Protection by a transdermal patch containing eserine and pralidoxime chloride for prophylaxis against (±)-Anatoxin A poisoning in rats

European Journal of Pharmaceutical Sciences 56 (2014) 28–36

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Protection by a transdermal patch containing eserine and pralidoxime chloride for prophylaxis against (±)-Anatoxin A poisoning in rats Subham Banerjee a,b, Pronobesh Chattopadhyay a,⇑, Animesh Ghosh b, Manash Pratim Pathak a, Jyotchna Gogoi a, Vijay Veer a a b

Division of Pharmaceutical Technology, Defence Research Laboratory, Tezpur 784 001, Assam, India Department of Pharmaceutical Sciences, Birla Institute of Technology, Mesra, 835 215 Ranchi, Jharkhand, India

a r t i c l e

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Article history: Received 26 August 2013 Received in revised form 27 January 2014 Accepted 28 January 2014 Available online 14 February 2014 Keywords: Eserine Pralidoxime chloride Prophylactic Transdermal patch (±)-Anatoxin A Neuroprotection

a b s t r a c t The prophylactic and neuroprotective impact of a transdermal patch containing eserine and pralidoxime chloride (2-PAM) against (±)-Anatoxin A poisoning was investigated using Wistar strain albino rats. Rats were smooth-shaved on the dorsal side, attached with a drug-in-adhesive matrix type prophylactic transdermal patch for 72 h and challenged with subcutaneous injection of three doses (1.0, 1.5 and 2.0  LD50) of (±)-Anatoxin A. The LD50 value of (±)-Anatoxin A was determined to be 1.25 mg/kg, and at this particular dose (1.0  LD50) of toxin induced severe clinical symptom including extreme seizures in rats, resulting acute brain injuries in discrete brain regions, leading to 100% mortality within 5 min. The anticonvulsant effect, antiarrythmic effect, nerve conduction study, clinical observations and mortality, neuroprotective effect as well as skin histopathology of the prophylactic transdermal patch against (±)-Anatoxin A poisoning were investigated systematically. It was found that seizures, tachycardia, nerve damage, clinical symptoms, brain injuries and mortality induced by such lethal toxin were effectively prevented by the prophylactic patch treatment up to certain LD50 level. Hence, it could be a choice of potential therapeutic regimen against such lethal poisoning. Ó 2014 Elsevier B.V. All rights reserved.

1. Introduction Cyanobacteria can produce several potent toxins as secondary metabolites presenting hazard to human and ecosystem health. These organisms can proliferate in freshwater systems forming blooms, dominating freshwater habitats of rivers, lakes and reservoirs. Toxicity of these blooms has concerned health authorities and the scientific community due to their health risk and ecotoxicological implications. It is estimated that 50% of cyanobacterial blooms are toxic. Regarding their mode of action, cyanotoxins are classified into four major groups: neurotoxins, hepatotoxins, cytotoxins and irritants and gastrointestinal toxins (Codd et al., 2005). Among these physiologically distinct alkaloidal neurotoxins, isolated from strains of cyanobacterium (blue–green algae) produced by the filamentous freshwater cyanophyte, Anabaena flos-aquae, have been termed as anatoxins-a,-b,-d,-a(s) and-b(s) (Carmichael and Gorham, 1978). Apart from those neurotoxins, (±)-Anatoxin A (formerly called very fast death factor), a white solid, having ⇑ Corresponding author. Tel.: +91 9435183212; fax: +91 3712 258534. E-mail addresses: [email protected] (P. Chattopadhyay), aghosh@ bitmesra.ac.in (A. Ghosh). http://dx.doi.org/10.1016/j.ejps.2014.01.013 0928-0987/Ó 2014 Elsevier B.V. All rights reserved.

molecular weight (Mw – 281.3) is an alkaloidal potent neurotoxin produced by several genera of cyanobacteria, which are found in waters throughout the world. It is one of the most common worldwide, being reported in countries like USA, Canada, Scotland, Germany as well as in Africa (Osswald et al., 2007). This toxin has caused several poisoning episodes involving wildlife, livestock, and domestic animals (Carmichael, 2001). Additionally, there is a growing concern that humans may be vulnerable to (±)-Anatoxin A poisoning (Stone and Bress, 2007; Dittmann and Wiegand, 2006). Exposure to (±)-Anatoxin A can occur through ingestion, inhalation, or at high concentrations through the skin (Devlin et al., 1977; Patocka and Streda, 2002). It was observed that it is rapidly absorbed after oral administration and can enter the brain from the systemic circulation, which possibly contributes to its rapid lethal effect (Carmichael et al., 1977; Stolerman et al., 1992). Considering the possible exposure routes of all organisms, it is rather curious to verify that most of the experiments about toxicology of (±)-Anatoxin A were done using intra-peritoneal route in rodents. Subcutaneous injection was administered only in one work where 0.2 mg/kg was lethal to one rat only (Stolerman et al., 1992). (±)-Anatoxin A produces its effects primarily by binding selectively and stereospecifically to nicotinic cholinergic receptors of

