Designing and Preclinical Evaluation of a Molecular Imprint Polymer-Based...

[Full title: Designing and Preclinical Evaluation of a Molecular Imprint Polymer-Based Cocaine Odor Mimic for Conditioning Detection Dogs]

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OPEN ACCESS International Journal of Pharmacology ISSN 1811-7775 DOI: 10.3923/ijp.2022.171.181 Research Article Designing and Preclinical Evaluation of a Molecular Imprint Polymer-Based Cocaine Odor Mimic for Conditioning Detection Dogs 1 Alonso Sierra-Resendiz, 2 Berenice Robles-Heredia, 3 María J. Bernad-Bernad, 4 Roberto Díaz-Torres, 1 Sheila I. Peña-Corona, 1 Dinorah Vargas-Estrada and 2 Jesús Gracia-Mora 1 Department of Physiology and Pharmacology, Veterinary Medicine School, National Autonomous University of Mexico, Mexico 2 Department of Inorganic Chemistry, Faculty of Chemistry, National Autonomous University of Mexico, Mexico 3 Department of Pharmaceutical Technology, Faculty of Chemistry, National Autonomous University of Mexico, Mexico 4 Multidisciplinary Research Unit, Faculty of Chemistry, FES Cuatitlan, National Autonomous University of Mexico, Mexico Abstract Background and Objective: The use of trained dogs to detect illegal or dangerous substances and goods is an increasingly used tool The present work proposed to design a polymer that imitates the odor of cocaine HCl using molecular printing technology, with a mixed release to maintain a balance of the volatile compound between the medium and the MIP, intended for the conditioning of detector dogs. Materials and Methods: Design of a Molecular Imprinting Polymer (MIP) based on the monomer 4-vinyl-pyridine and ethylene glycol-dimethacrylate (EGDMA) crosslinker for the controlled desorption of methyl benzoate to mimic the "headspace" of cocaine- HCL, comparing its behaviour with a preparation based on the non-specific adsorbent most used in commercial products, impregnated with the same desorbent at the saturation point of each material. Its function was tested in Wistar rats and assessed its acute toxicity Results: Odor desorption was tested over seven days of environmental exposure, considerably longer desorption was achieved than with the challenged commercial product (49.117% MIP vs. 90.810% challenge compound). The rats were trained with the designed prototypes and no toxicity was found in the blood chemistry results. Conclusion: The designed MIPs are innocuous and has better performance, reaching a faster and more sustained equilibrium of the supernatant vapour, showing a quality, intensity and duration of the odor stimulus useful for the conditioning of biosensors, demonstrating that the MIP design technology represents a broad, versatile and solid option for the design of odor imitators Key words: Molecular imprint polymers, odoriferous signature, odor mimics, selective adsorption, controlled desorption Citation: Sierra-Resendiz, A., B. Robles-Heredia, M.J. Bernad-Bernad, R. Díaz-Torres, S.I. Peña-Corona, D. Vargas-Estrada and J. Gracia-Mora, 2022. Designing and preclinical evaluation of a molecular imprint polymer-based cocaine odor mimic for conditioning detection dogs. Int. J. Pharmacol., 18: 171-181 Corresponding Author: Dinorah Vargas, Department of Physiology and Pharmacology, Veterinary Medicine School, National Autonomous University of Mexico, Mexico Copyright: © 2022 Alonso Sierra Resendiz et al. This is an open access article distributed under the terms of the creative commons attribution License, which permits unrestricted use, distribution and reproduction in any medium, provided the original author and source are credited. Competing Interest: The authors have declared that no competing interest exists Data Availability: All relevant data are within the paper and its supporting information files.

