Caramiphen edisylate: an optimal antidote against organophosphate poisoning.

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Contents lists available at ScienceDirect

Toxicology journal homepage: www.elsevier.com/locate/toxicol

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Review

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Caramiphen edisylate: An optimal antidote against organophosphate poisoning

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Raveh Lily a, * , Eisenkraft Arik b,c,d, Weissman Ben Avi e,1

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Department of Pharmacology, Israel Institute for Biological Research, PO Box 19, Ness Ziona 74100, Israel Israel Defense Forces, Medical Corps, Israel c NBC Protection Division, Ministry of Defense, Hakiria, Tel Aviv 61909, Israel d The Institute for Research in Military Medicine, The Faculty of Medicine, The Hebrew University of Jerusalem, PO Box 12272, Jerusalem 91120, Israel e Casali Institute of Applied Chemistry, The Institute of Chemistry, The Hebrew University of Jerusalem, Jerusalem 91904, Israel b

A R T I C L E I N F O

A B S T R A C T

Article history: Received 3 July 2014 Received in revised form 21 August 2014 Accepted 4 September 2014 Available online xxx

Potent cholinesterase inhibitors such as sarin, induce an array of harmful effects including hypersecretion, convulsions and ultimately death. Surviving subjects demonstrate damage in specific brain regions that lead to cognitive and neurological dysfunctions. An early accumulation of acetylcholine in the synaptic clefts was suggested as the trigger of a sequence of neurochemical events such as an excessive outpour of glutamate and activation of its receptors. Indeed, alterations in NMDA and AMPA central receptors’ densities were detected in brains of poisoned animals. Attempts to improve the current cholinergic-based treatment by adding potent anticonvulsants or antiglutamatergic drugs produced unsatisfactory results. In light of recent events in Syria and the probability of various scenarios of military or terrorist attacks involving organophosphate (OP) nerve agent, research should focus on finding markedly improved countermeasures. Caramiphen, an antimuscarinic drug with antiglutamatergic and GABAergic facilitating properties, was evaluated in a wide range of animals and experimental protocols against OP poisoning. Its remarkable efficacy against OP exposure was established both in prophylactic and post-exposure therapies in both small and large animals. The present review will highlight the outstanding neuroprotective effect of caramiphen as the optimal candidate for the treatment of OP-exposed subjects. ã 2014 Published by Elsevier Ireland Ltd.

Keywords: Caramiphen Antidote Nerve agents Sarin Neuroprotection Organophosphates

Contents 1. 2.

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The history of caramiphen as an antitussive . . . . . . . . . . . . . . . . . . . . Animal studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Human studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. The pharmacological profile of caramiphen – mechanism of action . . Caramiphen and the cholinergic system . . . . . . . . . . . . . . . . . . 3.1. Caramiphen and non-cholinergic systems . . . . . . . . . . . . . . . . 3.2. Toxicity of caramiphen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Caramiphen in the treatment against OP poisoning . . . . . . . . . . . . . . Caramiphen in prophylaxis against OP poisoning . . . . . . . . . . . 5.1. Caramiphen in post exposure treatment against OP poisoning 5.2. Concluding remarks and future prospects . . . . . . . . . . . . . . . . . . . . . . Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Transparency document . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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* Corresponding author. Tel.: +972 8 9381747; fax: +972 8 9381559. E-mail addresses: [email protected] (R. Lily), [email protected] (E. Arik). 1 Deceased on January 2014. http://dx.doi.org/10.1016/j.tox.2014.09.005 0300-483X/ ã 2014 Published by Elsevier Ireland Ltd.

Please cite this article in press as: Lily, R., et al., Caramiphen edisylate: An optimal antidote against organophosphate poisoning. Toxicology (2014), http://dx.doi.org/10.1016/j.tox.2014.09.005

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1. Introduction Organophosphates (OPs) are extremely toxic substances, some of which are categorized as chemical warfare agents and are found in the arsenals of several armed forces as well as of terrorist groups and have created a substantial threat on the battlefields of the world. Their destructive effects have been repeatedly demonstrated in recent decades. Sarin, one of the most potent toxic chemical warfare agents, was employed twice against peaceful city dwellers in Japan, resulting in more than 6000 victims (Okumura et al., 2003, 2005; Yanagisawa et al., 2006). Previously during the Iran–Iraq war, sulfur mustard and nerve agents were used against Iranian soldiers and Iraqi Kurdish civilians, causing many casualties (Balali-Mood et al., 2005). Recent events in Syria floated the issue Q2 of chemical weapons (Editorial, 2013; Patrick and Stanbrook 2013; Eisenkraft et al., 2014; Rosman et al., 2014) and emphasized the need to address the issue of effective treatment against OP poisoning (Dolgin, 2013). The deleterious action of nerve agents stem from their ability to potently inhibit cholinesterases (ChEs) (Cannard, 2006; Taylor, 2011). As a result, large quantities of acetylcholine (ACh) accumulate in synaptic clefts (Shih, 1982; Shih et al., 1990; Lallement et al., 1992a,b). It is the prevailing concept that this excessive outpour triggers an array of toxic effects such as hypersecretion, respiratory distress, convulsions, and finally death. The accumulation of ACh is accompanied by a rapid increase in the activity of the central glutamatergic system, including robust activation of glutamate receptors (McDonough and Shih, 1997; Solberg and Belkin, 1997; Eisenkraft et al., 2013). The notion of a stepwise intoxication mechanism led to a treatment paradigm consisting of pretreatment with pyridostigmine, a reversible inhibitor of ChE, and therapy with an oxime and atropine sulfate. This approach which is currently considered as the standard care in OP poisoning, markedly improves survival rates after soman exposure (Berry and Davies, 1970; Leadbeater et al., 1985). However, this regimen does not prevent the OP-mediated central nervous system (CNS) sequel of convulsions, brain damage, and behavioral deficiencies (Shih and McDonough, 1997; Raveh et al., 2003). Similar results were obtained following sarin exposure (0.8–1.2xLD50) and treatment with oxime (TMB4) and atropine (Grauer et al., 2008; Raveh et al., 2008). In fact, soman- or sarin-evoked seizures progress to status epilepticus, and thus generate a complex problem for medical management (de Araujo Furtado et al., 2012; Eisenkraft et al., 2013). Moreover, when left unchecked, this condition may lead to cognitive and behavioral deficits including long-term learning and memory (Filliat et al., 1999; Weissman and Raveh, 2008; Moffett et al., 2011; de Araujo Furtado et al., 2012) and cardiovascular deficits (Allon et al., 2005). To improve existing therapeutic regimens, simultaneous administration of an adjunct compound with anticonvulsant activity such as a benzodiazepine was proposed (Lipp, 1972; Dunn and Sidell, 1989). Nevertheless, this strategy was proven to provide insufficient protection against the ensuing brain injury (Shih, 1990). To improve medical protocols against chemical agents, better insights into their mechanism of action is required. The methodical examinations performed by Shih and McDonough (1997) and Solberg and Belkin (1997) led to the conclusion that the glutamatergic system is intimately involved in OP poisoning. Furthermore, glutamate receptors’ antagonists in general, and NMDA blockers in particular, were recommended as potential antidotes against nerve agents (Braitman and Sparenborg, 1989; Figueiredo et al., 2011). Despite an extensive effort in search for a comprehensive treatment for subjects exposed to OPs, current medical paradigms are limited and controversial (Connors et al., 2013; Thiermann et al., 2013).