S. Banerjee et al. / European Journal of Pharmaceutical Sciences 56 (2014) 28–36

the nervous system in rat brain membranes, where it acts as a potent agonist (Soliakov et al., 1995), but it is not hydrolyzed by cholinesterase (Thomas et al., 1993). Typical cholinergic toxic signs can be observed within 5 min of administration, such as loss of muscle coordination, gasping, irregular breathing, tremors, altered gait, twitching, rapid paralysis of the peripheral skeletal and respiratory muscles, convulsions before death due to respiratory arrest can occur within minutes to few hours (Carmichael and Gorham, 1978; Viaggiu et al., 2004; Rogers et al., 2005). Such toxicological signs are quite similar to organophosphate poisoning that also causes cholinergic toxicities such as epileptiform seizures, resulting in brain and cardiac injuries and acute death (Shih et al., 1991a; McDonough and Shih, 1993; Tryphonas and Clement, 1995; Tryphonas et al., 1996; Kim et al., 1999, 2000). The prophylactic efficiency of eserine (carbamate) against organophosphate poisoning is well established (Harris et al., 1984; Leadbeater et al., 1985). Likewise, in vivo pretreatment with eserine/physostigmine and high concentrations of pralidoxime chloride (2-PAM) were the only effective antagonists against a lethal dose of Anatoxin-a(s) poisoning (Patocka et al., 2011). Again, the protective effects of a nonselective muscarinic antagonist (atropine), a cholinesterase-reactivating oxime (2-PAM), and two reversible acetyl cholinesterase inhibitors (physostigmine and pyridostigmine) against fonofos and phosphamidon induced lethality in 24 h Artemia is well documented (Barahona and Sa´nchez-Fortu´n, 2007). Furthermore, it is well known that the pretreatment with carbamates greatly improve the efficacy of antidotes in reducing the lethality induced by diverse organophosphates (Berry and Davies, 1970; Gordon et al., 1978; Dirnhuber et al., 1979). For this reason, eserine has been suggested as an alternative prophylactic against such lethal poisoning because it is an unquaternized carbamate which penetrates the central nervous system (Solana et al., 1990) and its high protective potential in combination with antidotes like oxime derivatives (Dunn and Sidell, 1989). However, eserine has a short plasma half-life and narrow therapeutic window (Becker and Giacobini, 1988; Mohs et al., 1985) which limited its clinical use in humans (Somani and Khalique, 1987; Hartvig et al., 1986) and needs the use of a sustained release formulation (Jenner et al., 1994). In order to overcome this difficulty, we developed a novel sustained release prophylactic drug-in-adhesive matrix type transdermal therapeutic system for the delivery to the skin. Hence, in the present study, a prophylactic drug-in-adhesive matrix type transdermal patch composed of eserine and 2-PAM along with other ingredients was fabricated and transdermally attached to rats for 72 h prior to challenge with various dose levels of (±)-Anatoxin A. Effectiveness of the patch, were evaluated based on anticonvulsant activity, antiarrythmic effect, nerve response study, clinical observation, neuroprotective studies and skin histopathology analysis following (±)-Anatoxin A exposure in rats. The concentrations of eserine and 2-PAM were selected based on the clinically available plasma concentrations in human, without considerable dermal irritation, skin sensitization and acute dermal toxicity (Banerjee et al., 2013a).

