| Excessive exposure to manganese (Mn) can often lead to Mn-induced neurotoxicity with companion symptoms similar to those of Parkinson’s disease. The mechanisms of Mn-induced neurotoxicity are quite complicated and as a result, there still lacks effective clinical treatments. Therefore, there is a pressing need to establish an effective and viable therapeutic agent for the treatment of Mn-induced neurotoxicity. On the other hand, Para-aminosalicylic acid (PAS), being an anti-tuberculosis drug since 1959’s, has recently been reported as an effective agent for the treatment of Mn. However, the drug metabolism and pharmacokinetics of PAS and its metabolite N-acetyl-para-aminosalicylic acid (AcPAS) in brain is still largely unknown. This study used in vivo and in vitro methods to quantify the rat brain regional pharmacokinetics as well as the cerebral absorption, distribution, metabolism and excretion of PAS and AcPAS. In addition, it will provide guide for the practical usage of both PAS and AcPAS, as well as theoretical and practical foundations for the development and design of drugs for Mn-induced neurotoxicity.1. Developing an HPLC-fluorescence analytical method for simultaneous measurement of PAS and AcPASPurpose:This study was aimed to develop a simple and effective method to assay PAS and its major metabolite AcPAS in rat plasma, cerebrospinal fluid (CSF) and brain homogenate. Method:Biological samples underwent one-step protein precipitation. The supernatant was determined using a reversed-phase C18 column with a gradient elution system, followed by on-line fluorescence detection. Results:No endogenous fluorescent interferences were observed during the measurement of PAS, AcPAS and internal standard compound in bio-samples. The calibration curves for both PAS and AcPAS spiked into blank plasma showed an excellent linearity in the concentration range of 0.05～500μg/mL. The calibration curves for both PAS and AcPAS in brain homogenate and artificial CSF (aCSF) showed an excellent linearity in the concentration range of 0.017～166.7μg/g of tissue or CSF weight. The lower limits of quantification for both PAS and AcPAS were 50ng/mL of plasma and 17ng/g of tissues. PAS and AcPAS in samples have the intra-day and inter-day precision values between 1～8%, with the accuracy between 93～109%. The absolute recoveries of PAS from plasma and brain homogenates were between 64-67% and 67-69%, respectively, while the absolute recoveries of PAS from aCSF were between 94-97%. For AcPAS, the absolute recoveries from plasma, brain tissue and aCSF were between 65-66%,77-85%, and 94-97%, respectively. Studies on the freeze-thaw stability and the long-term stability revealed no significant decrease in concentrations of both PAS and AcPAS. The results confirmed no stability-related problems during routine analysis of the samples.Conclusion:In this study we established a simple and effective method to quantify PAS and AcPAS in bio-samples. The method has been proven to be sensitive, reproducible, and practically useful for laboratory and clinical investigations of PAS in treatment of Mn Parkinsonism.2. Distribution, protein (tissue) binding and metabolism studies of PAS and AcPASPurpose:We evaluated the capabilities of PAS and AcPAS for entering brain parenchyma from rat blood, and provided scientific justifications for the understanding and utilization of detoxicity mechanism of PAS. We conducted protein binding experiments to determine the free, unbound PAS and AcPAS in plasma and brain regions, and conducted metabolism experiments to determine if PAS was converted to AcPAS by N-acetyltransferases-1 expressed in targeted brain regions in the hope of predicting the possible factors which could affect the absorption, distribution and elimination of PAS and AcPAS in rat brain. Method:The male Sprague-Dawlcy rats received a femoral artery intravenous injection of 200mg/kg PAS. Plasma, CSF, and brain tissues were collected and quantified for PAS and AcPAS by HPLC after 45 minutes with 5-aminosalicylic acid as the internal standard. We collected and purified the fraction of potential metabolite of brain sample from the HPLC and analyzed the sample by HPLC and the proton NMR spectroscopy. We conducted protein binding experiments to measure the protein binding ratio of PAS and AcPAS in plasma and 6 selected brain regions. The in vitro metabolism study was carried out by brain homogenate incubation. Results:Except for choroid plexus and CSF, the concentrations of AcPAS in most brain regions are higher than those of PAS, and in particular, we can only detect AcPAS in brain capillary. Choroid plexus has the highest PAS concentration, followed by CSF, with thalamus the lowest; for AcPAS, choroid plexus has the highest concentration, followed by hippocampus, with CSF the lowest. Most of PAS molecules existed in a very high percentage (>90%) as the free, unbound PAS, whereas the unbound AcPAS fractions were between 80-87%. The in vitro metabolism study confirmed that arylamine N-acetyltransferase in rat brain was not capable of converting PAS to AcPAS. Conclusion:45 minutes after PAS administration, both PAS and AcPAS can be detected in rat brain regions, and AcPAS has higher concentrations than PAS in brain parenchyma. Both PAS and AcPAS appeared to exist in plasma and brain tissues mainly in the free, unbounded form. AcPAS had a higher plasma and tissue binding than PAS. The AcPAS in brain was from blood cycle, but not from the bio-conversion