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Int. J. Pharmacol., 18 (1): 171-181, 2022 INTRODUCTION The use of trained dogs to detect illegal or dangerous substances and goods is a tool used by government institutions in charge of public safety 1 . The origin of this activity dates back to the sixties of the last century 2,3 , to achieve conditioning as a consequence of the simultaneous connection of an odoriferous stimulus emanating from the “aid substances” (conditioned stimulus) and a satisfier that triggers an emotion from which a determine behavior or is molded. At the beginning of the conditioning, the smell has a neutral physiological meaning but in the end, the close association between the two elements will induce the nervous system to give it a particular meaning, the odorant must guarantee an appropriate quality, constant intensity and purity that avoid confusion in the learner and consequently a successful change of emotion, so that the succession of exposures, causes the individual to spontaneously repeat the learned behavior, until the consolidation of conditioning 4 To date, a sector dedicated to this activity is inclined towards using real substances, which represents several inconveniences: Risks of ingestion and intoxication of canines, presence of contaminants, special storage requirements, legal risks derived from its possession, transfer and use. Another sector shows a marked interest in developing "aid odorant substances" that allow inducing optimal conditioning with less complex conditions of use and storage, avoiding the risks of using real substances 5 . There are different “helper substances” on the market, designed for the conditioning of different specialties, such as the search and location of drugs, explosives, people and pests: bed bugs ( Cimex sp.), moths ( Hylotrupes bajulus ), etc 6 . A first challenge in the design of mimicking substances is to identify "the characteristic odor" or "odoriferous signature" of the substance to be detected, which is made up of a set of “dominant volatile compounds” that are part of the “supernatant gas,” these have the ability to produce a response in instrumental and biological methods 6 . Cocaine, the drug of interest in this study, is composed of an ester of benzoic acid and a base that contains nitrogen, its maximum stability is presented at a pH close to 3.0, so depending on the temperature and pH, it undergoes hydrolysis reactions 6-9 . In various works of gas chromatography and mass spectrometry, it is reported as the main product of the decomposition of cocaine hydrochloride to methyl benzoate, which is highly detectable since it has a very high vapor pressure, 0.38 Torr at 25 E C, a dissipation rate of 62 ng min G 1 at 40 E C and with 80% relative humidity 10,11 The second challenge for the design of aid substances are the so-called "matrices" whose characteristics are the preservation and control of odor release, its main function is to allow the emanation of a constant and unmistakable odorous stimulus, the approaches to solve this problem are directed to the choice of non-specific adsorbent materials such as silica, cellulose derivatives or non-specific polymers, alone or in combination 12-14 , the quality that in some cases allows the design of products with compound odors, with imitators with one and with several odors contained in the same product 15-17 Molecular Imprint Polymerization (MIP) is a technology that has existed since 1931 18 which consists of the design of compounds that have the ability to adsorb and desorb specific molecules, through the design of high-crosslinking macromolecular structures for the selective recognition of a substrate, its design is based on the functioning models of biological molecular recognition systems such as the antigen-antibody ratio (Ag / Ac), enzyme-substrate, hormonereceiver, etc 19,20 . MIPs are obtained by the polymerization of a monomer around a template molecule that, when extracted, generates dimensionally and structurally compatible cavities as well as a distribution of functional groups, which makes possible a selective recognition, so that molecules with different molecular weight, which do not have the geometric characteristics or functional groups of the template molecule, they are not capable of being incorporated into these spaces, so the MIP and the template molecule are in optimal interaction conditions, generally reversible because the most frequent design is through non-covalent relationships The molecule to be imprinted acts as a complement to one or more functional monomers through reversible noncovalent electrostatic relationships, such as the relationship between dipoles, electrostatics or hydrogen bonding, etc 21,22 that interacts with functional monomers, in the manner of a preorganized structure, which is exposed to a crosslinker and a polymerization initiator The relationship between free radicals forms structures, when the template molecule is removed, the spaces or pores specifically to the molecule used will remain. Its selectivity allows to know the maximum retention capacity and through specific procedures, it is possible to know the desorption behavior with high reliability and precision the desorption behavior. MIPs are adsorbent materials par excellence due to their affinity, specificity and generally reversible binding with the substrate, they allow efficient adsorption and prolonged or controlled desorption through specific washes, in addition to offering high chemical stability, they are generally inert to acids, bases, metal ions and organic solvents, high stability to physical changes such as temperature and pressure, giving them a long half-life with reuse capacity 23,24 . MIPs are solid compounds that can be 172