2. Materials and methods

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2.2. Test articles Drug-in-adhesive matrix type prophylactic transdermal patches composed of eserine and 2-PAM were dissolved in suitable solvent and mixed with a mixture of pressure sensitive adhesives. The mixture was then casted on drug impermeable backing membrane, dried and finally attached with a protective release liner. (±)-Anatoxin A was dissolved in 0.89% w/v physiological saline solution to make a stock solution (1000 lg/mL) and stored at 4 °C. The stock solution was further diluted in physiological saline solution immediately before use, and administered by subcutaneous injection. The best optimized prophylactic transdermal patch was tested for anticonvulsant effect, antiarrythmic effect, nerve conduction study, clinical observations, mortality, neuroprotective effect and skin histology. Detailed in vitro physico-chemical evaluations, ex vivo skin permeation study on Wistar rats, skin adhesion capability (Chattopadhyay et al., 2013), mutagenicity (Banerjee et al., 2013b) and accelerated stability testing studies (Banerjee et al., 2014) were performed prior to start pharmacodynamic evaluations. 2.3. Animals and maintenance Healthy, adult male Wistar strain albino rats (weighing 200– 228 g, 5–7 weeks of age) were obtained from Defence Research Laboratory (DRL), Tezpur (Reg. No. 1127/bc/07/CPCSEA). The animals were placed in polypropylene cages, with free access to standard laboratory diet (Pranav Agro Industries Limited, Maharastra, India) and provided water ad libitum. Animals were allocated into three groups namely control group (n = 3), toxin treated group (n = 9) and prophylactic transdermal patch treated group (n = 9). Animals were housed in an environmentally-controlled room with temperature of 22 ± 3 °C, 60–70% relative humidity with a 12 h light/dark cycle and ventilation of 15–21 air changes/h for an acclimation period of 7 days to laboratory conditions prior to the beginning of the experiment in order to adjust the new environment and to overcome stress incurred during their transit. Approval to carry out these studies was obtained from the Institutional Animal Ethics Committee (IAEC/DRL/01/JULY/2011) under a subproject and an experiment was performed in compliance with the Principles of Laboratory Animal Care (NIH Publication 85-23, revised 1985). All animal experimental protocols were in accordance with the guidelines of the committee for the purpose of control and supervision of experiments on animals (CPCSEA), Ministry of Forest and Environment, Govt. of India. 2.4. Transdermal application of patch 24 h prior to dermal application of the patch, the hair at the dorsal test sites was closely clipped. The dorsal test site of the animal was depilated by hair remover cream, which was applied to the skin for 3 min, and subsequently the depilator was removed with a spatula and wiped with paper towels, followed by washing under running water and careful drying. Patches were applied to a predetermined area of the depilated test side using a double coated polyethylene tape (9766, 3M Co, St. Paul, MN, USA).

2.1. Materials 2.5. (±)-Anatoxin A challenge Eserine and pralidoxime chloride (2-PAM) were procured from Sigma–Aldrich Chemical, Co. (St. Louis, MO, USA). (±)-Anatoxin A was purchased from Enzo Life Sciences (New York, USA). Formalin, Hematoxylin, xylene and eosin were obtained from HiMedia Laboratories Pvt. Ltd. (Mumbai, India). All biochemical kits were obtained from Coral Clinical Systems (Verna, Goa, India). All other reagents were of analytical grade.

The animals were challenged with (±)-Anatoxin A whose toxicity was assessed on the same day in rats. The LD50 value of this toxin administered subcutaneously to rats was determined to be 1.25 mg/kg (1.0  LD50) and at this particular dose of (±)-Anatoxin A, induced extreme clinical symptoms leading to 100% mortality within 5 min.

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2.6. Anticonvulsant effect Transdermal patch was administered to rats for 72 h prior to subcutaneous poisoning with low (1.0  LD50), medium (1.5  LD50) and high (2.0  LD50) dose levels of (±)-Anatoxin A to reduce the mortality without influencing the seizure activity. The intensity of seizures was evaluated using five-point scores (Kim et al., 1997; Kim et al., 2002; Choi et al., 2004) as follows: 0 = no response; 1 = myoclonic jerks of the contralateral forelimb; 2 = mouth and facial movements and head nodding with or without mild forelimb clonus; 3 = severe forelimb clonus; 4 = rearing and severe forelimb clonus; 5 = rearing and falling. 2.7. Antiarrythmic effect Electrocardiogram (ECG) recordings of control group, toxin treated group and prophylactic transdermal patch treated group were recorded using ECG-100C (NICO-100C, BSLSTMB, Biopac Systems, Inc., California, USA) on a data acquisition system connected to transducers and amplifiers and monitoring electrodes placed on the surface of the skin on the lower chest in anesthetized rats. The incidences of arrhythmias on different groups were evaluated in accordance with the criteria of arrhythmia (Curtis and Walker, 1988) as follows: 0 = no arrhythmia; 1 = <10 s premature ventricular contraction (PVC) and/or ventricular tachycardia (VT); 2 = 11–30 s PVC and/or VT; 3 = 31–90 s PVC and/or VT; 4 = 91–180 s PVC and/or VT, or reversible ventricular fibrillation (VF) of <10 s; 5 = >180 s PVC and/or VT, >10 s reversible VF; 6 = irreversible VF. 2.8. Nerve conduction (NC) study NC is the speed at which an electrical stimulus signal passes through the nerve system. Fresh nerve was attached with the low voltage stimulator (SS58L). In this study NC of toxin treated group and prophylactic transdermal patch treated group were recorded on a data acquisition BSLCBL4B (Biopac Systems, Inc, California, USA) system connected to transducers and amplifiers to record the nerve response. 2.9. Clinical observations and mortality Rats were monitored for clinical symptoms such as seizures, tremors, gasping, arrhythmia, fasciculation, acute asphyxiation, decrease in locomotors activities, latency followed up by twitching, altered gait and physical incapacitation such as incoordination and recumbency. In addition, mortality response ratio, alive/dead and mean time to death were also recorded. The statistical differences for toxin treated group with patch treated group were calculated using unpaired t-test (two-tail P value) the computer software package GraphPad InStat Version: 3.05 (Graph-Pad Software Inc., USA). 2.10. Necropsy Dead and surviving animals were euthanized by cervical dislocation under anesthesia. Necropsy of brain and skin were carried