Scheme 1. The chemical structure of caramiphen.

Caramiphen (Scheme 1), a nonopioid antitussive with anticholinergic and antiglutamatergic properties (Weissman and Raveh, 2008) was one of the drugs evaluated as an antidote against OP poisoning. It was successfully used against diisopropyl fluorophosphate (DFP) poisoning more than six decades ago (Essig et al., 1950). It was later demonstrated as an effective antidote and neuroprotectant against various OPs (e.g., Shih et al., 1991; Raveh et al., 1996, 1999, 2002, 2003; Levy et al., 2007). Recent studies provided additional support for the notion that caramiphen is an optimal candidate to be used against OP poisoning (Figueiredo et al., 2011; Schultz et al., 2012). The purpose of this report is to review the available information on this drug and demonstrate its superiority as a potential antidote against OP poisoning.

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2. The history of caramiphen as an antitussive

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Caramiphen was first described by scientists of the Geigy Company in 1942 and later patented in the USA (Martin and Hafliger, 1946). It was used as an antispasmodic, both as a muscle relaxant and as an anti-Parkinson treatment (Dunham and Edwards, 1948; Schwab and Leigh, 1949; Sciarra et al., 1949; Jersild, 1950; Grünthal, 1995). It had an atropine-like effect in this respect, yet without atropine’s side-effects. It was later introduced as an over-the-counter (OTC) antitussive drug for several decades and approved for human use from the age of two years and above (Todd, 1967; PDR, 1988; Reynolds, 1989). Caramiphen edisylate was approved as a New Drug Application (NDA) by the FDA in 1973 (published as a notice in the Federal Register of December 14, 1973, 38 FR 34,481). However, in 1976 the OTC drug review panel for cold, cough, allergy, bronchodilator, and antiasthmatic drugs of the FDA (what was then the National Center for Drugs and Biologics) concluded that there were no well-controlled, objective, clinical studies documenting the effectiveness of caramiphen edisylate as an antitussive (41 FR 38,312, September 9, 1976) and proposed to withdraw approval of the original formulations (48 FR 40,322, September 6, 1983). Later, data in support of the use of the drug in two new applications was presented (NDA 12-903 and NDA 13-068), but the FDA determined that there was not substantial evidence of the effectiveness of caramiphen edisylate as an antitussive compound, and approvals were withdrawn in 1984 (Food and Drug Administration, 1984). The drug was still in use as an OTC in Europe until the end of the 1990s’.

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2.1. Animal studies

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Several research groups studied the central effects of caramiphen on cough. A cat model of centrally-evoked cough was used and three differential effects of the drug on respiration were tested: cough, sustained inspiration (apnea), and pulmonary ventilation (Toner and Macko, 1952; Chakravarty et al., 1956). Caramiphen was found to have direct central nervous system (CNS) action in the inhibition of cough. The depression of cough was characterized by an increase in latency of onset, with no change in amplitude and frequency. Complete cough

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suppression was the result of a rise in threshold with no significant effect on pulmonary ventilation. It also reduced the magnitude of centrally-elicited sustained inspiration. They have concluded that the latency of the cough elicited was increased in a dose-related fashion, and that the observed effects cannot be explained by a single action of the drug in the respiration complex (i.e. probably several separate mechanisms are involved). Intravenous administration of caramiphen had a pronounced antitussive effect on cough induced by inhalation of ammonia vapor in cats and by electrical stimulation of the trachea in dogs. However, p.o. administration of caramiphen was ineffective (Stefko et al., 1961). Caramiphen (i.v. or i.p.) was effective in relieving bronchoconstriction caused by histamine, acetylcholine or furfuryl trimethyl ammonium iodide in guinea pigs (Toner and Macko, 1952; Plisnier, 1954, 1975). In addition, caramiphen relieved the intestinal contraction produced by the latter agent (a spasmolytic effect). Wang et al. (1977) found that the minimal effective dose of caramiphen for suppressing cough response in cats is 3.18 mg/kg (i.v.), making it a relatively weak antitussive compound, with no effect on respiration or blood pressure (Wang et al., 1977). Caramiphen was tested in cats using a direct electrical stimulation of the cough center and was found to be far more effective when given directly into the left vertebral artery than when given i.v., strengthening the notion that caramiphen has a central rather than a peripheral site of antitussive action (Domino et al., 1985).

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2.2. Human studies

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The antitussive effect of caramiphen was studied in healthy volunteers (Bickerman, 1962; Eddy et al., 1969). The cough threshold was determined as the effective concentration of ammonia inhalation. Caramiphen and codeine were administered to 30 volunteers, at doses of 4–20 mg/day. It was concluded that there was an optimal dose beyond which no further improvement occurred. Caramiphen was less effective than an equal dose of codeine. Sporadic reports of the beneficial effects of caramiphen in the relief of cough were published but only few of them were double-blind controlled trials (Liechti, 1950; Svoboda, 1950; Birkner, 1952; Hudson et al., 1952; Sporn, 1952; Segal and Dulfano, 1953; Segal et al., 1953; Snyder, 1953; Abelmann et al., 1954; Cass and Frederik, 1956; Banyai, 1960; Dreyer, 1961; Glick, 1963). For example, in a cross-over study, Abelmann et al. (1954) showed caramiphen to be an effective antitussive in patients suffering from severe chronic irritating cough. It was given p.o. (n = 23, 3 10 mg/day), with partial to considerable relief in 57% of the patients. Caramiphen also decreased the amount of sputum in 61% of these patients. 18 out of 23 subjects had no adverse effects, three had nausea and two suffered from dizziness. This study also indicated that caramiphen was less effective than codeine. In summary of these human studies, it was concluded (Eddy et al., 1969) that caramiphen is more effective in treating chemically-induced cough than codeine in healthy volunteers, it was found to have little or no effect on respiration, and both decreased and increased expectoration had been reported. It proved to be similar to codeine with respect to circulatory effects, and has no abusive effect. We have not found any report of dependence or tolerance to caramiphen. For the purpose of following blood levels in humans during the clinical trials, a sensitive detection method for caramiphen, in whole blood, was developed. Blood levels were monitored following administration of 20 mg at 0, 4, and 8 h daily for four consecutive days to healthy volunteers. No side effects were reported (Levandoski and Flanagan, 1980).

