1-Benzyl-1,2,3,4-tetrahydroisoquinoline passes through the blood–brain barrier of rat brain: An in vivo microdialysis study

Neuroscience Letters 395 (2006) 63–66

1-Benzyl-1,2,3,4-tetrahydroisoquinoline passes through the blood–brain barrier of rat brain: An in vivo microdialysis study Yaru Song a , Yangzheng Feng b , Michael H. LeBlanc b , Neal Castagloni Jr. c , Yi-Ming Liu a,∗ a

b

Department of Chemistry and Biochemistry, Jackson State University, 1400 Lynch Street, Jackson, MS 39217, USA Department of Pediatrics, University of Mississippi Medical Center, 2500 North State Street, Jackson, MS 39216, USA c Department of Chemistry, Virginia Tech, Blacksburg, VA 24061, USA Received 3 September 2005; received in revised form 18 October 2005; accepted 19 October 2005

Abstract Previous work has established that 1-benzyl-1,2,3,4-tetrahydroisoquinoline (1-BnTIQ) causes a parkinsonian syndrome in rats. The present study reports the blood–brain barrier (BBB) permeability of 1-BnTIQ in freely moving rats with the aid of in vivo microdialysis-based measurements. The microdialysis probe was implanted in the frontal cortex of rat brain. Brain dialysate samples were analyzed using an HPLC-MS/MS assay. 1-BnTIQ, when administered i.p., dose-dependently appeared in brain extracellular fluid (ECF), reaching a maximum concentration after about 40 min. Two other tetrahydroisoquinoline derivatives, 1,2,3,4-tetrahydroisoquinoline (TIQ) and 6,7-dihydroxy-1-methyl-1,2,3,4-tetrahydroisoquinoline [salsolinol (SAL)], served as positive and negative controls, respectively. The results confirmed an earlier report that SAL does not reach the brain after i.p. administration. In contrast, TIQ readily passed through the BBB. The brain dialysate concentration of 1-BnTIQ was about 24% that of TIQ when administered i.p. at the same dose. Both of them decreased quickly with a half-life of about 50 min. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Tetrahydroisoquinoline; 1-Benzyl-1,2,3,4-tetrahydroisoquinoline; Neurotoxin; Blood–brain barrier; Liquid chromatography-mass spectrometry

Many tetrahydroisoquinoline derivatives (TIQs) are neurotoxic [10,11,15]. Due to their overlapping structural features, these compounds may possess neurochemical properties similar to those of 1[N]-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a well known neurotoxin that induces symptoms similar to that of parkinsonism in humans, monkeys, and other susceptible animals [4]. MPTP causes the selective destruction of nigrostriatal dopaminergic neurons resulting in dopamine deficiency in the striatum and hence the symptoms of parkinsonism. Studies on the neurotoxicity of the TIQs’ have been extensive, particularly since the discovery of MPTP’s damaging effects on the nervous system. Dopamine-derived 6,7dihydroxy-1-methyl-1,2,3,4-tetrahydroisoquinoline [salsolinol (SAL)] has been reported to be cytotoxic to rat PC 12 cells [17], human dopaminergic SH-SY5Y cells [16], and melanoma cells [2]. 1-Methylsalsolinol (1-MeSAL) has been shown to cause parkinsonism in rats [12]. The CSF levels of 1-benzyl1,2,3,4-tetrahydroisoquinoline (1-BnTIQ) in patients suffering

∗

Corresponding author. Tel.: +1 601 979 3491; fax: +1 601 979 3674. E-mail address: [email protected] (Y.-M. Liu).

0304-3940/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2005.10.050

from Parkinson’s disease were found to be two times higher than those of a control cohort [7]. It has been shown that chronic administration of 1-BnTIQ induces parkinsonism in mice and primates [1,8]. Although TIQs can be formed in vivo from the condensation of catecholamines with aldehydes, they also exist in various foods such as cheese, banana, broiled beef, wine, and milk [3,6,9,19]. Therefore, it is important to examine the extent to which the blood–brain barrier (BBB) may prevent brain access of these types of neurotoxins following environmental exposures. Origitano et al. reported that SAL was unable to pass through the rat BBB [13]. On the other hand, TIQ, 1-MeTIQ, and N-methyl-norsalsolinol were found to pass through the BBB [5,18]. As far as we know, the question of the BBB permeability of 1-BnTIQ has not been addressed. Therefore, we have undertaken studies to investigate whether this important neurotoxin passes through the BBB by using in vivo microdialysis in combination with a highly sensitive and selective HPLC-MS/MS assay. All animal experiments were performed in strict accordance with the protocol approved by the Institutional Animal Care and Use Committee at Jackson State University. Male Sprague-