out for the investigation of neuroprotective effect and patch toxicity evaluation respectively. 2.11. Neuroprotective effect Wistar rats challenged with toxin for the investigation of pharmacodynamic study was subjected to the evaluation of neuroprotective effect of the patch. At the time of death only for the toxin treated group animals (1.5  LD50) after toxin exposure, then for the control group, and finally for the prophylactic transdermal patch treated group (1.5  LD50), whole brain was harvested, washed with normal saline and fixed in 10% neutral buffered-formalin solution by dissolving formaldehyde in phosphate buffer saline (pH-7.4). The brains were dehydrated in different grades of alcohol and embedded in paraffin block, for the evaluation of neural injuries. The sections were cut frontally 5 lm in thickness by using motorized rotary microtome (Spencers India Pvt. Ltd., New Delhi, India) and then stained with hematoxylin and eosin (HE). Neuronal death and integrity of neutrophils in hippocampus, the most-susceptible region, were examined under a phase contrast light microscope (Carl Zeiss Microimaging, Germany) and photographed using a camera (AxioCam, ERc5s, Carl Zeiss Microscopy, Germany) controlled by software (Axio Vision, Release-4.8.2, SP2, Germany) for any histopathological changes. The degree of brain injury was evaluated using 5-point scores based on the approximate percentage of tissue involvement according to the grading system of McDonough et al. (1995) with a slight modification (Kim et al., 1999, 2000). 0 = no lesion; 1 = minimal (1–10%); 2 = mild (11–25%); 3 = moderate (26–45%); 4 = severe (46–60%); 5 = extreme (>60%). The experiments performed here were conducted according to the ‘‘Guide Principles in the Use of Animals in Toxicology’’ which had been adopted by the Society of Toxicology in 1989 (Society of Toxicology, 1989). 2.12. Skin histology Skin samples, before and after 72 h patch application, were taken from the application site and were washed with normal saline and preserved in 10% neutral buffered-formalin solution. Samples were dehydrated in ascending degrees of ethyl alcohol (70%, 80%, 90% and 100%), cleared in xylene, and embedded in paraffin. A 7 lm tissue sections were cut and stained with H&E, examined, photographed under a phase contrast light microscope (Carl Zeiss Microimaging, Germany). 3. Results 3.1. Anticonvulsant effect Rats challenged with low (1.0  LD50), medium (1.5  LD50) and high (2.0  LD50) dose levels of (±)-Anatoxin A exhibited extreme seizures by falling and rearing (score 5.0), displaying early tremors followed by death (Table 1). Interestingly, the convulsant effect induced by toxin was protected by the administration of prophylactic transdermal patch to a significant extent. Note-worthy, the animals pretreated with patch did not show any considerable convulsive signs with low (score 1.0) and medium (score 2.0) LD50 dose levels, but failed to prevent at high (score 4.0) LD50 dose levels

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Table 1 Effects of different groups on the incidences of clinical signs, mortalities and other effects occurred in male Wistar strain rats induced by (±)-Anatoxin A administered subcutaneously. Treatment group

Toxin dose (LD50) (n = 3)

Duration of patch attachment (h)

Observed clinical signs

Mortality response ratio

Survived (S)/died (D)

Time to death in min (mean, ±SD)

Anticonvulsant effect score

Antiarrythmic effect score

Neuroprotective effect score

Control group/ 0.89% w/v Normal saline (n = 3) Toxin treated group (±)-Anatoxin A (n = 9)

Not given

Not applied

No clinical signs were observed

0/3

S

–

0

0

0

1.0 1.5 2.0

Not applied

3/3 3/3 3/3

D D D

4.63 ± 0.07 3.97 ± 0.09 3.01a ± 0.03

5

6

5

Prophylactic transdermal patch treated group (n = 9)

1.0

72

Extreme seizures, tremors, tachycardia, gasping, fasciculation, acute asphyxiation, latency followed up by twitching, decrease in locomotors activities, coma and death. Minimal seizures, tremors and slight decrease in locomotors activities. Mild seizures, tremors, gasping, fasciculation and slight decrease in locomotors activities. Severe seizures, tremors, tachycardia, gasping, fasciculation, acute asphyxiation, latency followed up by twitching, decrease in locomotors activities and death.

