehyde through a multistep bioconjugation strategy. An aldehyde functional group was first attached to interior tyrosine residues through a diazonium coupling reaction. The aldehyde was further elaborated to an alkoxyamine functional group, which was then condensed with n.c.a. [18F]fluorobenzaldehyde. Biodistribution of the radioactive MS2 conjugates was subsequently evaluated in Sprague-Dawley rats. RESULTS: Relative to fluorobenzaldehyde, fluorine-18-labeled MS2 exhibited prolonged blood circulation time and a significantly altered excretion profile. It was also observed that additional small molecule cargo installed inside the capsids did not alter the biodistribution. CONCLUSIONS: These studies provide further insight into the pharmacokinetic behavior of nanomaterials and serve as a platform for the future development of targeted imaging and therapeutic agents based on mtMS2.     

Reactions of methylphosphonic difluoride with human acetylcholinesterase and oximes – Possible therapeutic implications

Highly toxic organophosphorus (OP) nerve agents are well characterized regarding chemical, biological and toxicological properties and the effectiveness of standard atropine and oxime therapy. Open literature data on the key nerve agent precursor methylphosphonic difluoride (DF) are scarce. To fill this gap the reactions of DF and its main degradation product methylphosphonofluoridic acid (MF) with human acetylcholinesterase (AChE) and the oximes obidoxime, HI-6 and 2-PAM were investigated in vitro. DF and MF were found to be weak inhibitors of human AChE being at least five orders less potent compared to the nerve agent sarin. Incubation of human AChE with millimolar DF and MF and subsequent addition of obidoxime and HI-6 resulted in a concentration-dependent decrease of AChE activity. This effect was not observed when incubating highly diluted AChE with oximes. The most likely explanation for this phenomenon is an inhibitory effect of phosphonyloximes formed by direct reaction of DF or MF with obidoxime and HI-6. These data indicate that high DF doses, resulting in millimolar blood and tissue DF/MF concentrations, are necessary to induce cholinergic signs and that under these conditions treatment with obidoxime and HI-6 may even worsen the poisoning.     

Therapeutic and reactivating efficacy of oximes K027 and K203 against a direct acetylcholinesterase inhibitor

As oxime-based structures are the only causal antidotes to organophosphate (OP)-inhibited acetylcholinesterase (AChE), the majority of studies on these have been directed towards their synthesis and testing. In this study, experimental bispyridinium oximes K027 and K203, which have shown promising results in the last decade of research, were examined in vivo for their therapeutic and reactivating ability in acute poisoning by the direct AChE-inhibitor dichlorvos (DDVP), used as a dimethyl OP structural model. Additionally, the efficacy of oximes K027 and K203 was compared with the efficacy of four oximes (pralidoxime, trimedoxime, obidoxime and HI-6), already used in efficacy experiments and human medicine. To evaluate therapeutic efficacy, groups of Wistar rats were treated with equitoxic doses of oximes (5% LD₅₀, i.m.) and/or atropine (10 mg/kg, i.m.) immediately after s.c. DDVP challenge (4–6 doses). Using the same antidotal protocol, AChE activity was measured in erythrocytes, diaphragm and brain 60 min after s.c. DDVP exposure (75% LD₅₀). The oxime K027 was the most efficacious in reducing the DDVP induced lethal effect in rats, while the oxime K203 was more efficacious than trimedoxime, pralidoxime and HI-6. Significant reactivation of DDVP inhibited AChE was achieved only with oxime K027 or its combination with atropine in erythocytes and the diaphragm. Moreover, the acute i.m. toxicity of oxime K027 in rats was lower than all other tested oximes. The results of this study support previous studies considering the oxime K027 as a promising experimental oxime structure for further testing against structurally-different OP compounds.     