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Int. J. Pharmacol., 18 (1): 171-181, 2022 designed in-ground particles, regular spherical particles, nanoparticles or monoliths of relatively simple, reproducible synthesis, with excellent versatility and robustness 25 This technology is used in the design of sensors, catalysts, modified release pharmaceutical forms, chromatography, among others, even the adsorption of residues resulting from the decomposition of illicit drugs such as cocaine, this work represents an opportunity for innovation in using this technology for the controlled desorption of odorants useful for the odorant conditioning of biosensors 26,27 The proposed MIP is considered an imitator because the odorant emanated is the product of the isothermal decomposition of real drugs, so the lack of legal restrictions, the absence of psychoactive or toxic effects, represents extremely interesting elements for its use 28,29 In the present study, a Molecular Imprinting Polymer (MIP) based on the monomer 4-vinyl pyridine was designed to control the desorption of methyl benzoate to imitate the “headspaces” of cocaine, it was validated and characterized the desorption of methyl benzoate and was compared against that obtained from silica, to determine the differences between a specific material and a non-specific adsorbent MATERIALS AND METHODS Study area: The design of the molecular imprinting polymer as a cocaine odor mimic was carried out in Laboratory 100 of the Department of Inorganic and Nuclear Chemistry of the Faculty of Chemistry of UNAM, The rheological study Department of Pharmacy, Faculty of Chemistry, National Autonomous University of Mexico, Mexico City, Mexico, from January, 2018-December, 2019 The release study was carried out at the Department of Physiology and Pharmacology, Faculty of Veterinary Medicine, National Autonomous University of Mexico, Mexico City, Mexico, from January-November, 2020 Materials Reagents: 4-vinyl pyridine (4-VP) 97%, Silica gel 60, brand products Sigma Aldrich, (DEU. GER), ethylene glycol dimethyl acrylate 98%, (EGDMA), Acetonitrile 99.5% (ACN), Methanol (MeOH) QP grade, brand products Merck, (DARM, GER), acrylamide 98% (AA), Fluka analytical, ethanol (EtOH) reagent grade, brand products J.T Baker, (NJ USA), Azobisisobutyronitrile 98% (AIBN) Akzonobel (CAU, COL), Methyl benzoate 99% (MB) Alfa Aesar, (MA, USA), acetone, phosphoric acid 85%, Supelco, (DEU. GER) were used. The reagents and solvents were not purified before use Equipment: Multi-shaker IKA, RO-5-P-S 1(ILM, USA), centrifuge Gilson GMC Lab, model F (WI, USA), Grinder Cole Palmer (IL, USA), Spectrophotometer UV/vis Ocean optics, DT-mini 2 GS(NY, USA), Thermo shaker Biobase, BJPX-1008 (Braunschw, GER) Experimental method Synthesis of MIP: The polymerization method was carried out by free radicals, using the stoichiometry of monomer, crosslinker and last (4: 20: 1), the synthesis was carried out by the following reaction:     4 vp AIBN 65 C 24 hrs 4 4 VP 20 EGDMA 1 MB MIP        The 4-vinyl pyridine polymer was prepared from the solution of the monomer (4-vinyl pyridine) (0.181 µL), with a template molecule (Methyl benzoate) (66 µL), cross linker (Ethylene glycol dimethyl acrylate) (1.84 µL) and initiator of polymerization (Azobisisobutyronitrile) (11.62 mg). In a 10 mL test tube, 5 mL of acetonitrile were added, it was closed with a screw cap, the sample was sonicated for 10 min and oxygen was removed by including nitrogen for 5 min. It was incubated for 24 hrs at 65 E C in Fig. 1 Fig. 1: Reaction of a molecular impression polymer based on the 4-VP monomer to control the desorption of MB to mimic the “headspaces” of cocaine MB: Methyl benzoate, (4-VP) 4-vinylpyridine, EGDMA: Ethylene glycol dimethyl acrylate, AIBN: Azobisisobutyronitrile, MIP: molecular impression polymer 173 Assemble Monomer Crisscrosser Molecule mold Initiator MIP loaded Desorption and/or washing AIBN 65°/24 hrs MB (1 M) 4-VP (4 M) EGDMA (15 M) Experience reaction addition Free radical initiator ±70°C releases nitrogen 6 times° Polymerization

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Int. J. Pharmacol., 18 (1): 171-181, 2022 The result of the process is a monolith, requiring the rupture of the test tube to obtain it, the stone was crushed in a mill, the pulverized produced was washed with 20 mL of ethanol and acetone alternately to eliminate the residues that did not react. Finally, the powder obtained was sieved through a 250-micron mesh Successive washes removed the template molecule with ethanol at room temperature for 3 hrs, the presence of methyl benzoate was monitored by UV spectrometry. Finally, the powder obtained was dried at 50 E C. As part of the MIP adsorption evaluation methodology, an unprinted polymer (NIP) was prepared with a similar procedure in the absence of the template molecule Maximum retention capacity: The adsorption capacity of MIP and NIP was carried out in an aqueous system in a magnetic multi-stirrer with temperature control at 25 E C, determining the equilibrium concentration over time, according to the following reaction:       4 vp 4 vp s ac s MIP MB MIP MB         Samples of 20 mg of MIP and NIP with 9 mL of a solution in distilled water of methyl benzoate at a concentration of 0.6 µL mL G 1 were deposited in a beaker for 3 