0/3

S

–

1

1

1

0/3

S

–

2

2

2

3/3

D

97.43a ± 1.5

4

4

4

1.5

2.0

a

Significantly different (P 6 0.05).

displaying severe seizures, tremors followed by prolonged epilepsy, which ultimately leads to death. Such protective effects at low and medium LD50 levels might be due to the observations that 2-PAM potentiated the level of the protective effect of eserine. Note-worthy, this prophylactic transdermal patch attenuated seizures to certain LD50 levels, and thereby markedly reduced the degree of brain injuries as evidenced by the histopathological findings (1.5  LD50), suggestive of the seizure-related hypoxicischemic encephalopathy (Fig. 3). Moreover, the seizures and brain injuries were fully prevented by the combined action of eserine and 2-PAM. Such effects might be supported by the observations that eserine potentiated the antidotal and anticonvulsant actions of anticholinergics (Dunn and Sidell, 1989; Kim et al., 1998). In addition for control group animals, arrhythmia score was found to be zero (Table 1).

markedly reduced by the application of transdermal patch. Such protective effects at low and medium LD50 dose levels might be due to the observations that 2-PAM potentiated the prophylactic and cardiovascular actions of eserine. In addition toxin treated group and control group rats, arrhythmia score were 5.0 and 0.0 respectively (Table 1). 3.3. NC analysis NC recordings of toxin treated group (1.5  LD50) showing no nerve response (Fig. 2a) in comparison to prophylactic transdermal patch treated group rats (Fig. 2b), indicating (±)-Anatoxin A causes severe damage in nerve terminals to generate nerve response against stimulation. But the application of transdermal patch in medium LD50 level considerably generated nerve response of toxin treated group at the same LD50 level.

3.2. Antiarrhythmatic effect 3.4. Clinical observation and mortality To define the protective effect of the transdermal patch on cardiac arrhythmias, standard lead ECG was recorded to check the electrocardiogram profile and score of arrhythmias performed on Wistar rats induced by (±)-Anatoxin A administered subcutaneously. The electrocardiogram profile of toxin treated group (Fig. 1b) was significantly abnormal in comparison to control group rats (Fig. 1a), indicating that this toxin is causal factor to serious cardiac arrhythmias. Administration of prophylactic transdermal patch in medium LD50 dose level considerably compensated electrocardiogram profile of toxin treated group at the same LD50 level (Fig. 1c). Moreover, arrhythmia score, a commonly used parameter to evaluate the severity of arrhythmia, was decreased in low (score 1.0) and medium (score 2.0) LD50 dose levels in prophylactic transdermal patch treated group as compared with high (score 4.0) LD50 dose level. Hence, it is observed that the score of arrhythmia was

In the present study, rats exposed to various LD50 dose of toxin exhibited extreme seizures, tremors, tachycardia, gasping, fasciculation, acute asphyxiation, latency followed up by twitching, decrease in locomotors activities, coma and death in 100% rats within 5 min after (±)-Anatoxin A challenge. Effects of the patches on the incidences of toxic clinical signs and mortalities of rats are summarized in Table 1. In contrast, the rats attached with the prophylactic transdermal patch containing eserine and 2-PAM for 72 h before challenge with low (1  LD50) and medium (1.5  LD50) dose levels of toxin did not show any mortality in rats. Only mild transient seizures were observed. The patch exerted a full protection against seizures and mortality of rats poisoned with low and medium LD50 dose level, but no effect on high (2.0  LD50) dose level of toxin, although markedly delayed the time to death of animals.

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Fig. 1. ECG recordings of (a) control group, (b) toxin treated group (1.5  LD50) and (c) prophylactic transdermal patch treated group (1.5  LD50).

3.5. Necropsy Following necropsy, no visual macroscopic changes were observed in the external and internal organs of all treated groups. 3.6. Neuroprotective effect The severities and degrees of brain injuries induced by (±)-Anatoxin A poisoning were investigated and compared between control groups (Fig. 3a), toxin treated group (Fig. 3b) and prophylactic transdermal patch treated group (Fig. 3c) in experimental rats. Two brain regions, cerebral cortex and hippocampal CA1 region which has been known to be the most susceptible against neurotoxin, the neurodegeneration was found to spread to all hippocampal formation (CA1–CA4 and dentate gyrus) in severe cases were inspected. (±)-Anatoxin A induced seizures in rats led to severe neuropathological changes likewise, dark shrinkage degeneration of neurons, forming a pericellular halo and microglial cell infiltration were observed to be more serious in hippocampal CA1 region and brain injuries in hippocampus, cortices, amygdala and thalamus within 5 min (score 5.0), showing dark degeneration of pyramidal neuronal cells and malacia of neutrophils. It is worthy of note that such neuroprotective effects remarkably reduced by the pretreatment with patch at low (score 1.0) and medium (score 2.0) LD50 dose level in brain regions examined, exhibiting normal features such as shrunken neurons with eosinophilic cytoplasm and nucleic condensation, in comparison with intact feature of cerebral cortex of the control group animal (Fig. 3a). In toxin treated group, the rats exposed to all LD50 level of toxin exhibited extreme seizures and 100% mortality with mean survival time of 3.87 min, leading to score 5.0 of brain lesions (Table 1). In contrast, the patch-attached animals survived the challenge with

