Reagents and Chemicals
PT-31 was synthesized by the Research Nucleus in Therapeutic Innovation Suely Galdino of the Federal University of Pernambuco (Recife, Brazil) (Sudo et al., 2010), while clonidine hydrochloride (internal standard) was purchased from Sigma-Aldrich Chemie (Steinhem, Germany). HPLC grade methanol, ammonium acetate and ethyl acetate were purchased from Merck (Darmstadt, Germany), J. T. Baker (NJ, USA) and Macron Chemicals (PA, USA), respectively. Reagent grade formic acid was obtained from Scharlau Chemie S.A. (European Union). Water was obtained from a Millipore Milli-Q System and used throughout the analysis.
Log P determination
Log P of PT-31 was evaluated by in silico and in vitro methods. The software Chem Draw ultra 12.0 (Cambridge) was used for in silico determination, and the experimental method was based on the OECD Guideline for the testing of chemicals - Partition Coefficient (n-octanol/water): Shake Flask Method (1995).
In the experimental method, the partition of the compound in oil/water was evaluated using n-octanol as organic phase in three different oil/water proportions according to OECD guideline 107 (1995). The quantification of the compound was performed by a Power Wave HT - BioTek plate reader using a wavelength of 205 nm.
After the analysis of each phase, the concentration of PT-31 in the n-octanol phase was divided by the concentration in the water phase to determine the partition. The analytical curves in water and octanol were constructed at concentrations of 15.62, 31.25, 62.5, 125, 250, 500 µg/mL in triplicate, and showed correlation coefficients (R) above 0.99.
In vitro stability
For in vitro hydrolysis studies, an appropriate amount of PT-31 was weighed and dissolved in methanol to obtain a concentration of 200 µg/mL (stock solution) followed by dilution in aqueous buffers (pH 3.0 and 7.4) at a concentration of 20 µg/mL. Plasma samples were spiked with stock solution of PT-31 to get a concentration of 20 µg/mL. Afterwards, buffer solution or spiked plasma was mixed constantly at 37ºC during the assay. The plasma and pH 3.0 buffer samples were collected at the times points zero, 0.25, 0.5, 1, 2, 4, 8 and 12 h, while the pH 7.4 buffer samples were collected at the times zero, 0.25, 0.5, 1, 1,5, 4 and 12 h. The collected samples were analyzed by LC-MS. All analyses were performed in triplicate, and the results were expressed as the means of the concentrations obtained.
Chromatographic and mass spectrometry conditions
The analysis was carried out using a Waters Alliance HPLC 2695 series (Waters, UK) with separation on a reversed-phase Symmetry C18 (4.6 x 75 mm, 3.5 µm) column, which was protected by a Symmetry C18 (3.9 x 20 mm, 5 µm) guard column at a temperature of 30ºC. The mobile phase consisted of methanol:5 mM ammonium acetate buffer (pH 2.8 adjusted with formic acid) at a proportion of 90:10 (v/v), and the flow rate was 0.5 mL/min. The injection volume was 20 µL and the run time was 5 min.
Detection and quantitation were performed using a Quattro-Micro™ triple-quadrupole mass spectrometer (Micromass, USA) equipped with an electrospray ionization (ESI) source in positive ion mode. After optimization, the following conditions were used for mass spectrometry to perform the analysis: capillary voltage 3.5 kV, source temperature 120ºC, desolvation temperature 450ºC; cone voltage 20V and collision energy 25 were for PT-31, while cone voltage 30V and collision energy 25 were for clonidine (internal standard, IS). Multiple reaction monitoring (MRM) was used to detect the analytes using m/z 243.45→143.02 for PT-31 and the m/z 230.05→172.00 for the IS, where they were the parent ion mass and product ion mass, respectively. All data acquisition was carried out using MassLynx 4.1 software (Waters, Milford, MA, USA).
