Novel mimetic tissue standards for precise quantitative mass spectrometry imaging of drug and neurotransmitter concentrations in rat brain tissues

Abstract

Understanding the relationship between the concentration of a drug and its therapeutic efficacy or side effects is crucial in drug development, especially to understand therapeutic efficacy in central nervous system drug, quantifying drug-induced site-specific changes in the levels of endogenous metabolites, such as neurotransmitters. In recent times, evaluation of quantitative distribution of drugs and endogenous metabolites using matrix-assisted laser desorption/ionization (MALDI)-mass spectrometry imaging (MSI) has attracted much attention in drug discovery research. However, MALDI-MSI quantification (quantitative mass spectrometry imaging, QMSI) is an emerging technique, and needs to be further developed for practicable and convenient use in drug discovery research. In this study, we developed a reliable QMSI method for quantification of clozapine (antipsychotic drug) and dopamine and its metabolites in the rat brain using MALDI-MSI. An improved mimetic tissue model using powdered frozen tissue for QMSI was established as an alternative method, enabling the accurate quantification of clozapine levels in the rat brain. Furthermore, we used the improved method to evaluate drug-induced fluctuations in the concentrations of dopamine and its metabolites. This method can quantitatively evaluate drug localization in the brain and drug-induced changes in the concentration of endogenous metabolites, demonstrating the usefulness of QMSI.

Keywords: Brain; Clozapine; Matrix-assisted laser desorption/ionization; Neurotransmitters; Quantitative mass spectrometry imaging.

Fig. 1

The workflow for construction of a mimetic model. a Mimetic tissue models construction step for Clozapine (CLZ) using syringes. b Mimetic tissue models construction step for dopamine (DA), 3-methoxytyramine (3-MT), and homovanillic acid (HVA) using molds

Fig. 2

Calibration curves (Calc) on day 1 and day 7 of CLZ with mimetic tissue models. a Calculated image of day 1. b Calculated image of day 7. c Calibration curves of CLZ on day 1. d Calibration curves of CLZ on day 7. Images of CLZ (m/z 327.136, scaled from 0 to 30%) were acquired with a spatial resolution of 70 μm and normalized to the signal of the isotope-labeled standard (CLZ-d4, m/z 331.165). The region by white dashed lines indicates the respective concentration layer used for constructing the calc. weighted least squares calibration curves (solid lines) was applied to correlate the average ion intensity of each layer of the mimic model to its corresponding setting concentration

Fig. 3

Images of quality control (QC) samples on days 1 and 7 of clozapine (CLZ) treatment of mimetic tissue models. QC image on a day 1 and b day 7. Images of CLZ (m/z 327.136, scaled from 0 to 30%) were acquired with a spatial resolution of 70 μm and normalized to the signal of the isotope-labeled standard (CLZ-d4, m/z 331.165). The region bordered by white dashed lines indicates the respective concentration layer used for constructing the QC samples

Fig. 4

MSI image of clozapine (CLZ) and optical image of rat brain tissue sections after CLZ or vehicle treatment. Representative images were measured with a spatial resolution of 70 μm. The signals are normalized to the signal for the isotope-labeled standard (CLZ-d4). a Distribution of CLZ in the brain tissue sections from rats A, B, C, and D as determined using MSI at 70-μm resolution. b Optical images of the brain tissue sections from rats A, B, C, and D visualized using hematoxylin staining after MSI measurement. Corresponding selected regions of interest (ROIs 1, 2, and 3) show the ion distribution, localized to features of the regions defined using the Brain Atlas. The CLZ ion distribution was localized to a feature of the tissue

Fig. 5

Comparison of clozapine (CLZ) quantification result in three regions of interest (cortex, thalamus, and cerebellum) in the brain tissue of rats (N = 3) using MSI (white) and LCM-LC/MS (black). The concentrations are shown as means ± SD

Fig. 6

MSI images for dopamine (DA), 3-methoxytyramine (3-MT), and homovanillic acid (HVA) and optical images of rat brain tissue sections after treatment with clozapine (CLZ) or vehicle. Representative images were acquired with a spatial resolution of 70 μm. Images for DA, 3MT, and HVA (m/z 421.191, m/z 435.206, and m/z 450.170, scaled from 0 to 8%) were acquired and normalized to the signal for the isotope-labeled standard (m/z 425.217, m/z 439.232, and m/z 455.202). a–d Ion distribution of DA in the rat brain after administration of CLZ or vehicle. e, f Ion distribution of 3-MT in the rat brain after administration of CLZ or vehicle. i–l Ion distribution of HVA in the rat brain after administration of CLZ or vehicle. m–p Optical images of the rat brain tissue sections after administration of CLZ or vehicle visualized using hematoxylin staining after MSI measurement. Corresponding selected regions of interest (white dashed lines) show the ion distribution, localized to the striatum defined using the Brain Atlas

Fig. 7

Calibration curves (Calc) of DA, 3-MT, and HVA with mimetic tissue models. a Calculated image of DA. b Calculated image of 3-MT. c Calculated image of HVA. d Calibration curves of DA. e Calibration curves of 3-MT. f Calibration curves of HVA. Images of DA, 3MT, and HVA (m/z 421.191, m/z 435.206, and m/z 450.170, scaled from 0 to 50%) were acquired and normalized to the signal for the isotope-labeled standard (m/z 425.217, m/z 439.232, and m/z 455.202). The region by white dashed lines indicates the respective concentration layer used for constructing the calc. weighted least squares calibration curves (solid lines) was applied to correlate the average ion intensity of each layer of the mimic model to its corresponding setting concentration