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3. The pharmacological profile of caramiphen – mechanism of action

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3.1. Caramiphen and the cholinergic system

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The concept that caramiphen (or in its original trade name Parpanit or Panparnit) is a multifunctional drug was known a short time after its introduction to the clinic as an antispasmodic and as an anti Parkinsonian agent (Domenjoz et al.,1946). In a later publication, Bovet and Longo (1951) described two ‘preparations’ (Diparcol and Panparnit) which were used for the treatment of Parkinson’s disease and stated: “With respect to their pharmacodynamic action, these preparations cannot be classified as a single, discrete pharmacological group. They exhibit spasmolytic, ganglionic blocking, and parasympathetic properties.” While these authors pointed three distinct activities of caramiphen, further research attributed additional actions to this compound (see below). Caramiphen was recognized as an anticholinergic compound shortly after its synthesis (Kraatz et al., 1949). The authors reported that the novel drug antagonized the effects of acetylcholine on several animal preparations such as the rabbit intestine and presented a “curare-like” action in a frog nerve–muscle preparation. Antagonistic action was demonstrated by relatively low doses of panparnit (2–5 mg/kg) to the convulsive effects elicited by nicotine (Bovet and Longo, 1951). In their communication regarding Diparcol and caramiphen, Heymans and Estable (1949) stated: “These compounds also very actively protect against high doses of acetylcholine, pilocarpine, DFP, strychnine, and metrazol” (Heymans and Estable, 1949). Notably, these authors claimed that caramiphen exhibits synaptolytic, parasympatholytic, and anticonvulsant properties. In fact, a year later, Essig and his colleagues evaluated the effects of caramiphen on DFP-evoked convulsions (Essig et al., 1950). Based on EEG and ECG patterns they concluded that the drug possesses a pharmacodynamic profile similar to atropine. They also noted that its anticonvulsant activity was independent of the DFP-inhibited brain cholinesterase activity (Essig et al., 1950). Caramiphen’s mechanism of action as a blocker of nicotine-provoked seizures was evaluated in curarized animals (Fleisch and Baud, 1948). The administration of nicotine provoked a grand mal-like EEG discharge and this convulsive seizure was completely abolished by caramiphen treatment. A probable cholinergic mechanism of action was proposed by the authors. Further investigations in models of nicotine-induced tremors and convulsions revealed that while the potent antimuscarinic drug atropine is devoid of any effect, caramiphen exerted marked inhibition (Heymans and Estable, 1949; Bovet and Longo, 1951; Gao et al., 1998). Gao et al. (1998) compared a series of anticholinergic drugs used in the treatment of Parkinson’s disease as blockers of nicotine-induced convulsions. From the six compounds examined including trihexyphenidyl and benactyzine, caramiphen was the least active and procyclidine and trihexyphenidyl the most effective with ED50 values of 7.8, 3.1, and 3.3 mg/kg, for caramiphen, procyclidine and trihexyphenidyl, respectively. They also suggested that caramiphen may have both a competitive and a non-competitive component in antagonizing nicotine-induced convulsions in mice (Gao et al., 1998). The anticholinergic activity of caramiphen was also assessed by its ability to antagonize oxotremorine-induced salivation and tremors and to produce mydriasis in mice (Brimblecombe et al., 1970). Similar evaluation was performed by Madill et al. (1968) who determined the effective doses to block methacholine-induced bradycardia, to prevent seizures after intracerebral carbachol and to cause mydriasis in mice. In both laboratories caramiphen was 50–100 fold a less potent anticholinergic, at equimolar doses, than atropine.

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Studies of the interactions of caramiphen and some of its analogs with muscarinic cholinergic receptors found that caramiphen demonstrate M1/M2 selectivity (26-fold) and a lesser M1/M3 selectivity (6-fold) (Hudkins and DeHaven-Hudkins, 1991; Hudkins et al., 1991, 1993). In addition, these studies reported that its affinity to M1 receptors was lower than that of atropine (Ki values of 1.2 and 0.26 nM against [3H]Pirenzepine binding, respectively) (Hudkins and DeHaven-Hudkins, 1991). Some studies explored the possibility that caramiphen exerts its pharmacological effects via the inhibition of cholinesterases (ChE) (Starita, 1955; Bannard et al., 1969). Data from these reports indicated that caramiphen and its analogs are weak reversible inhibitors of ChEs and no correlation was found between their inhibitory potencies and the protection against sarin poisoning.

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3.2. Caramiphen and non-cholinergic systems