Method Validation
The validation procedure was based on the (Brazilian) ANVISA resolution 899 [10]. To assess linearity, analyses of the analyte at 9 concentrations were conducted using five replicates. The calibration curve was generated from the PT-31 to I.S. peak area ratios, by least-squares linear regression. The range of the calibration curve in plasma was from 50 to 20,000 ng/mL. Correlation coefficient (R > 0.99) and accuracies were used to test the linearity of the calibration curve.
Intra- and inter-run precision and accuracy were assessed for three different concentrations or quality controls (QC), namely the lower limit of quantification (LLOQ) as low QC, mid QC, and high QC, where the chosen levels were 50, 1000 and 10,000 ng/mL. Accuracy was defined as the deviation of the mean analytical result from the theoretical value, while precision was represented by the relative standard deviation (R.S.D.) of measurements at each level. Intra-run precision and accuracy consisted of five replicates on the same day, while inter-run precision and accuracy were calculated using 15 replicate determinations, on three different days with five replicates per day. The QC plasma samples were prepared daily. The data acceptance criterion for precision was R.S.D. less than 15%, except for LLOQ, where it should not exceed 20%. For accuracy, acceptable values were 85–115% of the theoretical value, except at LLOQ, where 80–120% was permissible.
Recovery was assessed by comparing the analytical results for extracted samples at three concentrations with a blank final extract spiked to the equivalent final concentration, which represented 100% recovery. The recovery of the method was assessed at low QC, mid QC, and high QC for PT-31 .
The stability of PT-31 was assessed by comparing the measured analyte concentration of fresh samples to measured analyte concentration in stored samples under several conditions, such as freezing/thawing (-20ºC), short-term room temperature (25ºC), long-term frozen (-20ºC), and post-preparative (10ºC). All stability tests were performed at the low and high QC levels. The PT-31 was considered stable when the analyte concentration measured in the stored samples was within 85–115% of the analyte concentration measured in fresh samples.
Animals
The pharmacokinetic profile, locomotor activity, hepatotoxicity and nephrotoxicity studies were carried out in male Wistar rats (weighing 200–250 g). They were housed in wire cages, five animals per cage, the bedding was woodchips, with free access to food and water. the The animals were kept in controlled conditions of temperature (23 ± 1°C), relative humidity (55 ± 5%) and light (12/12 h cycle, lights on at 7 am). The preclinical study protocol was approved by The Research Ethics Committee of the School of Pharmaceutical Sciences, UNESP, Araraquara (Process #24/2010). In these studies, 118 animals were used, distributed as follows: 30 to locomotor activity test, 40 to toxicity assays and 48 to pharmacokinetic profile.
In vivo Biochemical Evaluation
The biochemical evaluation was performed using aspartate aminotransferase (AST), alanine aminotransferase (ALT), urea, and creatinine kits, which were purchased from LabTest Diagnostica SA (Lagoa Santa, Minas Gerais, Brazil). The analyses were performed in a Power Wave HT plate – Biotek reader.
To perform these studies, rats were divided into 4 groups (n = 10 per group) and treated intraperitoneally (ip) as follows: Group I or control group with only DMSO, Group II with 10 mg/kg PT-31 in DMSO, Group III with 10 mg/kg PT-31 plus 6 mg/kg morphine in DMSO, and Group IV 6 with mg/kg morphine in DMSO.
Blood samples were collected (by decapitation) after 8 h for the determination of the aforementioned biochemical parameters, except for the DMSO group, where samples were also collected before administration. The samples from the DMSO group were compared before and after (8 h) administration to verify that DMSO had no effect on the parameters, so that this group could be used as a control group, since DMSO was used as vehicle for the compounds.
Doses of 10 mg/kg for PT-31 and 6 mg/kg for morphine in rats were established by allometric extrapolation, considering that Sudo et al. (2010) used 15 and 10 mg/kg, respectively, in mice.
Locomotor Activity
Locomotor activity was measured in commercially available activity monitoring chambers (Columbus Instruments, CA), consisting of Plexiglas cages. The chambers, measuring 44 (width) X 44 (length) X 20 (height) cm, have 10 pairs of infrared photocells, which were used to measure the horizontal locomotor activity. The consecutive interruption of two beams was recorded as one unit of locomotion count.