1.5  LD50 of toxin did not show extreme brain lesions (score 2.0), and the rats exposed to 1.0  LD50 of toxin exhibited only a minimal neuronal injuries (score 1.0). Interestingly, such prophylactic transdermal patch fully prevented death and brain injuries of the rats exposed to 1.0 and 1.5  LD50 of toxin, although the prophylactics did not exert remarkable neuroprotective effect in rats challenged with 2.0  LD50 of toxin. Similar type of observation was also found by Kim et al. in dogs against soman poisoining (Kim et al., 2005). 3.7. Skin histology The histopthological changes in the excised rat skin before and after (72 h) application of the patch were given in Fig. 4a and b respectively. Normal skin (before application of the patch) had clearly defined stratum corneum, with well-woven structures and no inflammatory cell infiltration or change in the skin appendages. On normal observation, a very slight sub-epidermal edema and collagen fiber swelling was observed, which was ascribed to the removal of the hair from the skin but no significant change was observed histologically. In spite of the relatively large duration patch applied on the rat skin, no toxic signs and symptoms were observed based on skin histology findings at a fixed magnification. 4. Discussion In the present study, we investigated the prophylactic, anticonvulsant, antiarrythmic, nerve conduction velocity, clinical signs, neuroprotective effect and dermal toxicity of our fabricated matrix-type transdermal therapeutic system composed of eserine and 2-PAM as a basic research in order to develop a non-invasive prophylactic patch addressed to populations at risk of poisoning

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Fig. 2. NCV recordings of (a) toxin treated group (1.5  LD50) and (b) prophylactic transdermal patch treated group (1.5  LD50).

Fig. 3. Histopathological findings of (a) control group, (b) toxin treated group (1.5  LD50) and (c) prophylactic transdermal patch treated group (1.5  LD50) on hippocampal and neocortical brain injuries. Note the pyramidal neurons exhibiting dark degeneration, leading to pericellular halo, in hippocampus of a rat intoxicated with 1.5  LD50 dose (±)-Anatoxin A (b), in contrast to mild injuries in a rat attached with a patch (c), but no neural injuries in a rat observed by control group showing normal features of neural region (a). The arrows represent the points of the neuropathological changes. [HE stain  100].

with such lethal toxin. It was established that the fabricated transdermal patch exerted a protective effects against such poisoning up to certain LD50 level. In addition, it was also observed from the earlier studies that the possible adverse effects of carbamates and anticholinergics used prophylactically might be offset by each other (Berry and Davies, 1970; Lim et al., 1991; Philippens et al., 1996, 2000a⁄⁄⁄). For example, the doses of anticholinergics influencing the behavioral, learning and memory, and physiological performances of rats were increased by the combination with carbamates to some extent (unpublished data). Taken together, it is suggested that carbamates and anticholinergics could be promising mate antidotes for the prophylaxis against organophosphate poisoning. Again it is well known that eserine readily penetrated the skin enough to reach the blood concentration by exerting high antidotal, anticonvulsant and neuroprotective effects (Kim et al., 1997, 1998, 2000). It was found that a combination of eserine plus

2-PAM is of high value significantly increases the level of the protective effect of 2-PAM alone against fonofos and phosphamidon exposures (Barahona and Sa´nchez-Fortu´n, 2007). They also examined whether the presence of 2-PAM, a cholinesterase-reactivating oxime, applied together with physostigmine or pyridostigmine, would show a synergic effect. Their data lead us to suggest that the use of eserine plus 2-PAM is of high value for the protection to populations at risk of (±)-Anatoxin A poisoning. Our investigation also demonstrated that in fabricated prophylactic transdermal patch treated group showed almost normal electrocardiogram profile in comparison to control group rats. In this study patch showed obvious protective actions against cardiac arrhythmias caused by such lethal poisoning in rats. Moreover, this prophylactic therapeutic regimen effectively restricted the incidence of cardiac arrhythmias and electrocardiogram profile observed in toxin treated group, which may attributed to the

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Fig. 4. Histopathological findings of excised rat skin (a) before application and (b) after 72 h application of the prophylactic transdermal patch. Note the intact structure of the epithelial line and reticular dermal layer (a), in contrast to the passage of drugs permeation through the slight disruption of epithelial line to reticular dermal layer (b). The arrows represent the points of the dermatological changes. [HE stain  100].