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The capacity of drugs with mixed anticholinergic and antiglutamatergic pharmacological profile to block NMDA toxicity in vitro was demonstrated (Olney et al., 1987). Caramiphen and several related compounds had been shown to possess the ability to interact with components of the glutamatergic system. In an early research, caramiphen and its analog carbetapentane were bath-applied to brain slices before or after epileptiform activity was induced (Apland and Braitman, 1990). While both these antitussive drugs were effective against epileptiform activity evoked by Mg2+ -free medium, no blockade of activity was obtained when NMDA was administered. Atropine, even at very high doses, was ineffective in blocking this epileptiform activity (Apland and Braitman, 1990). The same drugs as well as the antitussive dextromethorphan significantly reduced the release of glutamate from potassium-stimulated rabbit hippocampal slices (Annels et al., 1991). Examination of the activity of caramiphen (40 mM) in cultured rat hippocampal pyramidal neurons exposed to high K+ concentrations showed a marked reduction of the evoked rise in intracellular Ca2+ and this effect was greater than its action on NMDA evoked rise (Church et al., 1991). Pontecorvo et al. (1991) reported that when administered at or below the rotorod TD50 dose (162 mg/kg), caramiphen considerably increased survival time in a hypoxic environment (Pontecorvo et al., 1991). Caramiphen also blocked maximal electroshock-induced seizures (ED50, 52 mg/kg), and antagonized seizures and lethality (ED50, 95 mg/kg) induced by administration of NMDA (250 mg/kg) to mice (Pontecorvo et al., 1991). The latter observation was supported by a more recent report (Raveh et al., 1999). A substantial interaction with the glutamatergic system was established when caramiphen was tested against the behavioral and EEG effects elicited by NMDA antagonists (Diana et al., 1993). The NMDA antagonists MK-801 and phencyclidine (PCP) induced dose-dependent changes in the locomotor/exploratory activity of mice; caramiphen potentiated these effects. Fletcher et al. (1995) also demonstrated the ability of caramiphen to block NMDA-evoked responses in cultured hippocampal neurons (Fletcher et al., 1995). Under whole-cell voltage-clamp, caramiphen, and carbetapentane reversibly attenuated NMDA-, but not kainate- or AMPA-evoked currents (Fletcher et al., 1995). The attenuation of NMDA-evoked responses was not mediated through interactions with the agonist glycine or polyamine binding sites on the NMDA receptor-channel complex (Fletcher et al., 1995). Interestingly, Raveh et al. (1999) showed that caramiphen interacts with central NMDA receptors via the extracellular zinc binding site. Recently, Figueiredo et al. (2011) demonstrated that caramiphen facilitates GABA-evoked currents at 100 and 300 mM concentrations, but reduces currents at 1 mM, which suggests the possibility of a high affinity binding site that potentiates currents through the GABAa channel and a low affinity site that has the

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opposite effect (Figueiredo et al., 2011). Additionally, caramiphen has been shown to antagonize voltage-gated Ca2+ channels when applied at micromolar concentrations (Church and Fletcher, 1995; Fletcher et al., 1995; Thurgur and Church, 1998) and to inhibit glutamate release (Annels et al., 1991). Besides its antitussive effect (Snyder, 1953; Glick, 1963; Chou and Wang, 1975; Wang et al., 1977), and like other non-opioid antitussives, caramiphen binds with high affinity to central dextromethorphan sites (Craviso and Musacchio, 1983). Additionally, it has protective effects against convulsions induced by maximal electroshock in animal models (Tortella et al., 1988) and it enhances the anticonvulsant properties of diphenylhydantoin, lowering its ED50 33-fold. Another pharmacological feature of caramiphen, as well as of its analogs and trihexyphenidyl, is its interaction with central s 1 receptors (Hudkins and DeHaven-Hudkins, 1991; Fletcher et al., 1995). Additionally, caramiphen and other s 1 ligands demonstrate a broad repertoire of anticonvulsant (Pontecorvo et al., 1991; DeHaven-Hudkins et al., 1995) and neuroprotective properties (Leonardo et al., 2010). It is not clear which of the above described characteristics of caramiphen contributes more to its anticonvulsant effects. It appears that a combination of its diverse activities is responsible for being a very efficient anticonvulsant. Chen et al. (2010) described another pharmacological activity for caramiphen, producing dose-dependent effects (at micromole/kg doses) of spinal anesthesia in rats (Chen et al., 2010). Caramiphen was more potent than lidocaine. Co-administration of caramiphen with lidocaine produced an additive effect. Additionally, caramiphen was recently shown to produce a dose-dependent cutaneous analgesia (Hung et al., 2012). Together, these findings reveal that caramiphen, like trihexyphenidyl, procyclidine, benactyzine and its analogs possess a distinct glutamatergic component as well as a considerable s 1 binding element, in their pharmacological repertoire. It is suggested that these aspects of its profile may explain, at least in part, its unique efficacy against nerve agent poisoning.

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Kraatz et al. (1949) examined the intravenous and intraperitoneal toxicity of caramiphen hydrochloride (parpanit) in large groups of rabbits, mice, and rats and described values of LD50 in the tens and hundreds of mg/kg for these routes of administration, respectively, indicating relatively low order of toxicity. These results are similar to values obtained by others: 12.0 and 67.5 mg/ kg intravenously in rabbits and mice, respectively (Domenjoz, 1946) and 36.5 mg/kg intravenously in mice (Jovic and Milosevic, 1970). Only minor side effects were observed in toxicity studies in rats and dogs (Toner and Macko, 1952). Sub-acute administration (100 mg/kg and 10 mg/kg daily for 30 days in rats and in dogs, respectively) and chronic toxicity studies (with the same doses and animal models) showed no significant changes in hematological parameters and no visible side effects besides a slight loss of weight in some of the dogs in the sub-acute study. Histopathological examination of animals from all groups found no changes (Toner and Macko, 1952). In a study performed by SKF laboratories (Toner and Macko, 1952) the acute oral toxicity of caramiphen edisylate was determined in rats. Low mortality was witnessed at very high doses of 800–2000 mg/kg. Toxic signs observed in some of the animals included salivation, ataxia, restlessness, tremors, dyspnea, and convulsions. Sub-acute and chronic toxicity assessment in rats and dogs revealed no changes in hematological parameters or in weight gain, following oral administration of 100 and 10 mg/kg daily for periods of 1 and 3 months. Similar results were described