hrs, 2 mL samples of the supernatant were taken at each time and they were centrifuged for 3 min at 6000 RPM, filtered with 0.22 µm sterile syringe filter. Changes were monitored over time by absorbance by UV visible spectroscopy. To determine the concentration, a calibration curve was prepared that ranged between 0.075 and 0.018 µL mL G 1 at a wavelength of 273.75 nm and a coefficient of determination (r²)>99.87 Desorption: The release of methyl benzoate from the polymeric matrix was evaluated over time as shown in the following reaction:       4 vp s 4 vp s s MIP MB MIP MB          With the understanding that the amount released or desorbed is the difference that existed between the initial amount adsorbed and the amount adsorbed at a time "n" The 70% methyl benzoate based on the weight of the polymer sample dissolved in 3 parts of ethanol was poured onto the MIP 4-VP , it was left to evaporate for 24 hrs until the matrix was dry Samples of 40 mg of MIP 4-VP were placed, under the following conditions: Room temperature in an open container, in which the desorption was monitored for 0-9 hrs on the first day and the rest every 24 hrs until completing one week and in refrigeration, at room temperature in closed containers, desorption was evaluated for 7 days Each loaded MIP 4-VP sample was placed in a cellulose bag, It was introduced into a 50 mL beaker where 20 mL of a methanol: Water solution (1: 1) were added, Subsequently, the glass was sealed with parafilm and placed on an orbital shaker at 100 rpm at room temperature, a 2 mL sample was taken every two hrs, the concentration of methyl benzoate was determined by UV spectrophotometry and the medium was replaced, repeating the procedure until no substrate is detected. Repeating the procedure until no substrate is detected Preparation of the confrontation compound: Silica gel 60 was impregnated by adding a solution of methyl benzoate in acetone at a concentration similar to that of MIP, samples were prepared to determine desorption under the same conditions of MIP To benefit the interaction between MIP-methyl benzoate, a concentration close to the solubility limit (0.6 µL mL G 1 in distilled water) was chosen, the adsorbed amount was interpreted in mg of methyl benzoate per gram of MIP 4-VP, determined by the subtraction of the final concentration from the initial concentration of the solution, using the following Eq 25 :   n Initial concentration Concentration a t 10.8 mg mb Volume in glass Adsorbed mg 1 mL NIP/MIP (g) MIP/ NIP (g)           The volume must be corrected according to the volume in the tube prior to sampling To avoid erroneous results, it was standardized to three washes of the polymer for the complete extraction of the solute, determining the amount adsorbed from the substrate by the following equation:       3 i 1 i V MB mg MB desorbed MIP g MIP g      where, i is the concentration of each wash 174

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Int. J. Pharmacol., 18 (1): 171-181, 2022 Fig. 2(a-c): Specially designed rat drinker to expose odors to rats during conditioning, (a) Whatman Grade 41 filter paper with odorous sample, (b) Plastic mesh to protect the filter and (c) The sprue is placed in the center of the wooden base SDRD: Specially designed rat drinker The methyl benzoate desorption percentage was calculated using the following Eq.:   n Total concentration a t Lost % 100 100 Initial concentration a t 0     Acute toxicity assessment Subjects: 20 adult male Wistar rats 300-360 g of body weight, donated by the Institute of Cellular Physiology of the National Autonomous University of Mexico, Mexico, were randomly divided into two groups of seven rats (Conditioning groups) and another two groups of three specimens per box (Control groups). They were housed in a clean, dry and ventilated area under environmental conditions under the following parameters: 18-26 E C and 40-70% relative humidity, with unmodified natural light. Were kept the rats in boxes provided with a bed (Aspen type), the space requirements were respected (387 cm 2 and 18 cm in height). The nutritional needs were to provide the balanced commercial food BDL-7100 Abene ® (Mexico) and free access water, except when the rats were deprived for testing as described below. The rats were kept in this space 14 days before the experiment to achieve a period of adaptation to the new environment, The housing and feeding conditions maintained were by animal experimentation guidelines and Mexican regulations on animal welfare (NOM-062-ZOO-1999) Apparatus Drinkers with a special design (SDRD) for rats: For the present study, the molecular imprinting polymer to mimic cocaine HCl "headspaces" was placed in drinkers for rats with a special design (SDRD) which had a circular low relief that surrounds the outlet orifice of the hydration source, where was placed a cellulose bag with the MIP protected by a plastic mesh that prevented direct contact or ingestion of the product, so that every time the animal hydrates it has contact with the smell in Fig. 2(a-c) Exposure to odorants: For the exposure of the odorants, a wooden support was used that holds the hydration source enriched with sodium saccharin at a concentration of 0.005 M, the outlet of the hydration tube is surrounded by a circular low relief of 4 cm in diameter and 2 mm deep where the odoriferous MIPs were placed, each one contained in an envelope of Whatman Grade 41 filter paper, the low relief was protected with a polyethylene mesh with 3×3 mm openings to avoid the risk of direct