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Dawley rats (280–320 g) rats were anesthetized with sodium pentobarbital. The microdialysis cannulae were implanted in the frontal cortex. The coordinates for the implantation were +3.2 mm anterior to the bregma, −3.5 mm from the midline, and –1.5 mm from the dura surface [14]. Animals were allowed to recover for >2 days before dialysis experiments were performed. The dialysis probe (CMA/12, cut-off 20 kDa, o.d. 0.5 mm) was inserted without any visible distress or pain while the rats were conscious. Artificial cerebral spinal fluid (aCSF) consisting of 148 mM NaCl, 3.0 mM KCl, 0.8 mM MgCl2 , and 1.4 mM CaCl2 , pH 7.4, was perfused through the probe at a flow rate of 1.0 ␮L/min using a microdialysis pump (CMA/102, CMA Microdialysis, North Chelmsford, MA, USA). Dialysis samples were collected at 20 min intervals and analyzed immediately by LC-MS/MS without further purification. At the end of the experiment, the animals were euthanized and the probe placement was verified histologically. Rats were randomly allocated to groups (n = 4 for each group). Since the water solubility of 1-BnTIQ is low, all of the test compounds were dissolved in a mixture of DMSO and 9% NaCl solution (5:95). Animals of group 1 received TIQ (5 mg/kg body weight) as a positive control. Group 2 received SAL (5 mg/kg body weight) as a negative control. Groups 3–6 received 1-BnTIQ at doses ranging from 1 to 20 mg/kg body weight. The control group (group 0) received the vehicle (0.35 mL/kg body weight). The LC-MS/MS system consisted of two pumps (LC10ADvp, Shimadzu, Kyoto, Japan), an on-line degasser (DGU12A, Shimadzu) and an ion trap mass spectrometer equipped with an electrospray ionization (ESI) source (LCQ Deca, ThermoFinnigan, San Jose, CA, USA). Capillary LC columns (5 cm × 0.25 mm) packed with 5 ␮m phenyl C18 silica particles were used for the separation. A flow splitter was used to carry approximately 80% of the mobile phase delivered by the pumps to waste. The flow rate of the mobile phase passing through the capillary column was adjusted to 25 ␮L/min by changing the flow setting of the pumps. Mobile phase A used for loading samples was water. Mobile phase B used for eluting samples was a mixture of acetonitrile and water (70:30, v/v) containing 0.1% formic acid. Sample loading and isocratic elution were programmed as following: time 0.00–5.00 min, 100% mobile phase A (during this time period the sample was loaded onto the column and the eluent was directed to waste); time 5.10–15.00 min, 100% mobile phase B (during this time period the sample was eluted and the eluent was monitored by the MS detector); 15.10–20 min, 100% mobile phase A (to equilibrate the column); 20.10 min, stop. Injections were made by means of a Rheodyne 8125 injector (Supelco, Bellefonte, PA, USA) equipped with a 20-␮L sampling loop. The mass spectrometer was operated in positive ion mode. Multiple mass spectrometry (MS/MS) experiments were performed to isolate and fragment the targeted ions. The operating conditions of the MS detector were optimized with a solution of TIQ infused into the detector with a syringe pump at a flow rate of 10 ␮L/min. The signal intensity of [M + 1]+ was maximized as follows: sheath gas flow, 40 arbitrary units; auxiliary gas, 0 arbitrary units; monitored precursor ion precursor isolation