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in a later publication (Coleman et al., 1968) that evaluated the oral toxicity of caramiphen in weanling rats and found it to be 2.9 g/kg. In a chronic toxicity study where daily oral doses as high as 0.2xLD50 were applied for 30 days, no evidence of cumulative effects could be found nor was there any evidence that the protective capacity of caramiphen against sarin was affected by the extended application of the drug (Coleman et al., 1968). When high dose of caramiphen edisylate (300 mg/kg) was applied intraperitoneally to mice 25% of the animals died (Pontecorvo et al., 1991). No untoward effects were reported for doses greater than 175 mg/kg that provided protection against NMDA-induced seizures. The effects of caramiphen alone and in combination with phencyclidine on behavior were studied in rats (Szekely et al., 1994). No effect was noted on locomotion; however, significant increased stereotypy behavior was observed at all doses tested (15–120 mg/kg). A wide range of doses (0.63–100 mg/kg) of caramiphen was used and found to afford efficient protection, in various animal studies, against several OPs (Jovic and Milosevic, 1970; Sparenborg et al., 1990; Shih et al., 1991; Raveh et al., 1996, 1999, 2002, 2003, 2008; Myhrer et al., 2008b, 2011; Figueiredo et al., 2011; Schultz et al., 2014). The protective dose (PD50) and the protective index (PI50) of caramiphen and other drugs were evaluated against nerve agent poisoning (Jovic and Milosevic, 1970). It was found that the values of PD and PI are 3.45, 4.4, 2.05 and 10.6, 8.3, 17.8, respectively against 1.3xLD50 of sarin, soman and tabun. In the study described by Raveh et al. (1996) the effects of caramiphen in rats were assessed by sensitive physiological and cognitive tests. Doses of 2.5–20 mg/kg were evaluated and the minimal effective dose was found to be 10 mg/kg. The highest dose used in several laboratories was 100 mg/kg i.m. (Sparenborg et al., 1990; Mikler et al., 2007; Figueiredo et al., 2011; Schultz et al., 2014) and no adverse effects were reported in any of these investigations. Close observation, for one week, of rats administered intramuscularly 100 mg/kg of caramiphen did not reveal any signs of toxicity (our lab unpublished data). Moreover, behavioral and physiological evaluation of parameters that are associated with cholinergic activity emphasized that caramiphen is a very safe drug compared with other anticholinergics. The sign free dose recommended for prophylactic treatment against OP poisoning, in rats, was 10 mg/kg (Raveh et al., 1996). Jovic and Milosevic (1970) stated that it is safer to give repeated therapeutic doses of caramiphen than atropine probably because of the different mechanisms of action and a better safety profile for caramiphen. Considering the above described toxicological data it can be concluded that among the known anticholinergic compounds and among various antidotes in use against OP poisoning, caramiphen is a relatively nontoxic and safe drug. Together with its wide therapeutic range, it seems justified to test its application both in prophylactic and in post exposure antidotal treatments against nerve agent poisoning.

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The first report on the protective effect of caramiphen in nerve agent poisoning was in 1948. Intravenous injections of 20–30 mg/kg of parpanit (caramiphen commercial formulation) suppressed all toxic actions of high doses of nicotine (0.06–0.63 mg/kg) and DFP (6 mg/kg) (Heymans and De Vleeschhouwer, 1948). Caramiphen (4 mg/kg) was also shown to protect against seizure activity in rabbits following exposure to 1.5 mg/kg of DFP (Essig et al., 1950). When compared with atropine, caramiphen hydrochloride, in conjunction with TMB4, conferred significantly higher protection than atropine at equimolar dosages

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against sarin (1.2xLD50), in mice and in guinea pigs. Yet it was less effective when assessed for its peripheral and central anticholinergic activity (Madill et al., 1968). Evaluation of various anticholinergics (Coleman et al., 1968) revealed a marked protection efficacy of caramiphen, in conjunction with P2S and TMB4, against sarin, tabun, CMPF, and DSDP poisoning in mice, rats, hamsters, guinea pigs, and rabbits. Likewise, the superiority of both caramiphen and benactyzine was demonstrated, alone or in a mixture with 2-PAM, against poisoning by 1.3xLD50 of sarin, soman, and tabun in mice (Jovic and Milosevic, 1970). The authors noted that two different mechanisms may operate in the protective action of atropine and caramiphen against OPs, thus postulated that it is safer to give repeated therapeutic doses of caramiphen and other synthetic cholinolytics than atropine. Another report demonstrated the efficacy of caramiphen and other anticholinergics against high sarin doses of 228–517, 422–1215, 44–744 mg/kg in mice, rats, and guinea pigs, respectively (Brimblecombe et al., 1970). The results of this study showed clearly that no correlation exists between either peripheral or central anticholinergic activities of the tested drugs (as measured either by inducing a mydriatic effect or by blockade of oxotremorine-induced salivation and tremors) and their efficacy, when used alone or in conjunction with P2S, to protect against sarin poisoning. The authors concluded that a pharmacological action other than anticholinergic only is involved, in part, in the protective action against sarin. Most of the studies evaluated the efficacy of caramiphen as a component in a prophylactic mixture or an adjunct to standard antidotal treatment against OPs. The level of intoxication was generally low (1.2–1.4xLD50) though producing central nervous system injury, and caramiphen’s efficacy was measured by assessment of the attenuation or prevention of these damages. In a few studies caramiphen afforded protection against higher levels of intoxication: prophylactic treatment with caramiphen and an oxime provided high protection against various OPs in several animal species (e.g. protective ratio (PR) = 6.1 and 29 against sarin in rats and guinea pigs, respectively, Coleman et al., 1968). In Raveh et al. (1996), prophylaxis with caramiphen (10 mg/kg), in conjunction with pyridostigmine, afforded a PR of 2.5 against sarin and increasing the dose of caramiphen to 20 mg/kg resulted in a PR value close to 4. Furthermore, a mixture of caramiphen and aprophen provided remarkable synergistic efficacy PR 6.5 (Raveh et al., 1996). Similar results (Leadbeater et al., 1985) were reported in guinea pigs against the lethal and incapacitating effect of sarin and soman. Additionally, post exposure treatment (1 min following sarin exposure) with of 20 mg/kg of caramiphen, in a mixture with an oxime and atropine, resulted in PR higher than 5. The ranking order of prophylactic and post-exposure neuroprotective efficacy of various drugs will be detailed in the following sections. The safe pharmacological and behavioral profile of caramiphen (Coleman et al., 1968; Jovic and Milosevic, 1970; Levandoski and Flanagan, 1980) led to a more intensive evaluation of its protection efficacy against OP poisoning both in prophylactic and post exposure therapy.

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During the 1980s’, non-opioid antitussives such as dextromethorphan, carbetapentane, and caramiphen were found to block convulsive activity in the rat (Tortella and Musacchio, 1986; Tortella et al., 1988) and to attenuate epileptic activity in brain slices (Wong et al., 1988; Aram et al., 1989). These results prompted the evaluation of their efficacy against OP-induced convulsions, when administered together with the carbamate pyridostigmine bromide prior to soman poisoning (Sparenborg et al., 1990). Caramiphen edisylate (10, 18, 30, or 100 mg/kg) combined with