partial reversal action on the accelerated depolarization and shortened plateau in our experimental animal model. This reveals the incapability of patch to show the severe incidence of cardiac arrhythmias in comparison to toxin treated group of rats. Thus, it explains our understanding of anti-arrhythmic action from the pharmacological point of view. Moreover, it is also noted that patch administered in medium LD50 dose level considerably compensated ECG profile of toxin treated group at the same dose level (Fig. 1c), but not fully compensated the ECG profile of control group. It might be due to seizure-related cardiac changes induced by toxin effects but it does not appear to cause clinically significant abnormalities for the rats (Maromi Nei, 2009). Finally at this particular dose level all the animals were survived and showed zero mortality with a mild intensity of PVC and/or VT. The generation of nerve response in prophylactic transdermal patch treated group in medium LD50 dose level might be due to eserine, which is a powerful sialogogue, stimulates almost all involuntary muscles in the body and reversible acetylcholine esterase inhibitor, which effectively increases the concentration of acetylcholine at the sites of cholinergic transmission. Its carbamate functional group binds to the active site of cholinesterase in a similar manner to the binding of cholinesterase inhibitors to cholinesterase (Somani and Dube, 1989). The carbamylation of the active site of cholinesterase ensures that the active site is protected from attack by this potent neurotoxin, which binds to the enzyme permanently. The result is a continual stimulation of receptor cells that causes intense spasms of muscles due to the prevention of a breakdown of acetylcholine. This reversible carbamylation accounts for the prophylactic properties of eserine. It was found that toxin treated group animals were died within 5 min after the subcutaneous administration of (±)-Anatoxin A. Therefore, the death might come from acute brain injury due to severe hypoxic-ischemic encephalopathy, rather than convulsions. The primary causes of this condition are systemic hypoxemia and continued oxygen deprivation to brain results in fainting, loss of consciousness, coma, seizures, cessation of brain stem reflexes and acute brain death. In this context, the developed matrix-type transdermal patch system, in which the drug concentrations were optimized to minimize skin irritancy, sensitization as well as acute dermal toxic effects of the patch. The patch fully protected seizures and mortality induced by low (1.0  LD50) and medium (1.5  LD50) dose of toxin in rats. The patch might have exhibited constant blood concentrations of eserine and 2-PAM over 72 h in hairless rats. The sustained release of these two drugs from the patch also might have led to stable profiles of blood concentrations. On the other hand, rats pretreated with the patch against high dose level (2.0  LD50) of toxin exposure exhibited profound symptoms of intoxication such as seizures, tremors, gasping, latency followed by twitching, tachycardia, acute asphyxiation, altered gait, decrease in locomotors activities, severe brain injury,

coma and ultimately leads to death in 97.43 min might be due to severe convulsions, thus making it difficult to stop the long-lasting seizures. Differences were considered to be significant at a probability level of P 6 0.05 between toxin treated group vs. patch treated group at high LD50 toxin dose level. Fig. 3b shows the hippocampal, neocortical brain injuries and neuropathological changes such as pyramidal neurons exhibiting dark degeneration, leading to pericellular halo, in hippocampus of a rat as a result of epileptic seizures. In contrast, the prophylactic transdermal patch treated rats underwent mild clinical signs with medium LD50 challenge of toxin showed generally normal features in brain region (Fig. 3c). Hence it can be said that the prophylactic transdermal patch fully protected rats from seizures and mortality against low and medium dose of toxin (Table 1) without giving any additional treatment under the condition that blood concentrations of the drugs are much lower than sign-free doses, but the patch treatment in high (2.0  LD50) dose of toxin not achieved the protection against this lethal poisoning. Again no significant damage to the epidermis was found in samples taken 72 h after application of the patch, but slight disruption was found in stratum corneum layer might be indicating the slow permeation of the drugs through epithelial line located in stratum corneum layer to reticular dermal layer in order to reach systemic circulation. No cellular infiltration within the bullas and in the papillary dermis was found. Although there are no severe morphological changes were observed in the skin structure following prolonged patch application, indicates the safety of the test formulation. Based on the displayed table and figures, the patch shows promising results in view of the prevention of rats from toxic clinical signs, seizures, brain injuries and mortality up to certain LD50 level. Thus, it is expected that a remarkable effect could be achieved with the patch in primates including human. Further studies, now in progress, will deal with the pharmacoscintigraphic and pharmacokinetic application of the presently reported findings in experimental animal model, indicating its clinical application. The doses of the patch applicable to human are expected to be further optimized in the clinical trials scheduled in the near future.