contact, ingestion, humidification of the MIPs in Fig. 1 For groups A and B of rats were administered sources of hydration as follows: An SDRD loaded with the MIP and reinforcer was added as well as four empty SDRDs. Reinforces were prepared with: Sweetened solution formulated with 99% saccharin sodium dihydrate from Merck (DARM, GER), (0.005 M) 30,31 For control groups: C and D, an SDRD loaded with the MIP's was placed, water was added to all the SDRD of group C, to those of group D, a reinforcing solution (sweetened solution) To evaluate the acute toxicity, after exposing them, during training for a period of 14 days to the odors emitted by the MIP, the animals were anesthetized with sodium pentobarbital to obtain blood by a direct puncture. Then they were induced to euthanize with an overdose of barbiturates. The institutional subcommittee reviewed and approved the study for the care and use of experimental animals of the National Autonomous University of Mexico (SICUAE.DC- 2019/1-3) RESULTS Molecular Printing Polymers (MIP) were developed, whose function is to mimic the "headspaces" of cocaine, Fig. 1: Schematizes the reaction of MIP based on the monomer 4-vinyl pyridine (4-VP) to control desorption methyl benzoate (MB) 175 (a) (b) (c)

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Int. J. Pharmacol., 18 (1): 171-181, 2022 Table 1: Desorption (%) of methyl benzoate included in a molecular imprint polymer mimicking the odoriferous imprint of cocaine HCl for 7 days (168 hrs) Time (hrs) Desorptión (%) 9 10.15±1.137 24 12.42±1.772 48 36.32±2.248 72 36.94±3.101 96 46.35±3.613 168 49.12±2.536 Percentage±SD of the desorption of methyl benzoate included in a Molecular Imprint Polymer (MIP) designed from 4-vinyl pyridine Figure 3(a-c) showed the possible interactions between monomer (4-VP) and methyl benzoate, they are marked in blue color. Different types of bonds are observed between molecules: C π-π type bonds C Interaction between electron pair of nitrogen and carbon with positive delta of the carboxyl group of methyl benzoate C Interaction of the electronic pairs of nitrogen that of 4-VP with the benzene ring, favored by the existing electrostatic environment Table 1 showed the percentage of desorption during 168 hrs that methyl benzoate had from a molecular fingerprint polymer designed from 4-vinyl pyridine. Desorption analysis was performed at room temperature (25 E C). Desorption was 10.15% at 9 hrs, 12.42% (24 hrs), 36.32% (48 hrs), 36.94% (72 hrs), 46.35 (90 hrs), 49.12 (168 hrs). These results show equilibrium in desorption from 24 hrs and only desorption of 49.12% reached at seven days (168 hrs), which suggests a long useful life Figure 4 showed the comparative test of the release percentage of methyl benzoate from two matrices: MIP and a silica gel-based preparation at 168 hrs, under different storage conditions: Refrigeration (7 E C), room temperature (25 E C) and under the open or closed system (open or closed bottle). This evaluation was carried out Fig. 3(a-c): Possible interactions between methyl benzoate and 4-vinyl pyridine in the polymer matrix, (a) p-p type bonds, (b) Interaction between electron pair of nitrogen and carbon with positive delta of the carboxyl group of methyl benzoate and (c) Interaction of the electronic pairs of nitrogen that of 4-VP with the benzene ring, favored by the existing electrostatic environment Fig. 4: Methyl benzoate desorption (%) occurred at 168 hrs under different storage conditions from a designed MIP and a non-specific adsorbent (a silica gel preparation) 4-VP: 4-vinylpyridine, MIP: Molecular impression polymer 176 O O O O O O (a) (b) (c) N N 21.77 30.88 90.81 17.49 20.64 49.12 Refrigeration/closed system Room temperature/closed system Room temperature/open system Desorption (%) Storage conditions MIP-4 VP desorption Silica gel desorption 0 20 40 60 80 100

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Int. J. Pharmacol., 18 (1): 171-181, 2022 simultaneously with the desorption study and with the same determination techniques described in the present study During the first 168 hrs, a desorption rate of 49.12% was achieved, demonstrating a continuous release of MIP 4-VP odor, representing half of what was performed with the Silica gel (90.81%) in the "open system" at room temperature. In the "closed system," MIP desorption (%) was lower, at room temperature and refrigeration (20.64 and 17.49%), respectively, compared to that obtained from the gel of silica 30, 88 and 21.77%, respectively. Thus, the results show that the designed MIPs performed better, reaching a faster and more sustained equilibrium of the supernatant vapor, showing more extended and controlled desorption compared to Silica Gel, fulfilling the purpose for which the compound was designed. The maximum loading capacity concentrations for MIP and NIP (unprinted polymer) were 103.633 and 106.818 mg g G 1 respectively, During the present study, factors such as agitation, temperature, which has a directly proportional effect with molecular kinetics and therefore the volatilization of methyl benzoate, were controlled The polymers were ground and sieved to control parameters such as surface area and the number of sites between the samples, the granule size was homogenized to favor reproducibility To benefit the interaction between MIP-methyl benzoate, a concentration close to the solubility limit (0.6 µL mL G 1 in distilled water) was chosen, the adsorbed amount was interpreted in mg of methyl benzoate per gram of MIP 4-VP, determined by