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pyridostigmine (0.11 mg/kg) was administered to guinea pigs 30 min before a 2xLD50 soman challenge. All animals were treated with atropine methylnitrate and PAM-Cl 30 sec following OP exposure. Caramiphen, in a dose-dependent manner, protected against lethality and either prevented or reduced the intensity of convulsions, electrographic seizure activity, and brain damage. Similar results were obtained in another study in rats pretreated with HI-6 and poisoned with 1.6xLD50 of soman (Shih et al., 1991). Caramiphen hydrochloride and caramiphen edisylate were found effective against soman-induced seizures in rats even without atropine while the edisylate salt was 2.5-fold more potent than the hydrochloride (Anticonvulsant ED50 of 0.63 and 1.53 mg/kg, i.m., respectively). The efficacy of a number of drugs against OP poisoning was evaluated in an extensive screening study. Some drugs possessed pure anticholinergic activity and others had a combined anticholinergic and antiglutamatergic activity profile (Raveh et al., 1996). Mixtures composed of pyridostigmine together with various doses of atropine, aprophen, scopolamine, trihexyphenidyl, benactyzine, or caramiphen were injected to rats before exposure to sarin. No additional post-exposure therapy was used. The evaluation of the prophylactic mixtures was based on determination of the dose conferring protection against sarin poisoning relative to minimal effective dose (MED) of each drug affecting behavioral and physiological parameters. The behavioral assessment included a number of tasks sensitive to cholinergic manipulation (Grauer and Kapon, 1993, 1996; Pizzo et al., 2009) and the physiological effect was estimated by antagonism of oxotremorine-induced hypothermia. The ratio between the dose that afforded protection (PR 2.5) and the MED was determined as the efficiency ratio and used as a quantitative parameter for ranking the efficacy of the tested drugs (Raveh et al., 1996). As low and as close to the value of 1, the tested drug is considered more potent and safer as antidotal treatment. The efficiency ratio for caramiphen was found to be lower (1.3–3.3) compared to scopolamine, benactyzine, and trihexyphenidyl (5–10, >16–32, 7.5–30, respectively). The ranking order obtained was: aprophen = caramiphen > scopolamine > trihexyphenidyl > atropine >> benactyzine. In additional study it was shown that prophylactic treatment of rats with pyridostigmine (0.1 mg/kg) either with caramiphen (10 mg/kg) or scopolamine (0.1 mg/kg) prevented the lethal effect of soman (1xLD50) and completely blocked the development of electrographic seizure activity (Raveh et al., 1999). In contrast, only caramiphen abolished somaninduced modifications in NMDA/ion channel characteristics indicated by a marked decrease in Bmax value of [3H]MK-801 binding to brain membranes (Raveh et al., 1999). In addition, caramiphen, but not scopolamine, blocked the somanevoked down-regulation of [3H]AMPA binding to forebrain membrane preparations. Moreover, caramiphen completely prevented the effects of soman poisoning on cognition as tested in the Morris water maze, while scopolamine exhibited only partial protection (Raveh et al., 2002). This set of results highlights the importance of the mixed antiglutamatergic and anticholinergic activity profile against nerve agents poisoning compared with pure anticholinergic activity. In this respect, it is worth mentioning that benactyzine and trihexyphenidyl (both, drugs with mixed activity profile similar to caramiphen), together with pyridostigmine, are approved as a prophylactic mixture called PANPAL by the Czech army against nerve agent exposure (Kassa, 2006). Evaluation of prophylactic treatment using caramiphen and pyridostigmine against sarin exposure (Levy et al., 2007) revealed that this combination confers substantial protection against 1.6xLD50 in monkeys and 1.8xLD50 in dogs at a concentration range of caramiphen of 70–100 ng/ml in plasma, which is regarded as acceptable for humans (Levandoski and Flanagan, 1980).

Prophylactic microinfusion of drugs at various areas of the brain was used to assess their ability to prevent seizures caused by soman. It was found that pretreatment with caramiphen by microinfusion into area tempestas (located deep in the piriform cortex) in rats exposed to soman (ca. 1xLD50) delayed the onset of epileptiform activity and convulsions longer than pretreatment with other drugs with similar pharmacological profiles (benactyzine, biperiden, and trihexyphenidyl) (Myhrer et al., 2008a). Additional prophylactic paradigms consisted of reversible ChE inhibitors that readily enter the brain and thus are more effective than pyridostigmine, were tested, including physostigmine, huperzine A and galantamine. Each was combined with either caramiphen or scopolamine, and their effects were analyzed both in vivo and in vitro, using tolerability and efficacy tests against soman exposure in rats (Mikler et al., 2007). Animals were pretreated l0 min prior to l.5xLD50 soman challenge (s.c.). Results showed that the combination of caramiphen (100 mg/kg, i.m.), with either huperzine A (0.05 mg/kg, i.p.) or galantamine (8.0 mg/kg, i.p.), protected animals with only minimal signs of toxicity, up to four hours following soman exposure. In vitro studies using phrenic nerve or diaphragm preparations suggest that the combination of caramiphen and huperzine A is more effective than caramiphen and galantamine at restoring contractile function following soman exposure. The authors suggested that the combination of huperzine A and caramiphen should be considered as a future pre-treatment regimen against nerve agents poisoning (Mikler et al., 2007). Taken together these results emphasize the superiority of drugs that exhibit both anticholinergic and antiglutamatergic activity such as caramiphen, benactyzine, and trihexyphenidyl, over the potent and specific antimuscarinic activity of drugs such as atropine or scopolamine (Lennox et al., 1992; McDonough et al., 1995; Raveh et al., 1999, 2002; Layish et al., 2005; Myhrer et al., 2008a). Furthermore, the finding that adding an antiglutamatergic activity contributes to the protection against OP when administered prophylactically, strongly suggest that the activation and the involvement of the glutamatergic system occurs immediately, at the very early stage of poisoning with organophosphates and not secondary to the cholinergic hyperactivity (McDonough and Shih, 1997).

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5.2. Caramiphen in post exposure treatment against OP poisoning

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The post-exposure antidotal efficacy of caramiphen was evaluated against organophosphates poisoning in studies conducted in the 1950s’ (Heymans and Estable, 1949; Essig et al., 1950) and showed that caramiphen protected against high doses of DFP (1.5 mg/kg) and effectively abolished the grand mal-like patterns produced by DFP. The article by Madill et al. (1968) describes the effects of caramiphen on sarin (1.2xLD50) poisoning and reports on the behavioral aspects of this drug. Interestingly, caramiphen was found to produce lower incapacitation (as measured by screen climbing and mydriasis) compared with scopolamine and atropine. Rats pretreated with the oxime HI-6, challenged with a convulsant dose of soman (180 mg/kg s.c.) and then treated with drugs such as atropine, trihexyphenidyl, and benactyzine (McDonough and Shih, 1993) revealed the advantage of the latter two compounds over the more selective anticholinergic drugs e.g., scopolamine. In contrast to atropine and scopolamine, benactyzine and trihexyphenidyl showed marked anticonvulsant activity and could even terminate seizures after 40 min. Together, these reports exhibit the ability of caramiphen, trihexyphenidyl, and benactyzine to afford a considerably better protection against nerve agent-evoked seizures and lethality compared with potent antimuscarinic compounds.