5. Conclusion Our study demonstrates the protective effect of the developed transdermal patch against (±)-Anatoxin A poisoning in rats. The mechanism of efficacy appears to be due to sustained and selective delivery of the two drugs into the brain in rats from a drug-inadhesive matrix type prophylactic transdermal patch in neutralizing the deleterious effect of (±)-Anatoxin A. Thus, the overall outcomes in patch system presented effective action on brain in experimental animal model which exhibits greater technique and

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offers a novel effective noninvasive drug delivery system for brain delivery through dermal route. Taken together, it is suggested that the matrix type prophylactic transdermal patch system composed of eserine and 2-PAM, could be a choice for the quality survival against such lethal poisoning.

Disclosure The authors declare that they have no conflicts of interest to declare in connection with the contents of this manuscript.

Contributors Subham Banerjee, Pronobesh Chattopadhyay, and Animesh Ghosh were responsible for macro/micro-planning, conception and design of the study. Jyotchna gogoi and Manas Pratim Pathak helped a lot in animal experiment, histopathological examination, data interpretation and in writing of manuscript. Vijay Veer critically reviewed the manuscript and approved the final version of the article submitted for publication. Acknowledgments Subham Banerjee is grateful to Defence Research and Development Organisation, Ministry of Defence, Govt. of India for providing necessary facilities and research fellowship for this work. The authors also thankful to the Birla Institute of Technology, Mesra, Ranchi, India for providing necessary administrative support for carrying out Ph.D work. References Banerjee, S., Chattopadhyay, P., Ghosh, A., Bhattacharya, S.S., Kundu, A., Veer, V., 2014. Accelerated stability testing study of a transdermal patch composed of eserine and 2-PAM for prophylaxis against (±) Anatoxin-A poisoning. J. Food Drug Anal. http://dx.doi.org/10.1016/j.jfda.2014.01.022. Banerjee, S., Chattopadhyay, P., Ghosh, A., Pathak, M.P., Singh, S., Veer, V., 2013a. Acute dermal irritation, sensitization, and acute toxicity studies of a transdermal patch for prophylaxis against (±)-Anatoxin A poisoning. Int. J. Toxicol. 32, 308. Banerjee, S., Singh, S., Policegoudra, R., Chattopadhyay, P., Ghosh, A., Veer, V., 2013b. Evaluation of the mutagenic potential of a prophylactic transdermal patch by Ames test. J. Immbio. 28, 322. Barahona, M.V., Sa´nchez-Fortu´n, S., 2007. Protective effect induced by atropine, carbamates, and 2-pyridine aldoxime methoiodide Artemia salina larvae exposed to fonofos and phosphamidon. Ecotox. Environ. Safe. 66, 65. Becker, R.E., Giacobini, E., 1988. Mechanisms of cholinesterase inhibition in senile dementia of alzheimer Type: clinical, pharmacological and therapeutic aspects. Drug Dev. Res. 12, 165. Berry, W.K., Davies, D.R., 1970. The use of carbamates and atropine in the protection of animals against poisoning by 1,2,2trimethylpropylmethylphosphonofluoridate. Biochem. Pharmacol. 19, 927. Carmichael, W.W., 2001. Health effects of toxin-producing cyanobacteria: ‘‘The CyanoHABs’’. Hum. Ecol. Risk Assess. 7, 1393. Carmichael, W.W., Gorham, P.R., 1978. Antitoxins from clones of Anabaena flosaquae isolated from lakes of western Canada. Mitt. Int. Verein. Llmnol. 21, 285. Carmichael, W.W., Gorham, P.R., Biggs, D.F., 1977. Two laboratory case studies on the oral toxicity to calves of the freshwater cyanophyte (blue-green alga) Anabaena flos-aquae NRC-44-1. Can. Vet. J. 18, 71. Chattopadhyay, P., Banerjee, S., Ghosh, A., Veer, V., 2013. Matrix type transdermal patch formulations. Patent Application No. 3033/DEL/2013. Choi, E.K., Park, D., Yon, J.M., Hur, G.H., Ha, Y.C., Che, J.H., Kim, J., Shin, S., Jang, J.J., Hwang, S.Y., Seong, Y.H., Kim, D.J., Kim, J.C., Kim, Y.B., 2004. Protection by sustained release of physostigmine and procyclidine of soman poisoning in rats. Eur. J. Pharmacol. 505, 83. Codd, G.A., Morrison, L.F., Metcalf, S.J., 2005. Cyanobacterial toxins: risk, management for health protection. Toxicol. Appl. Pharmacol. 203, 264. Curtis, M.J., Walker, M.J., 1988. Quantification of arrhythmias using scoring systems: an examination of seven scores in an in vivo model of regional myocardial ischaemia. Cardiovasc. Res. 22, 656.

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