No promising antidote 25 years after the Tokyo subway sarin attack: A review

On the battlefields of Syria, many innocent civilians have been killed or injured by sarin poisoning. In Malaysia in February 2017, a North Korean man was assassinated with VX at Kuala Lumpur International Airport. In the face of such threats, a more effective antidote against organophosphonate acetylcholinesterase (AChE) inhibitors is needed, one that can freely penetrate into the central nervous system (CNS) through the blood–brain barrier (BBB). In the 1995 Tokyo subway sarin attack, which produced more than 6,000 victims, 2-pyridinealdoxime methiodide was the most commonly used antidote in hospitals, but it was unable to prevent CNS damage and no other oximes have been approved for use in Japan. Ultimately, 12 people died, and many victims had severe neurological injuries or sequelae. Although more than 25 years have passed since the incident, progress has been slow in the development of a new antidote that can penetrate the BBB, restore AChE activity in the CNS, and definitely prevent brain injury. From the perspectives of countering terrorism and protecting innocent people from nerve agent attacks, the search for nerve agent antidotes should be accelerated with the goals of improving both survival and quality of life. This review gives an overview of a series of our studies on the development of a new antidote since the Tokyo subway sarin attack and emphasizes that there is unfortunately still no promising antidote for saving the CNS in Japan.     

Pharmacological actions of HS-6, an oxime, on the neuromuscular junction.

The effect of HS-6 [1-(2-hydroxyiminomethylpyridinium)-1-(3-carboxamidopyridinium)-dimethyl ether] on neuromuscular (NM) transmission was investigated using chick biventer cervicis (CBC), rat diaphragm (RD) and guinea pig ileum longitudinal muscle strip (GPLS) preparations. In the CBC preparation HS-6 did not cause a contraction, whereas it produced a dose-related contraction of the GPLS preparation. HS-6 produced a hemicholinium-like NM blockade in the CBC and RD preparations. This was supported by the fact that HS-6 inhibited choline uptake in erythrocytes. HS-6 inhibited the contractions of various nicotinic agonists in the CBC preparation and augmented the acetycholine contractions. The latter was due to AChE inhibition produced by HS-6.     

Schutzwirkungen von Oximen und Cholinolytica bei Somanvergiftungen

Zusammenfassung Bisher sind in der experimentellen Therapie von Somanvergiftungen nur geringe Erfolge erzielt worden, da somangehemmte Cholinesterase durch Oxime nicht reaktiviert wird. Deshalb versuchte man den Erfolg der Oximbehandlung dadurch zu verbessern, daß diese Substanzen in Kombination mit je zwei Cholinolytica angewendet wurden. Von den untersuchten Antidoten, PAM-2, TMB-4, LüH-6, HS-6, Atropin, d-Tuboourarin, Benactyzin, Hexamethonium und W-84 wies keines nennenswerte eigene Schutzwirkungen bei Soman-vergifteten Mäusen auf. Gewisse Kombinationen je zweier Cholinolytica (z.B. Atropin und d-Tubocurarin, Atropin und Hexamethonium, Benactyzin und W-84) setzten jedoch die Toxicität des Soman bedeutend herab, und diese Schutzwirkung konnte durch LüH-6, bzw. HS-6, noch etwas gesteigert werden. PAM-2 und TMB-4 ließen die Schutzwirkung der Cholinolytica unverändert. Es konnte festgestellt werden, daß die mit LüH-6, bzw. HS-6 erhaltene Wirkungssteigerung nicht auf einer Potenzierung des cholinolytischen Effekts beruht, sondern daß sie sich additiv aus beiden Einzelwirkungen zusammensetzt.     

Y124 at the peripheral anionic site is important for the reactivation of nerve agent-inhibited acetylcholinesterase by H oximes

The toxicity of organophosphorus (OP) nerve agents is manifested through irreversible inhibition of acetylcholinesterase (AChE) at the cholinergic synapses, which stops nerve signal transmission, resulting in a cholinergic crisis and eventually death of the poisoned person. Oxime compounds used in nerve agent antidote regimen reactivate nerve agent-inhibited AChE and halt the development of this cholinergic crisis. Due to diversity in structures of OP nerve agents, none of the currently available oximes is able to reactivate AChE inhibited by different nerve agents. To understand the mechanism for the differential activities of oximes toward AChE inhibited by diverse nerve agents in order to aid the design of new broad-spectrum AChE reactivators, we undertook site-directed mutagenesis and molecular modeling studies. Recombinant wild-type and mutant bovine (Bo) AChEs were inhibited by two bulky side-chain nerve agents, GF and VR, and used for conducting reactivation kinetics with five oximes. A homology model for wild-type Bo AChE was built using the recently published crystal structure of human AChE and used to generate models of 2-PAM and HI-6 bound to the active-sites of GF- and VR-inhibited Bo AChEs before nucleophilic attack. Results revealed that the peripheral anionic site (PAS) of AChE as a whole plays a critical role in the reactivation of nerve agent-inhibited AChE by all 4 bis-pyridinium oximes examined, but not by the mono-pyridinium oxime 2-PAM. Of all the residues at the PAS, Y124 appears to be critical for the enhanced reactivation potency of H oximes.     