width 1 u; capillary temperature, 220 ◦ C; spray voltage, 4.0 kV. For the MS/MS experiments, the relative collision energy used was 33%. Other parameters were optimized by the autotune program. Data processing was performed using XCalibur software. TIQ, 1-BnTIQ, SAL, formic acid, isopropanol, and acetonitrile (HPLC grade) were obtained form Sigma–Aldrich Chemicals (St. Louis, MO, USA). Sodium pentobarbital was purchased from Abbott Laboratories (North Chicago, IL, USA). The HPLC-MS/MS assay used for the quantitative measurements of TIQ, 1-BnTIQ, and SAL in aCSF was thoroughly validated. These compounds were quantified by using the following ion transitions: m/z 134 → 117 for TIQ, m/z 224 → 207 for 1BnTIQ, and m/z 180 → 163 for SAL. Peak heights were used for the quantification. Calibration curves (5-points) were prepared with authentic chemicals dissolved in aCSF at concentrations ranging from 1 to 200 ng/mL. Regression analysis was used to calculate the slope and correlation coefficient. The calibration curves obtained were linear for all the three test compounds (r2 > 0.998). From the calibration curves, limits of detection were estimated to 0.2 ng/mL for all the three test compounds in aCSF (signal/noise = 3). Method precision was evaluated by repeatedly analyzing aCSF samples spiked with authentic chemicals at 10 ng/mL. Good reproducibility of the assay (intraday R.S.D.s ≤ 2.2%, and interday R.S.D.s ≤ 5.1%) was obtained. Further, microdialysis recovery was investigated for the three test compounds. Individual compound was dissolved in aCSF at concentrations ranging from 10 to 150 ng/mL. A microdialysis probe was placed in the solution and the compound in the dialysate was determined. As a result, no significant difference in recovery was observed for these compounds (i.e. TIQ, 1-BnTIQ, and SAL). The recovery was estimated to be 16%. Fig. 1 shows typical extracted ion chromatograms obtained from the determination of the test compounds in brain dialysates. Ions corresponding to TIQ, SAL, and 1-BnTIQ were not detected in the control group of animals that received an injection of vehicle (DMSO: 9% saline 5:95). After i.p. administration at a dose of 5 mg/kg body weight, 1-BnTIQ and TIQ were detected in brain dialysate. The mean maximum concentration was reached after c.a. 40 min for both compounds (Fig. 2). However, the concentration of TIQ was about five times higher than that of 1-BnTIQ, indicating that TIQ reached the brain much more easily than 1-BnTIQ did. SAL was not detected in brain dialysate following i.p. administration throughout the monitoring time window of 6 h (Fig. 2). Furthermore, the level of 1-BnTIQ following i.p. doses of 1, 8, and 20 mg/kg was examined. It was found that 1-BnTIQ level in brain extracellular fluid (ECF) was clearly dependent on the administered dose (Fig. 3). The HPLC-MS/MS assay used in this study was very sensitive and selective. Therefore, low levels of the test compounds in brain dialysate could be determined accurately and reliably even following a small dose of a test compound or when the compound did not reach the brain. The high sensitivity was particularly significant since high doses of a test compound might cause toxic effects that could impact on its ability to pass through the BBB. As can be seen from Fig. 1, the levels of TIQ and 1BnTIQ could be quantified without any interference from other endogenous compounds in this sample matrix at sub ng/mL con-

Y. Song et al. / Neuroscience Letters 395 (2006) 63–66

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Fig. 1. HPLC-MS/MS determination of the test compounds in rat brain dialysate: (A) extracted ion chromatogram of m/z 224 → 207 from a dialysate sample collected before 1-BnTIQ injection (note that 1-BnTIQ was not detected); (B) extracted ion chromatogram of m/z 224 → 207 from a dialysate sample collected 40 min after 1-BnTIQ injection (5 mg/kg body weight, i.p.); (C) extracted ion chromatogram of m/z 134 → 117 from a dialysate sample collected 40 min after TIQ injection (5 mg /kg body weight, i.p.); and (D) extracted ion chromatogram of m/z 180 → 163 from a dialysate sample collected 40 min after SAL injection (5 mg/kg body weight, i.p., note that SAL was not detected).