Animals were randomly divided into five groups (n = 6 animals per group), and were injected ip with 3, 5, 10 or 20 mg/kg PT-31 in DMSO. The control group received an ip injection of DMSO (2.5 mg/kg). Immediately after injection, each rat was individually transferred to the activity chamber, and locomotor activity was recorded for 45 min. The experiments were performed during the light phase.
Pharmacokinetic Analysis
PT-31 pharmacokinetic parameters were determined using 48 male Wistar rats weighing 250 ± 10 g. The number of animals was calculated according to Chow and Wang (2001). The animals were housed in polypropylene cages and under standard laboratory conditions (20 ± 2ºC) with food and water ad libitum. Eight hours before drug administration the animals were deprived of food. In the PT-31 group (n = 16), 10 mg/kg PT-31 in DMSO [SUDO et al., 2010] were administered ip (0.125–0.150 mL). In the PT-31 plus morphine group (n = 16), 10 mg/kg PT-31 and 6 mg/kg morphine in DMSO were administered ip (0.125–0.150 mL). In the oral PT-31 group (n = 16), 10 mg/kg PT-31 in DMSO were administered (0.500–0.650 mL). All drug administrations were performed over a short period of time, less than thirty seconds. The doses were calculated by interspecific allometric scaling based on the mouse doses of 15 and 10 mg/kg for PT-31 and morphine, respectively (Sudo et al., 2010).
Blood samples (0.5 mL) obtained from the tail vein of the animals and by decapitation were respectively collected in heparinized plastic microtubes and glass tubes. The blood was centrifuged at 13,000 x g for 15 min at 8 ± 1°C and the plasma used for the laboratory tests. Serial blood samples were collected at 10 different time points (0, 15, 30, 45, 60 and 90 min and 3, 6, 12 and 24 h) and for each sampling time 5 animals were used. The plasma samples were analyzed by LC-MS to construct plasma concentration-time curves and to calculate the pharmacokinetic parameters.
PT-31 kinetic disposition was evaluated after administration of a single dose by gavage and ip route. The pharmacokinetic parameters were calculated on the basis of the plasma concentration vs time curves. Elimination half-life (t1/2) was determined by graphical method, and the elimination constant (kel) was calculated by the formula 0.693/t1/2. The area under the curve 0 to 10 h (AUC0 − 10) was determined by the trapezoidal method and the area under the curve 0 to ∞ (AUC0−∞) was calculated by the formula AUC0 − 10 + Cn/kel, where Cn was the last quantifiable plasma concentration (10 h). Oral bioavailability (F) of the PT-31 was calculated by the relation between the AUC obtained in the gavage administration and the AUC obtained for ip administration. Mean residence time (MRT) was calculated was according to the forumla AUMC/AUC, where AUMC is the area under the first moment curve. The maximum plasma concentration (Cmax) was obtained directly from the experimental data, along with the time of occurrence of Cmax (tmax) and minimum plasma concentration (Cmin).
Statistical Analysis of Data
The log P result and the chemical and ex vivo stabilities are presented as mean ± SD, but the stability results were tested over time against time 0 using the Kruskal-Wallis test followed by Dunn’s test. Biochemical parameters were presented as means, medians, and 95% confidence interval (CI95). They were compared by Kruskal-Wallis test followed by Dunn’s test, except for the comparison between DMSO before and after administration, where Wilcoxon’s test was used. Behavioral data were analyzed by two-way ANOVA, considering dose and time after injection as factors. Time was a repeated measure.
Plasma concentration versus time profile are presented as mean and CI95, while pharmacokinetic parameters are presented as mean, median, and CI95, and the parameters of PT-31 in the different groups were compared by the Mann-Whitney test, since they showed a non-parametric distribution.
For this purpose, GraphPad Prism 5 software was used. A significance level (α) of 0.05 was used for all experiments. The calculations of linear regression of standard curves and coefficient of variation (CV%) were performed using Origin ® program.