Synthesis and affinity for serotonin and dopamine transporters of some benzophenone oxime ether derivatives

In a previous study, we reported the synthesis of several 3-(methylenaminoxymethyl)-substituted piperidine derivatives and their ability to interfere with the transmission mediated by biogenic amines. The present study describes the preparation of some new oxime derivatives and their capacity to inhibit serotonin (SERT) and dopamine (DAT) transporters expressed at the level of the rabbit cortex and the striatal membranes, respectively. All the compounds showed K(i) values in the micromolar range on both transporters.     

Gaschromatographische Untersuchungen über die direkte Umsetzung von Soman und Paraoxon mit Pyridinium-Aldoximen in vitro

Zusammenfassung Es wurde die direkte Umsetzung von Soman und Paraoxon mit Pyridinium-Aldoximen in vitro untersucht. Der Reaktionsablauf wurde über die Abnahme der Somankonzentration und die Bildung von p-Nitrophenol, einem Reaktionsprodukt von Paraoxon, gaschromatographisch gemessen. Oximkonzentrationen von 5×10^–4 M (HS-3, Obidoxim) bzw. 10^–3 M (HS-6, 4-PAM) führen zu einer Halbwertzeit des Somanumsatzes von 14 bis 19 min bei pH 7.4 und 37 °C. Um entsprechende Halbwertszeiten des Paraoxonumsatzes mit Oximen zu erreichen, sind um 2 Zehnerpotenzen höhere Oximkonzentrationen nötig. Inwieweit diese Direktreaktion als Antidotprinzip therapeutische Bedeutung bei der Somanoder Paraoxonvergiftung hat wird diskutiert.

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Abkürzungen HS-3 [(2-Hydroxyiminomethyl)-pyridinium-(1)-methyl]-[(4-hydroxyiminoniethyl)-pyridinium-(1)-methyl]-äther-dichlorid - HS-6 [(2-Hydroxyiminomethyl)-pyridinium-(1)-methyl]-[(3-carbamoyl)-pyridinium-(1)-methyl]-äther-dichlorid Ich danke Frau N. Sittisomwong für ihre technische Mitarbeit     

A comparison of the efficacy of HI6 and 2-PAM against soman, tabun, sarin, and VX in the rabbit

This study compared the efficacy of HI6 and 2-PAM against nerve agent (soman, tabun, sarin, and VX) -induced lethality in the atropinesterase-free rabbits pretreated with vehicle (controls) or pyridostigmine. Treatment was administered at signs or 2 min after agent challenge and consisted of oxime (100 μmol/kg) + atropine (13 mg/kg) (alone or together with diazepam). Twenty-four-h LD₅₀, values were calculated for soman- and tabun-intoxicated animals, whereas 24-h survival was noted in animals given 10 LD₅₀s of sarin or VX. In pyridostigmine and control rabbits intoxicated with soman and treated with oxime + atropine (alone or together with diazepam), HI6 was 3–5 times more effective than 2-PAM. In contrast, HI6 was less effective than 2-PAM against tabun poisoning. In pyridostigmine-pretreated animals exposed to tabun, efficacy was increased more than 3-fold when compare to tabun-challenged animals treated with atropine + HI6 alone. Both oximes were highly effective against sarin and VX. These findings suggest that HI6 could replace 2-PAM as therapy for nerve agent poisoning, because it is superior to 2-PAM against soman, and when used in pyridostigmine-pretreated animals, it affords excellent protection against all four nerve agents when used in combination with atropine (alone or together with diazepam) therapy.