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The efficacy of caramiphen as an antidote was also evident when it was administered to rats at various time points after exposure to 1.2xLD50 of soman (Raveh et al., 2003) or sarin (Raveh et al., 2008). The presence of caramiphen in the mixture afforded complete protection. For example, no electrographic seizure activity or elevated levels of translocator protein (TSPO) binding capacity, a reliable marker for brain damage, were detected. In contrast, both parameters indicated brain damage when combinations of atropine and scopolamine (4 or 5 and 0.1 mg/kg, respectively) were employed. Similar results were obtained in the Morris water maze in which caramiphen-treated animals exhibited control-like behavior while scopolamine-injected rats exhibited memory deficits (Raveh et al., 2003). In a later study (Raveh et al., 2008) drugs were injected 5, 10, or 20 min after the initiation of convulsions following exposure to a lethal dose (1.2xLD50) of sarin. Comparison between scopolamine, trihexyphenidyl, benactyzine, and caramiphen revealed that scopolamine was the least effective and caramiphen the most effective antidotal therapy against sarin. The ranking order of the tested drugs with mixed pharmacological profile in post exposure treatment was shown to be: Caramiphen = aprophen > Benactyzine > trihexyphenidyl = procyclidine (Raveh et al., 2008 and our unpublished data). Another study (Katalan et al., 2013), with radio-telemetric electro-corticography monitoring and extensive histopathological evaluations, further confirmed the advantageous neuroprotection afforded by caramiphen to rats exposed to 1.2xLD50 sarin. One of the challenges in the development of antidotal treatment against nerve agents is to obtain efficacious treatment even when its administration is considerably delayed. In the study of Figueiredo et al. (2011) caramiphen (30–100 mg/kg) was given either 30 min or 60 min after exposure to 1.4xLD50 soman (Figueiredo et al., 2011). The treatment suppressed behavioral seizures within 10 min but required one to four and a half hours for complete cessation of seizures. Neuronal loss and degeneration were significantly reduced in the caramiphen-treated, somanexposed rats. Combinations of caramiphen with the benzodiazepine diazepam or levetiracetam (a drug with a unique profile shown to increase the potency of other antiepileptic drugs) following soman exposure were examined lately. In comprehensive studies (Schultz et al., 2012, 2014), caramiphen (20 or 100 mg/kg) and diazepam (10 mg/kg) were administered separately or in combination, at 10, 20, or 30 min after seizure onset following exposure of rats to soman (1.2xLD50) and immediate antidotal treatment with HI-6 and atropine. Results indicated that when treatment was delayed 20–30 min after seizure onset, the mixture of caramiphen and diazepam produced a synergistic effect, shortening seizure duration and reducing neuropathology compared with diazepam alone. Moreover, using a battery of behavioral test to assess motor coordination and function, sensorimotor gating and cognitive function, it was demonstrated that caramiphen, as adjunct to diazepam treatment, attenuated most of the cognitive and motor deficits (Schultz et al., 2014). Another study (McDonough et al., 2010) described the anticonvulsant efficacy, against soman-induced seizures, of various drugs (scopolamine, procyclidine, benactyzine, pentifin, and G-3063). The drugs were applied to the standard treatment of nerve agent poisoning – atropine, 2-PAM and diazepam 5–20 min after seizures started. Results indicated a time-dependent loss of anticonvulsant potential as the treatment delay was lengthened. Of the drugs tested, the anticonvulsant effect of caramiphen was significantly less affected by progressive treatment delay. In an additional study (Myhrer et al., 2011), levetiracetam (50 mg/kg), was combined with either procyclidine (10–20 mg/kg) or caramiphen (10–20 mg/kg). Rats were pretreated with pyridostigmine or HI-6

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(125 mg/kg) to enhance survival and treatment started 40 min after seizure onset following a soman dose of 1.6xLD50. Levetiracetam combined with either caramiphen or procyclidine terminated seizure activity, with survival rate considerably higher for levetiracetam + procyclidine than levetiracetam + caramiphen. It was suggested that the combination of levetiracetam with either procyclidine or caramiphen may be used in a military autoinjector, being effective regardless of the time of application. When comparing the efficacy of various drugs one has to take into consideration the variation in the pharmacological and behavioral effects of each compound. For example, in the study of Myhrer et al. (2011), equal doses of procyclidine and caramiphen were used. We would suggest evaluating more combinations of doses for comparison between these drugs. Procyclidine is more potent as an antiglutamatergic drug than caramiphen: the ED50 for antagonism of NMDA-induced lethality is 21 mg/kg (McDonough and Shih, 1995) compared to ED50 of 80–95 mg/kg for caramiphen (Pontecorvo et al., 1991; Raveh et al., 1999). Cognitive side effects in rats are observed at a dose of 6 mg/kg of procyclidine (Myhrer et al., 2004) whereas the minimal effective dose of caramiphen is 10 mg/kg (Raveh et al., 1996). In humans, 20 mg 3/day of caramiphen were not reported to induce toxic effects (Levandoski and Flanagan, 1980) while cognitive side effects were reported following procyclidine (Roesler et al., 2003). These effects were associated with the anti-NMDA profile of the drug. The recommended human dose of Q4 procyclidine is 2.5–5 mg 3/day (Kemadrin leaflet). Caramiphen was shown to be efficacious against intoxication with OP pesticides. Coleman et al. (1968) pointed out, in the introduction to their article, that caramiphen is effective against parathion poisoning in mice and efficacious against paraoxon, parathion, malathion, and schradan. The authors also presented results showing that caramiphen in conjunction with oximes protects five different animal species against amiton (DSDP). Another report (Jovic and Milosevic, 1970) showed the efficacy of caramiphen in mice poisoned by 1.3xLD50 of amiton and armin. The unique antidotal profile of caramiphen is most likely a result of its actual ‘blend’ of multiple mechanisms, pharmacokinetic, and pharmacodynamic properties. It is possible that in addition to the anticholinergic/antiglutamatergic activity, caramiphen affects other central neuronal systems that are damaged by OP poisoning. As described earlier, caramiphen had been shown to interact with s 1 receptors (Hudkins and DeHaven-Hudkins, 1991; Hudkins et al., 1991; Fletcher et al., 1995). In turn, ligands of sigma receptors had been shown to act as antagonists of the NMDA-ion channel (Pontecorvo et al., 1991; Fletcher et al., 1995) and block fast Ca2+ channels (Church and Fletcher, 1995). Thus, it is reasonable to assume that a considerable s 1 binding component of caramiphen’s activity may contribute to its efficacy against nerve agents. Another aspect of caramiphen’s multifaceted profile was recently reported (Figueiredo et al., 2011). In search for the mechanism of its neuroprotective efficacy, the authors noted that postsynaptic currents evoked by puff-application of NMDA on basolateral amygdala principal cells, were reduced by caramiphen in a dosedependent manner. This is in agreement with the reports by Fletcher and Pontecorvo mentioned earlier showing protection afforded by caramiphen against NMDA seizure and toxicity (Pontecorvo et al., 1991; Fletcher et al., 1995). Using the same system, caramiphen was shown to facilitate GABA-evoked currents which may explain the synergistic effect when given together with benzodiazepines (Schultz et al., 2012). Figueiredo et al. (2011) suggested that NMDA receptor antagonism and facilitation of GABAergic inhibition in the basolateral amygdala may play a key role in the anticonvulsive and neuroprotective properties of caramiphen.


