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Microbore LC with UV detection to study the in vivo passage of Compound 21, a non-peptidergic AT2 receptor agonist, to the striatum.
Book Contribution - Book Chapter Conference Contribution
Introduction: Compound 21 (C21) is a non-peptide angiotensin II type 2 (AT2) receptor agonist. It is used to study the role of the AT2 receptor in dopamine release and synthesis in the striatum and to study its possible neuroprotective effects in animal models of Parkinson's disease [1, 2]. In literature, there is no data available on the blood brain barrier (BBB) passage of this compound. In order to investigate its blood brain passage, a microbore liquid chromatography (LC) method coupled to UV detection has been developed and validated to quantify this compound in microdialysis samples. Preliminary in vivo experiments were performed to monitor the concentration of this AT2 receptor agonist in the striatal dialysates after intraperitoneal (ip) or intravenous (iv) injection.
Methods: All experiments were performed on an Ultimate 3000 Quaternary Micro system (Dionex, The Netherlands). Separations were performed on an UnijetTM C18 (Bioanalytical Systems, USA) column (15 cm x 1.0 mm I.D., 5 µm particles). The flow-rate was set at 50 µL/min. The injection loop was 20 µL and the detection wavelength was set at 271 nm. Gradient elution was performed using mobile phase A (water:acetonitrile:formic acid; 98:2:0.1 v/v/v) and mobile phase B (water:acetonitrile:formic acid; 20:80:0.1 v/v/v). The linear LC gradient was 0 - 100% B in 5 min, 100% B for 2 min, 100 - 0% B in 0.1 min and 0% B for 8 min. The total run time was 15 min. All LC parts were controlled by Chromeleon® software. 200 µL standards (10 - 200 ng/ml) were prepared by diluting 10 µL stock solutions with 100 µL mobile phase B and 90 µL water. The same procedure was applied to prepare the standards in Ringer's solution or dialysate. The in vivo experiments were carried out using the intracerebral microdialysis technique; 2 probes (MAB 2/6/9.14.IC with a membrane length of 4 mm) were implanted in the left and the right striatum. Ringer's solution (147 mM NaCl, 4 mM KCl, 1.1 mM CaCl2) was perfused at a flow-rate of 0.5 µL/min. The sampling time was 45 min, and each sample (22.5 µL) was collected in 22.5 µL of mobile phase B. The samples from both striata were taken together and mixed. 80 ?L was transferred to the injection vial and injected immediately. C21 was administered ip to the rats at a dose of 10 or 50 mg/kg or iv at a dose of 10 mg/kg.
Results and discussion: After optimization, the LC method was validated according to the guidelines for bioanalytical method validation [3]. The retention time was found to be 6.35 min. The calibration curve was linear between 10 and 200 ng/ml with a correlation coefficient larger than 0.999. No significant difference was observed between the regression curves obtained in water, Ringer's or dialysate. The limit of detection (LOD) and the limit of quantification (LOQ) were 2 and 10 ng/ml respectively. The intra-day and the inter-day precision complied with the guidelines and the RSD % was less than 8 % for LOQ and less than 4 % for larger concentrations. Accuracy was assessed by spiking blank dialysate and comparing the experimental values, calculated using the regression line constructed with standards in water, to the nominal values. An average recovery of 107 ± 5 % (mean ± SD) was obtained.
In vivo experiments were performed to investigate if this non-peptidergic AT2 receptor agonist could be detected in the striatum after injection of 10 or 50 mg/kg ip or 10 mg/kg iv of the compound. The obtained results suggest minimal passage of this compound to the striatum.