the subtraction of the final concentration from the initial concentration of the solution Figure 5(a-j) showed the blood chemistry results performed on the rats to detect signs that suggest the existence of acute toxicity, because of exposure for 14 days to MIP, using the specially designed Rat Drinker (SDRD) to expose odors (Fig. 2). To stimulate the rats to have a greater interest in seeking hydration, were removed conventional hydration sources for a period of 8 hrs before to exposure to the SDRDs was divided the lot into four groups: A and B subjected to conditioning associated with the odor emanating from MIP's and a sweetened solution, C with multiple SDRDs containing water with no source of odor emanation and one SDRD with MIP's, D with multiple SDRDs with a sweetened solution without MIP and one SDRD with MIP was evaluated values and their relationship within and between groups Figure 5 a measurement of blood cholesterol in rats was measured to detect signs of toxicity by exposure for 14 days to MIP. The values seen in the groups of rats were: A: 2.1, B: 2, C: 2.1, D 1.8 mmol L G 1 Figure 5 b measurement of triglycerides in blood in rats was observed to detect signs of toxicity. The values observed in the groups of rats were A: 1, B: 1.1, C: 1.2, D 0.9 mmol L G 1 The cholesterol and triglyceride results performed in rats do not suggest heart problems due to exposure for 14 days to MIP. In Fig. 5 c, measurement of total bilirubin in blood in rats was done. The values observed in the groups of rats were A: 3.1, B: 3.4, C: 2.4, D 2.5 µmol L G 1 Figure 5 d showed the measurement of conjugated bilirubin in blood in rats to detect signs of toxicity. The values observed in the groups of rats were A: 1.1, B: 1.1, C: 1.2, D 1.3 µmol L G 1 In Fig. 5 e, measurement of unconjugated bilirubin in blood in rats was detected. The values observed in the groups of rats were A: 2.5, B: 2.4, C: 1.25, D 2.4 µmol L G 1 The results bilirubin (Total, conjugated and unconjugated performed in rats do not suggest abnormalities related to oxidative metabolism and vascular conditions due to exposure for 14 days to MIP). Figure 5 f showed measurement of proteins in blood in rats to detect signs of toxicity. The values observed in the groups of rats were A: 81, B: 78, C: 63, D 72 g L G 1 . Figure 5 g showed the measurement of albumins in blood in rats to detect signs of toxicity. The values observed in the groups of rats were A: 36, B: 37, C: 31, D 33 g L G 1 Figure 5 h measurement of globulins in blood in rats was observed to detect signs of toxicity. The values observed in the groups of rats were A: 45, B: 40, C: 30, D 40 g L G 1 The results proteins and globulins performed in rats do suggest tissue degradation resulting from an increase in muscle activity due to competition between rats to reach hydration sources during the period of exposure to MIPʼs contained in the DRDS L G 1 Figure 5 i showed the measurement of urea in blood in rats to detect signs of toxicity. The values observed in the groups of rats were A: 7.9, B: 7, C: 7, D 7.9 mmol L G 1 Figure 5 j measurement of creatinine in blood in rats detected signs of toxicity. The values observed in the groups of rats were A: 49, B: 52, C: 50, D 49 µmol L G 1 The blood chemistry values found in the experimental rats do not suggest alterations related to oxidative metabolism, 177

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Int. J. Pharmacol., 18 (1): 171-181, 2022 Fig. 5(a-j): Complete blood analysis in rats to detect signs of toxicity (a) measurement of blood cholesterol in rats to detect signs of toxicity by exposure for 14 days to MIP, (b) measurement of triglycerides, (c) measurement of total bilirubin, (d) measurement of conjugated bilirubin, (e) measurement of unconjugated bilirubin, (f) measurement of proteins, (g) measurement of albumins, (h) measurement of globulins (i) measurement of urea (j) measurement of creatinine 4-VP: 4-vinylpyridine, MIP: molecular impression polymer. Average values of cholesterol in rats: 1.21-3.13 mmol L G 1 . Groups A and B subject to conditioning associated with the odor emanated by MIP's and a sweetened solution; group C with multiple water-based hydration sources and a SDRD with MIP's; group D with multiple solution-based hydration sources sweetened and a SDRD with MIP's. Typical values of triglycerides in rats: 0.41-2.19 mmol L G 1 , Average values of total bilirubin in rats: 2.0-9.0 µmol L G 1 , conjugated bilirubin (normal values in rats): 0.08-1.33 µmol L G 1 , average values of unconjugated bilirubin in rats: 1.92-7.67 µmol L G 1 , typical values of proteins in rats: 36-77 g L G 1 , typical values of albumins in rats: 21-46 g L G 1 , average values of globulins in rats: 3.0-35.0 g L G 1 , Average values of urea in this species: 4.64-7.49 mmol L G 1 , Average values of creatinine in rats: 27-35 µmol L G 1 178 Groups of rats (e) 3.0 2.5 2.0 1.5 1.0 0.5 0.0 Groups of rats A B C D Cholesterol (mmol L ) G 1 (a) 2.5 2.0 1.5 1.0 0.5 0.0 (c) 4 3 2 1 0 T otal bilirubin (µmol L ) G 1 A B C D 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 T riglycerides (mmol L ) G 1 (b) Groups of rats A B C D Groups of rats A B C D Conjugated bilirubin (µmol L ) G 1 (d) 1.5 1.0 0.5 0.0 Groups of rats A B C D Protein (g L) G 1 (f) Groups of rats A B C D 100 80 60 40 20 10 0 60 50 40 30 20 10 0 (h) Groups of rats A B C D 10 8 6 4 2 0 Urea (mmol L ) G 1 (i) Groups of rats A B C D 50 40 30 20 10 0 Globulins (g L ) G 1 (g) Groups of rats A B C D 54 53 52 51 50 49 48 47 46 Creatinine (µmol L ) G 1 (j) Groups of rats A B C D Unconjugated bilirubin (µmol L ) G 1 Albumins (g L ) G 1