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Exposure to chemical warfare nerve agents causes hyperactivation of the cholinergic system and recruitment of additional neurotransmitter systems. The glutamatergic system is most probably the foremost actor in the excitation process of the CNS and the origin of the epileptic activity that progresses to status epilepticus, resulting in massive brain damage (Weissman and Raveh, 2008, 2011; Eisenkraft et al., 2013). Involvement of other neurotransmitter systems is inevitable and only intervention with a multifunctional drug or a mixture of drugs may prevent or attenuate the ensuing massive central neuronal injury (Weissman and Raveh, 2011). Caramiphen, with its safe toxicological profile, unique pharmacological activity, and its substantial efficacy against OPs poisoning with minimal side effects, is thus a most suitable optimal antidotal treatment. The antidotal regimen against nerve agent poisoning differs in various countries. For instance, in Israel it is based on the immediate use of TAB autoinjector (TMB4, Atropine, and Benactyzine), where the prevailing centrally acting component is benactyzine. Benzodiazepines (diazepam or midazolam) are administered either in cases of a delay in TAB administration and/or appearance of convulsions (Markel et al., 2008). In most military medical protocols, the antidotal combinations consist of an oxime, atropine and a benzodiazepine as the anticonvulsant component. Based on the central pharmacological activities and the safety profile of caramiphen it may serve as a replacement of benactyzine or as an adjunct to benzodiazepines both in immediate and delayed therapy. This was recently addressed in comprehensive studies (Schultz et al., 2012, 2014) where caramiphen was demonstrated to attenuate the physiological, behavioral and neuropathological damage caused by soman when used as an adjunct to standard antidotal therapy. Pharmacokinetic data of caramiphen are scarce. Pulver (1951) reported that the half-life of caramiphen (parpanit) in the plasma of rabbits is 2.5 h and identical results were observed in rats and guinea pigs (our lab unpublished data). Similarly, half-life of 2.34 h was calculated (personal communication with Dr. MK Schultz.) from data of a human study (Levandoski and Flanagan 1980). Mean blood levels of caramiphen, two hours following an oral 20 mg 3/day dose, for four consecutive days, was found to be 37.7 ng/ml and plasma peak levels in part of the tested subjects ranged at 40–80 ng/ml (Levandoski and Flanagan, 1980). Similar plasma concentrations of caramiphen (in the range of 60–100 ng/ml) provided protection against 1.6–1.8xLD50 (i.m.) sarin in dogs and non-human primates (Levy et al., 2007). At these blood levels of caramiphen no side effects were observed. Attempts to find a method for extrapolation of the efficacy of caramiphen from large animals’ studies to humans was performed (Levy et al., 2007). Dogs and non-human primates appear to be more adequate for comparison of physiological, biochemical, and behavioral response to humans, than small animals. Thus, monitoring plasma levels of caramiphen that afford adequate protection against sarin poisoning might predict the needed in humans. The issue of extrapolation of effects and doses from animal to humans is raised frequently albeit it is very complex and intriguing. This is especially emphasized when animal data, as evidence of the effectiveness of a drug against life threatening pathological condition, cannot be ethically or feasibly tested in humans. Further, the issue of the most suitable animal model for OP poisoning and antidotal treatments should be considered carefully as comprehensively discussed by Pereira et al. (2014). Based on the above results one may assume that an oral dose of 30–50 mg will be both safe and efficacious in humans. Further assessments of human pharmacokinetics, pharmacodynamics as well as physiological and behavioral effects are required in order to evaluate the highest safe dose of caramiphen to be used as an OP antidote.

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Conflict of interest The authors declare that there are no conflicts of interest.

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Transparency document

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The Transparency document associated with this article can be found in the online version.

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Acknowledgments

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We wish to extend our appreciation to Dr. E. Grauer, Dr. G. Amitai and Dr. S. Katalan for their critical review of this manuscript.

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Duboisia myoporoides: native antidote against ciguatera poisoning.

Antidote for bromethalin poisoning.

Subacute poisoning with phosalone, an organophosphate insecticide.

Caramiphen edisylate as adjunct to standard therapy attenuates soman-induced seizures and cognitive deficits in rats.

Mania following organophosphate poisoning.

Organophosphate pesticide poisoning.

Organophosphate Poisoning and Terrorism.

Neurological aspects of organophosphate poisoning.

Transient opsoclonus in organophosphate poisoning.

An outbreak of organophosphate poisoning (Thimet) in cattle.

Midazolam as an anticonvulsant antidote for organophosphate intoxication--A pharmacotherapeutic appraisal.

Detoxification of Organophosphate Poisoning Using Nanoparticle Bioscavengers.

Adult respiratory distress syndrome from organophosphate poisoning.

Developments in alternative treatments for organophosphate poisoning.

Amitraz: a mimicker of organophosphate poisoning.

Re: acute abdomen associated with organophosphate poisoning.

Preclinical evaluation of injectable reduced hydroxocobalamin as an antidote to acute carbon monoxide poisoning.

Cases of Datura Poisoning, with Remarks on the Antidote.

Treatment of massive poisoning by the organophosphate pesticide methidathion.

Multi-organ Dysfunction Syndrome with Dual Organophosphate Pesticides Poisoning.

The Organophosphate Paraoxon and Its Antidote Obidoxime Inhibit Thrombin Activity and Affect Coagulation In Vitro.

Delayed Effects of Transcutaneous Organophosphate Poisoning in Four Children.

Two cases of organophosphate poisoning with development of intermediate syndrome.

Organophosphate poisoning with coronary artery vasospasm confirmed by angiography.