of PAS in brain tissues.3. Brain regional pharmacokinetic study of PAS and its major metabolitePurpose:We study the uptake and deposit role of different brain regions towards PAS and AcPAS, to further determine pharmacokinetic parameters of PAS and AcPAS following iv injection of PAS. Method:We used the well-developed HPLC-fluorescence analysis to determine pharmacokinetic characteristics of PAS and AcPAS in rat plasma, CSF and selected brain regions following PAS administration. The concentration-time profiles of PAS and AcPAS in plasma, CSF and brain tissues were analyzed by non-compartmental methods using DAS version 2.0 software. Results: After injection of PAS (200 mg/kg) in Sprague-Dawley rats, the concentration-time profile of PAS in plasma was consistent with a two compartment model with the t1/2 of 34mins. The metabolite AcPAS reached Cmax at 86 min after PAS administration. In the CSF, PAS concentrations arose rapidly and reached Cmax at 17 min following iv injection of PAS and was eliminated within 4 hrs. AcPAS reached Cmax at 44 min in CSF. The AUC0-∞of the parent PAS in plasma was about 3 fold as large as that of AcPAS. The AUC0-∞of CSF PAS was about 7.5 fold as large as that of AcPAS in the CSF. The concentrations of PAS and AcPAS in choroid plexus reached with Cmax of 42.81±4.90μg/g and 30.74±3.50μg/g respectively, and were much lower in other brain regions. AcPAS had much higher tissue concentrations and possessed longer t1/2 than the parent PAS in most tissues examined, indicating higher capabilities of AcPAS for getting across the brain barriers and entering brain parenchyma. Conclusion:PAS and AcPAS displayed distinct phannacokinetic characteristics. And the time profiles of the single specie in different brain regions distinctly differed from the pattern of those in plasma. Moreover, phannacokinetic parameters of AcPAS were significantly associated with PAS’s efficacy in reducing the tissue Mn. AcPAS seem to be more effective in chelating and mobilizing Mn in brain.4. Transport study of PAS and its major metabolitePurpose:This study was aimed at determining and comparing the potential trans-barrier uptake and transport mechanisms of PAS and AcPAS at blood-brain barrier and blood-CSF barrier. Method:We used MDCK-MDR1 cells as the in vitro model to mimic the drug transport at blood-brain barrier, and used Z310 cells as the in vitro model to mimic the drug transport at blood-CSF barrier, and applied the two-chamber Transwell system to study in vitro cell monolayer transport and to evaluate the uptake and transport of PAS and AcPAS at brain barriers, as well as the inhibition effect of PAS on efflux transporter P-glycoprotein (P-gp). Results:The apparent permeability coefficients of PAS in MDCK and MDCK-MDR1 cell monolayer are both less than 1×10-6cm/s, indicating that its permeability through blood-brain barrier is relatively poor. The transport of PAS in MDCK-MDR1 cell monolayer showed significant efflux phenomena with net efflux ratio 3.8. Furthermore, such efflux can be abolished by the classic inhibiters of P-gp such as Verapamil and Quinidine, which indicates that PAS is the substrate of P-gp and the latter is one of the main factors in causing the low cerebral bio-availability of PAS in brain. The permeatibility of AcPAS through MDCK-MDR1 is not affected by the efflux effect of P-gp. When using R123 as the substrate probe of P-gp, we found that PAS can affect the efflux of R123 by P-gp in MDCK-MDR1 cell monolayer with half maximal (50%) inhibitory concentration (IC50) of 10.29μg/mL, which revealed that PAS can inhibit the efflux activity of P-gp. Two-chamber Transwell studies with choroidal epithelial Z310 cells did not find significant differences between two compounds with regards to their influx or efflux permeability. The in vitro routes of PAS and AcPAS across Z310 cell monolayer were greater from the blood-facing membrane than from the CSF-facing membrane especially, indicating a bias for influx of both compounds at blood-CSF barrier. Conclusion:The in vitro transport studies of MDCK-MDR1 cells showed that PAS is the substrate of P-gp, and high dose PAS would inhibit the transport efficiency of P-gp, indicating PAS in brain tissues increased more sharply and then was eliminated very quickly. AcPAS is not the substrate of P-gp, and its permeability across the cells was greater from the blood-facing membrane to the CSF-facing membrane, indicating AcPAS in brain tissues increased more smoothly and steadily, and the concentration of AcPAS in brain parenchyma is significantly higher. The in vitro transport studies of Z310 cells showed that AcPAS was effluxed by MRP1 from CSF to choroid plexus at blood-CSF barrier, which revealed that the concentration of AcPAS is lower in CSF but higher in choroid plexus.In summary, after rat received a femoral artery intravenous injection of 200mg/kg PAS, PAS in blood can permeate across blood-CSF barrier into CSF and then be effluxed by blood-brain barrier back into blood. After getting across blood-brain barrier, AcPAS can stay in brain and eventually be eliminated from blood-CSF barrier. Based on the pharmacokinetics-pharmacodynamics analysis, it showed that both PAS and AcPAS can couple and eliminate Mn in rat brain regions and thus treat the Mn-induced neurotoxicity. Furthermore, AcPAS has been shown to have more important roles in the processing of coupling and eliminating Mn than PAS. |
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