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Int. J. Pharmacol., 18 (1): 171-181, 2022 vascular disorders, signs of liver disease or the presence of inflammatory processes. However, the increase in globulin, urea and creatinine values suggests a tissue degradation resulting from an increase in muscle activity due to competition between rats to reach sources of hydration during the period of exposure to the MIP contained in the DRDS DISCUSSION For the synthesis of MIP, the Brønsted-Lowry basic type 4-vinyl pyridine monomer was chosen, the nitrogen in its molecule can act as a donor of electron pairs and its benzene ring provides the necessary hydrophobicity to interact with the last molecule. The most common monomer: Crosslinker: Template ratio in molecular printing technology was selected 25 The literature suggests using protic or polar solvents 32 , selecting acetonitrile for the present work as a solvent of medium polarity, avoiding competition between the medium and most of the interactions between monomer and the template as well as the hydrophobic forces involved in the monomer-template relationship The results obtained suggest that the adsorption is due more to the affinity between the monomer and the template than to the imprinting process, an adsorption phenomenon is observed in the surface area of the polymer, which assumes a discrete cross linking of low porosity and therefore a reduced adsorption area in the interior 25 The concentration-dependent effect on the adsorption of MIPs was detected due to the existence of a wide surface area of nonspecific binding sites and limited molecular recognition sites In general, the effect of molecular imprinting is observed at low concentrations since the number of sites with molecular recognition capacity is always lower than the amount with which the polymer was molded, forming a large non-specific adsorption surface. Therefore, the process consists of two phases, the first consisting of the occupation of specific spaces and the second in the union of non-specific sites. Therefore, in low concentrations, the imprint effect can be appreciated 33 To observe the desorption behavior, a method for determining the template molecule was developed by removing the remaining substrate by exposing the MIP to a solution of H 2 O: Methanol (1: 1), improving the solubility of the substrate, prolonging evaporation time and preventing the generation of additional signals at the wavelengths 34 The desorption of the MIP in three different environments, aimed to determine the viability of the emanation of a clear odorant stimulus, unmistakable and constant that allows its use for the conditioning of detector dogs in field conditions, therefore, the compound was subjected to extreme storage environments for 7 days in order to record the degradation or modification of the qualities of the proposed compound, for the batch contained in an open system, it was observed that between 0 and 6 hrs no detectable changes in concentration were recorded,MIP 4-VP was able to retain in most of the adsorbed molecules, due to its monomer-template affinity, from 9 hrs the average desorption of methyl benzoate was 10%, at 24 hrs 12% of desorption, the highest proportion was up to 48 hrs with a value of 36%, subsequent desorption had a slower behavior as observed in Table 1 and Fig. 4, assuming that this behavior is attributed to desorption from the unspecified area as the solute was not associated, the acceleration of desorption was observed due to the incorporation of the substrate in the multilayer 35-37 During the first 168 hrs, a desorption rate of 49.12% was reached, in the period between 48 and 168 hrs, the reduction of desorption to only 12% was observed, attributed to greater stability in the interactions of the imprinted functional groups, in addition to the tendency to the equilibrium concentration of the released and desorbed substrate, demonstrating a continuous odorant emanation from MIP 4-VP fulfilling the purpose for which the compound was designed The results of the hematological analysis show normal values, except for the elevated levels of globulins and creatinine, values that can be attributable to fatigue or muscle injury, caused by handling during the conditioning and competition sessions in the group to reach the source of hydration during each session 38,39 CONCLUSION The present study results demonstrate that the "molecular imprint polymer" design technology is a robust option for the controlled adsorption and desorption of a wide variety of odor signatures. The adsorption equilibrium was reached relatively quickly, whereas the desorption is considerably longer concerning most existing commercial products. Due to their affinity, substrate specificity and chemical stability, in addition to making polymer refills with practically similar performance possible, reducing costs and waste generation The adsorption specificity of MIP's provides an excellent ability to remove methyl benzoate, giving MIP an alternative 179