centrations. The TIQ and 1-BnTIQ peaks were identified both by HPLC retention time and MS characteristics. In contrast to these results, SAL levels in the dialysate after i.p. injection at a dose of 5 mg/kg body weight was below the detection limit of the assay that was estimated to be 0.2 ng/mL. Analysis of the brain dialysate samples collected from the control group of animals showed that the basal levels of TIQ, 1-BnTIQ, and SAL were below the limit of detection of the assay. After i.p. injection at a dose of 5 mg/kg body weight, 1BnTIQ appeared in the brain dialysate (Fig. 2) confirming its ability to pass through the BBB. This result was examined by using a positive (i.e. TIQ) and a negative (i.e. SAL) control of BBB penetration. TIQ, SAL, and 1-BnTIQ share a common structural moiety. TIQ has been shown to pass through the BBB

Fig. 2. Levels of the test compounds in brain dialysate before and after the compounds were injected i.p. into rats. Dose of the test compound given: 5 mg/kg body weight; n = 4 for each compound.

of rat brain [5] while another study showed that a substantial BBB existed for SAL [13]. As can been seen from Fig. 2, the positive control, TIQ, was detected in brain dialysate after i.p. injection. In contrast, the negative control, SAL, did not appear in brain dialysate during a monitoring time window of about 6 h. These results validated the experimental protocols used in this work for studying the BBB permeability of 1-BnTIQ. By comparing the maximum concentration of TIQ (36.8 ± 4.0 ng/mL) with that of 1-BnTIQ in the dialysate (8.7 ± 1.0 ng/mL), the BBB permeability of 1-BnTIQ was estimated to be about 24% that of TIQ. However, it should be pointed out that other parameters such as metabolism and transport can also be different for TIQ and 1-BnTIQ, and contribute to the differences in the BCF concentrations. To further characterize the finding that 1-BnTIQ passes through the rat BBB, 1-BnTIQ con-

Fig. 3. 1-BnTIQ levels in brain dialysate after i.p. administration of 1-BnTIQ at different doses; n = 4 for each group.

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centration in brain dialysate was determined following increasing i.p. doses of 1-BnTIQ (i.e. 1, 8, and 20 mg/kg body weight). As can be seen from an inspection of Fig. 3, the mean maximum level of 1-BnTIQ measured in brain dialysate was directly dependent on the dose given to the rats. The concentration–time curves from all the three different doses indicated that the concentration of 1-BnTIQ in brain dialysate decreased quickly. Using semi-log plots of the concentration of 1-BnTIQ in brain dialysate versus time (min), the half-life of 1-BnTIQ was estimated to be 50 min. In summary, 1-BnTIQ, a parkinsonism-inducing neurotoxin, has been found to pass through the BBB of rat brain. This finding was confirmed by characterizing the behavior of positive and negative drug controls for BBB permeability. It was further verified by the fact that the mean-maximum concentration of 1BnTIQ measured in brain dialysate was directly dependent upon the i.p. dose given to the rats. As far as we know, this is the first report describing the BBB permeability of this neurotoxin. Acknowledgement Financial support from NIH grants (NS44177, and partially S06GM08047 and 12RR13459) is gratefully acknowledged. References [1] K. Abe, K. Taguchi, T. Wasai, J. Ren, I. Utsunomiya, T. Shinohara, T. Miyatake, T. Sano, Biochemical and pathological study of endogenous 1-benzyl-1,2,3,4-tetrahydroisoquinoline-induced parkinsonism in the mouse, Brain Res. 907 (2001) 134–138. [2] F. De Marco, M. Perluigi, M.L. Marcante, R. Coccia, C. Foppoli, C. Blarzino, M.A. Rosei, Cytotoxicity of dopamine-derived tetrahydroisoquinolines on melanoma cells, Biochem. Pharmacol. 64 (2002) 1503–1512. [3] H. Haber, A. Winkler, I. Putscher, P. Henklein, I. Baeger, M. Georgi, M.F. Melzig, Plasma and urine salsolinol in humans: effect of acute ethanol intake on the enantiomeric composition of salsolinol, Alcohol. Clin. Exp. Res. 20 (1996) 87–92. [4] R.E. Heikkila, A. Hess, R.C. Duvoisin, Dopaminergic neurotoxicity of 1methyl-4-phenyl-1,2,5,6-tetrahydropyridine in mice, Science 224 (1984) 1451–1453. [5] K. Kikuchi, Y. Nagatsu, Y. Makino, T. Mashino, S. Ohta, M. Hirobe, Metabolism and penetration through blood–brain barrier of

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