References
1. Wan Y, Wallinder C, Plouffe B, Beaudry H, Mahalingam A, Wu X, Johansson B, Holm M, Botoros M, Karlén A, Pettersson A, Nyberg F, Fändriks L, Gallo-Payet N, Hallberg A, Alterman M (2004) Design, synthesis, and biological evaluation of the first selective nopeptide AT2 receptor agonist. J Med Chem 47: 5995-6008
2. Mertens B, Vanderheyden P, Michotte Y, Sarre S (2010) Direct angiotensin II type 2 receptor stimulation decreases dopamine synthesis in the rat striatum. Neuropharmacology 58: 1038-1044
3. Guidance for Industry. Bioanalytical Method Validation. U.S. Department of Health and Human Services, Food and Drug Administration (2001)
(www.fda.gov/cder/guidance/index.htm)
Methods: All experiments were performed on an Ultimate 3000 Quaternary Micro system (Dionex, The Netherlands). Separations were performed on an UnijetTM C18 (Bioanalytical Systems, USA) column (15 cm x 1.0 mm I.D., 5 µm particles). The flow-rate was set at 50 µL/min. The injection loop was 20 µL and the detection wavelength was set at 271 nm. Gradient elution was performed using mobile phase A (water:acetonitrile:formic acid; 98:2:0.1 v/v/v) and mobile phase B (water:acetonitrile:formic acid; 20:80:0.1 v/v/v). The linear LC gradient was 0 - 100% B in 5 min, 100% B for 2 min, 100 - 0% B in 0.1 min and 0% B for 8 min. The total run time was 15 min. All LC parts were controlled by Chromeleon® software. 200 µL standards (10 - 200 ng/ml) were prepared by diluting 10 µL stock solutions with 100 µL mobile phase B and 90 µL water. The same procedure was applied to prepare the standards in Ringer's solution or dialysate. The in vivo experiments were carried out using the intracerebral microdialysis technique; 2 probes (MAB 2/6/9.14.IC with a membrane length of 4 mm) were implanted in the left and the right striatum. Ringer's solution (147 mM NaCl, 4 mM KCl, 1.1 mM CaCl2) was perfused at a flow-rate of 0.5 µL/min. The sampling time was 45 min, and each sample (22.5 µL) was collected in 22.5 µL of mobile phase B. The samples from both striata were taken together and mixed. 80 ?L was transferred to the injection vial and injected immediately. C21 was administered ip to the rats at a dose of 10 or 50 mg/kg or iv at a dose of 10 mg/kg.
Results and discussion: After optimization, the LC method was validated according to the guidelines for bioanalytical method validation [3]. The retention time was found to be 6.35 min. The calibration curve was linear between 10 and 200 ng/ml with a correlation coefficient larger than 0.999. No significant difference was observed between the regression curves obtained in water, Ringer's or dialysate. The limit of detection (LOD) and the limit of quantification (LOQ) were 2 and 10 ng/ml respectively. The intra-day and the inter-day precision complied with the guidelines and the RSD % was less than 8 % for LOQ and less than 4 % for larger concentrations. Accuracy was assessed by spiking blank dialysate and comparing the experimental values, calculated using the regression line constructed with standards in water, to the nominal values. An average recovery of 107 ± 5 % (mean ± SD) was obtained.
In vivo experiments were performed to investigate if this non-peptidergic AT2 receptor agonist could be detected in the striatum after injection of 10 or 50 mg/kg ip or 10 mg/kg iv of the compound. The obtained results suggest minimal passage of this compound to the striatum.
References
1. Wan Y, Wallinder C, Plouffe B, Beaudry H, Mahalingam A, Wu X, Johansson B, Holm M, Botoros M, Karlén A, Pettersson A, Nyberg F, Fändriks L, Gallo-Payet N, Hallberg A, Alterman M (2004) Design, synthesis, and biological evaluation of the first selective nopeptide AT2 receptor agonist. J Med Chem 47: 5995-6008
2. Mertens B, Vanderheyden P, Michotte Y, Sarre S (2010) Direct angiotensin II type 2 receptor stimulation decreases dopamine synthesis in the rat striatum. Neuropharmacology 58: 1038-1044
3. Guidance for Industry. Bioanalytical Method Validation. U.S. Department of Health and Human Services, Food and Drug Administration (2001)
(www.fda.gov/cder/guidance/index.htm)
Book: Monitoring Molecules in Neuroscience
Series: Monitoring Molecules in Neuroscience
Publication year:2010
Keywords:compound 21, AT2 receptor agonist, in vivo passage, microbore liquid chromatography