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Int. J. Pharmacol., 18 (1): 171-181, 2022 use as a sorbent in the removal of degradation products from real drugs, offering the opportunity of forensic use for the collection and identification of polluting residues even in aqueous media SIGNIFICANCE STATEMENT The present study achieved the design of a matrix to control the desorption of the most important odoriferous component of the isothermal decomposition of Cocaine HCl, which simulates the odorous stimulus of this drug, its desorption behavior was characterized in different environmental conditions, guaranteeing the intensity, purity and harmlessness. It was possible to design a new conditioning adjuvant with great potential for the training of drug-detecting animals REFERENCES 1 Yáñez-Sedeño, P., L. Agüí, R. Villalonga and J.M. Pingarrón, 2014. Biosensors in forensic analysis. A review. Analytica Chim. Acta, 823: 1-19 2 De Castro, A.C.V., Â. Araújo, A. Fonseca and I.A.S. Olsson, 2021 Improving dog training methods: Efficacy and efficiency of reward and mixed training methods. PLoS ONE, Vol. 16 10.1371/journal.pone.0247321 3 Haverbeke, A., B. Laporte, E. Depiereux, J.M. Giffroy and C. Diederich, 2008. Training methods of military dog handlers and their effects on the team's performances. Appl. Anim Behav. Sci., 113: 110-122 4 Kranz, W.D., N.A. Strange and J.V. Goodpaster, 2014 “Fooling fido”-chemical and behavioral studies of pseudoexplosive canine training AIDS. Anal. Bioanalytical Chem., 406: 7817-7825 5 Herz, R.S., 2005. Odor-associative learning and emotion: Effects on perception and behavior. Chem. Senses, 30: i 250-i 251 6 Furton, K.G. and L.J. Myers, 2001. The scientific foundation and efficacy of the use of canines as chemical detectors for explosives. Talanta, 54: 487-500 7 Sellergren, B. and C.J. Allender, 2005. Molecularly imprinted polymers: A bridge to advanced drug delivery. Adv. Drug Delivery Rev., 57: 1733-1741 8 Udo, M.S.B., M.A.A. da Silva, S. de Souza Prates, L.F. DalʼJovem and S. de Oliveira Duro et al ., 2021. Anhydroecgonine methyl ester, a cocaine pyrolysis product, contributes to cocaineinduced rat primary hippocampal neuronal death in a synergistic and time-dependent manner. Arch. Toxicol., 95: 1779-1791 9 Lapachinske, S.F., G.G. Okai, A. dos Santos, A.V. de Bairros and M. Yonamine, 2015. Analysis of cocaine and its adulterants in drugs for international trafficking seized by the Brazilian federal police. Forensic Sci. Int., 247: 48-53 10. Lai, H., I. Corbin and J.R. Almirall, 2008. Headspace sampling and detection of cocaine, MDMA, and marijuana via volatile markers in the presence of potential interferences by solid phase microextraction‒ion mobility spectrometry (SPME-IMS). Anal. Bioanal. Chem., 392: 105-113 11. Lorenzo, N., T. Wan, R.J. Harper, Y.L. Hsu, M. Chow, S. Rose and K.G. Furton, 2003. Laboratory and field experiments used to identify Canis lupus var. familiaris active odor signature chemicals from drugs, explosives and humans. Anal Bioanalytical Chem., 376: 1212-1224 12. Mayes, A.G. and M.J. Whitcombe, 2005. Synthetic strategies for the generation of molecularly imprinted organic polymers Adv. Drug Delivery Rev., 57: 1742-1778 13. Spivak, D.A., 2005. Optimization, evaluation, and characterization of molecularly imprinted polymers. Adv Drug Delivery Rev., 57: 1779-1794 14. Simon, A., L. Lazarowski, M. Singletary, J. Barrow and K.V. Arsdale et al ., 2020. A review of the types of training AIDS used for canine detection training. Front. Vet. Sci., Vol. 7 10.3389/fvets.2020.00313 15. Rice, S. and J.A. Koziel, 2015. Odor impact of volatiles emitted from marijuana, cocaine, heroin and their surrogate scents Data Brief, 5: 653-706 16. Rice, S. and J.A. Koziel, 2015. Characterizing the smell of marijuana by odor impact of volatile compounds: An application of simultaneous chemical and sensory analysis PLoS ONE, Vol. 10. 10.1371/journal.pone.0144160 17. DʼAurelio, R., I. Chianella, J.A. Goode and I.E. Tothill, 2020 Molecularly imprinted nanoparticles based sensor for cocaine detection. Biosensors, Vol. 10. 10.3390/bios 10030022 18. Turiel, E. and A.M. Esteban, 2020. Molecularly Imprinted Polymers. In: Solid-Phase Extraction, Poole, C.F. (Ed.)., Elsevier Inc., pp: 215-233 19. Alexander, C., H.S. Andersson, L.I. Andersson, R.J. Ansell and N. Kirsch et al ., 2006. Molecular imprinting science and technology: A survey of the literature for the years up to and including 2003. J. Mol. Recognition, 19: 106-180 20. Bahrani, S., R. Aslani, S.A. Hashemi, S.M. Mousavi and M. Ghaedi, 2021. Introduction to Molecularly Imprinted Polymer, Adsorption: Fundamental Processes and Applications. In: Interface Science and Technology, Ghaedi, M. (Ed.)., Elsevier, New York, pp: 511-556 21. Pardo, A., L. Mespouille, P. Dubois, P. Duez and B. Blankert, 2012. Targeted extraction of active compounds from natural products by molecularly imprinted polymers. Open Chem., 10: 751-765 180

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Real substance, Public safety, Nervous system, Environmental condition, Blood biochemistry, Characteristic odor, Hematological analysis, Chemical stability, Storage Requirements, Unconjugated bilirubin, Free Radical, Total bilirubin, Template molecule, Functional monomer, Sorbent, Aqueous media, Wistar rat, Polymeric matrix, Acute toxicity assessment, Normal value, Substrate specificity, Adsorption, Collection and Identification, Blood cholesterol, Functional group, Conjugated bilirubin, Adsorption equilibrium, Elevated level, Molecularly imprinted polymers, Blood chemistry result, Present study results, Affinity, Methyl benzoate, Vapor pressure, Legal risks, Design technology, Muscle injury, Open system, Conditioned stimulus, Illegal substances, Adsorbent material, Blood triglyceride, Adsorption and desorption, Desorption, Desorption behavior, Different environmental conditions, Great potential, Robust option, Dangerous substances, Adsorption phenomenon.