Virginia De Vito

Detection and quantification of the selective EP4 receptor antagonist CJ-023423 (grapiprant) in canine plasma by HPLC with spectrofluorimetric detection.

Grapiprant, a novel pharmacologically active ingredient, acts as a selective EP4 receptor antagonist whose physiological ligand is prostaglandin E2 (PGE2). It is currently under development for use in humans and dogs for the control of pain and inflammation associated with osteoarthritis. The aim of the present study was to develop an easy and sensitive method to quantify grapiprant in canine plasma and to apply the method in a canine patient. Several parameters, both in the extraction and detection method were evaluated. The final mobile phase consisted of ACN:AcONH4 (20 mM) solution, pH 4 (70:30, v/v) at a flow rate of 1 mL/min. The elution of grapiprant and IS (metoclopramide) was carried out in isocratic mode through a Synergi Polar-RP 80A analytical column (150 mm × 4.6 mm). The best excitation and emission wavelengths were 320 and 365 nm, respectively. Grapiprant was extracted from the plasma using CHCl3, which gave a recovery of 88.1 ± 10.22% and a lower limit of quantification (LLOQ) of 10 ng/mL. The method was validated in terms of linearity, limit of detection (LOD), LLOQ, selectivity, accuracy and precision, extraction recovery, stability, and inter-laboratory cross validation, according to international guidelines. The chromatographic runs were specific with no interfering peaks at the retention times of the analyte and IS, as confirmed by HPLC-MS experiments. In conclusion, this was a simple and effective method using HPLC-FL to detect grapiprant in plasma, which may be useful for future pharmacokinetic studies.

Synergistic interaction between tapentadol and flupirtine in the rat orafacial formalin test

Combination therapy with two or more analgesics is widely used for conditions associated with moderate to severe pain. Combinations of diverse analgesics with different modes of action can improve the risk-benefit ratio of analgesic treatments. The aim of this study is to evaluate the antinociceptive effect of tapentadol (TAP) and flupirtine (FLP), when administered separately or in combination, as well as their synergistic interaction in the orofacial formalin test in rats. After i.p. injection of TAP at different doses (2, 5, 10 and 15mg/kg), the biphasic nociceptive behavior was reduced in a dose-dependent manner in both phase I and II. Conversely, i.p. injection of FLP at different doses (0.6, 1.6, 3.3, 6.6, 16.6 and 22.2mg/kg) induced a dose-dependent antinociceptive effect in phase II only. TAP was found to be more effective than FLP. The interaction between TAP and FLP was synergistic in phase II with an interaction index (γ) of 0.50±0.24. The data reported in this study indicate that FLP enhances the antinociceptive effect of TAP and this drug combination might be potentially useful in the treatment of chronic pain.

BIOPHARMACEUTICS OF METOCLOPRAMIDE: INTRAVENOUS, INTRAMUSCULAR, SUBCUTANEOUS AND PER RECTUM ADMINISTRATIONS IN RABBITS

Introduction: The anatomy and physiology of pigs is closely related to human characteristics. Therefore, the use of pigs as an animal model to study the pharmacokinetic (PK) behavior of drugs in therapeutic subpopulations, including pediatrics, could be of interest. One of the key PK processes, biotransformation, is primarily mediated by the cytochrome P450 (CYP) enzyme system. Literature reports have demonstrated a high homology between human and porcine CYP3A, 2C and 2E in adults, namely at least 75, 62 and 79% amino acid sequence identity, respectively. However, data regarding the ontogeny of porcine CYP enzymes are lacking. Therefore, in order to assess whether piglets might serve as a model for pediatric PK studies, knowledge regarding the ontogeny of the CYP enzymes in pigs is mandatory. Materials and methods: Liver samples were collected immediately after euthanasia from 16 pigs (8 males and 8 females, Hybrid sow x Pietrain boar) of different ages (2 days, 4 and 8 weeks, 6 months-old). Samples were snap-frozen and stored at <-80°C until analysis. Microsomes were prepared by a differential centrifugation method. Midazolam, tolbutamide and chlorzoxazone probe drugs were used to determine the in vitro CYP3A, 2C and 2E catalytic activity, respectively. The corresponding metabolites, namely 1-hydroxy-midazolam, 4-hydroxy-tolbutamide and 6-hydroxy-chlorzoxazone, were quantified using a validated UHPLC-MS/MS method (1). Furthermore, the microsomal protein per gram of liver was determined as it is an important scaling factor in the extrapolation of the obtained in vitro enzyme activities to in vivo (2). Results and conclusions: The biotransformation of midazolam, tolbutamide and chlorzoxazone increased with age. The mean (±SD) CYP3A activity was 60.5 (±45.7) and 83.3 (±20.7) pmol/min/mg protein at the age of 2 days, 971.1 (±367.8) and 1072.7 (±371.7) pmol/min/mg protein at the age of 4 weeks and 723.4 (±146.3) and 1134.7 (±282.6) pmol/min/mg protein at 8 weeks of age for the barrows and sows, respectively. CYP2C activity at the same ages increased from 20.1 (±12.3) and 29.1 (±18.5) to 78.3 (±25.6) and 106.7 (±69.1) and 103.5 (±39.6) and 170.2 (±71.9) pmol/min/mg protein, while the activity of CYP2E was 539.3 (±251.0) and 643.3 (±220.3), 747.7 (±134.8) and 948.9 (±246.2) and 957.9 (±221.7) and 1549.8 (±345.0) pmol/min/mg protein, respectively for the barrows and sows. Significant sex differences (P<0.05) were only observed at 8 weeks of age. These data show similar trends with human CYP ontogeny.