Complementary pharmacological and toxicological characterization data on the pharmacological profile of N-(2,6-dichlorophenyl)-2-(4-methyl-1-piperidinyl) acetamide

This text presents complementary data corresponding to pharmacological and toxicological characterization of N-(2,6-dichlorophenyl)-2-(4-methyl-1-piperidinyl)acetamide (LIA) compound. These data support our research article entitled “Pharmacological profile of N-(2,6-dichlorophenyl)-2-(4-methyl-1-piperidinyl)acetamide, a novel analog of lidocaine” Déciga-Campos M., Navarrete-Vázquez G., López-Muñoz F.J., Librowski T., Sánchez-Recillas A., Yañez-Pérez V., Ortiz-Andrade R. (2016) [1]. Toxicity was predicted through the ACD/ToxSuite software and evaluated in vivo using brine shrimp larvae (Artemia salina L.) and mice. Also, we used the micronucleus assay to determine genotoxicity. We used the platform admetSAR to predict absorption properties of LIA and lidocaine.

Keywords: N-(2,6-dichlorophenyl)-2-(4-methyl-1piperidinyl)acetamide Toxicity Lidocaine Ortiz-Andrade R. (2016) [1]. Toxicity was predicted through the ACD/ ToxSuite software and evaluated in vivo using brine shrimp larvae (Artemia salina L.) and mice. Also, we used the micronucleus assay to determine genotoxicity. We used the platform admetSAR to predict absorption properties of LIA  LIA is not genotoxic compound in mice. This drug, in contrast to lidocaine, does not modify cardiovascular responses. LIA absorption (blood brain barrier permeability and intestinal absorption) predicted is 30% similar to that observed with lidocaine.
1. Data Table 1 shows theoretical predictive values of toxicity of lidocaine and LIA determined by the ACD/ ToxSuite software. Fig. 1 depicts preliminary data of toxicity assayed in Brine Ship (Artemia saline L.). Table 2 describes acute toxicity in two phases by the Lorke method in mice. Table 3 depicts acute genotoxicity in mice. Fig. 2 shows the cardiovascular effects of LIA and lidocaine in rats.

Artemia saline lethality test
LIA and lidocaine were evaluated for lethality in brine shrimp larvae (Artemia saina L.) according to the procedure described previously [2]. Each concentration of either LIA or lidocaine (100, 200, 400, 800 and 1000 ppm) was assayed in triplicate. The surviving shrimp were counted after 24 h and the percentage of deaths was determined by the computation of half lethal concentration 50 (LC 50 ) ( Fig. 1).

Toxicity in mice by Lorke method
Experiments were performed on ICR male mice by the Lorke method [3]. Doses were selected according this method. In both phases, mice were observed daily for 14 days for mortality, toxic effects and behavioral changes. Restlessness, respiratory distress, seizures, diarrhea, motor activity, posture and reflexes were qualitatively determined. Body weight was also monitored. The internal organs (including the stomach, heart, lung, liver, and kidneys) were removed at the end of experiment and visually examined for lesions. Neither LIA nor lidocaine produced visible macroscopically damage.

Determination of genotoxicity by the bone marrow micronucleus assay
This test was carried out following standard protocols [4,5]. Briefly, ICR male mice (25-30 g) were injected with cyclophosphamide (40 mg/kg, i.p.), as positive control, LIA (100 mg/kg, i.p.), lidocaine (100 mg/kg, i.p.) or vehicle (saline solution, 0.9%, i.p). Animals were sacrificed 24 h later and the bone marrow from both femurs was flushed out using 2 mL of saline and centrifuged for 5 min at 3000 rpm. The supernatant was discarded, and the pellet was re-suspended in 0.3 mL of saline. Of this smears were made on glass slides. The slides were fixed with methanol and stained with 10% Wright-Giemsa stain. Cells were blindly scored using a light microscope at 100 X magnification. For the analysis of micronucleated cells, 1000 polychromatic erythrocytes (PCE) per animal were scored. In order to assess the cytotoxic effects of compounds, the ratio of PCE to normochromatic erythrocytes (NCE) was determined in 1000 erythrocytes [4,5]. The results are presented as the mean number of micronucleated polychromatic erythrocytes (MNPCE) or the ratio PCE:NCE in individual mice (two smears per animal) 7 SEM for five animals per group (Table 3).

Cardiovascular response in rats
The hypotensive activity was determined using a standard protocol of Avila-Villarreal et al. [6]. This study was conducted in normotensive rats. Animals were allotted into four groups (of six animals) as follows: control rats (SS, group 1), and positive control (diltiazem, group 2), LIA (group 3) and lidocaine (group 4). Treated groups were administered with diltiazem (calcium channel blocker; 30 mg/kg, p.o.), LIA (50 and 80 mg/kg, p.o.) and lidocaine (50 and 80 mg/kg, p.o.). Systolic and diastolic blood pressure as well as heart rate were recorded before and after treatment at 0, 1, 3, 5 and 7 h by the tail cuff method using a Panlab non-invasive blood pressure system for rodents and dogs (Harvard Apparatus). Percent of reduction in heart rate (HR), systolic blood pressure (SBP) or diastolic blood pressure (DBP) were calculated using diltiazem as control (Fig. 2).

Pharmacokinetic parameters
We used the platform admetSAR [7] to calculate human intestinal absorption, blood-brain barrier penetration, Caco-2 permeability, renal organic cation transporter, P-glycoprotein substrate and inhibitor. These properties play key roles in the discovery/development of drugs ( Table 4). The software predicted high values of blood-brain barrier permeability and intestinal absorption for LIA. However, the software predicted a moderate permeability for LIA in Caco-2 cells. Furthermore, the software predicted that LIA is a P-glycoprotein substrate but not an inhibitor, indicating that this compound could have easily cleared from cells. The software also predicted that LIA is not an inhibitor of renal organic cation transporter.