A temporo-spatial pharmacometabolomics method to characterize pharmacokinetics and pharmacodynamics in the brain microregions by using ambient mass spectrometry imaging

Abstract

The brain is the most advanced organ with various complex structural and functional microregions. It is often challenging to understand what and where the molecular events would occur for a given drug treatment in the brain. Herein, a temporo-spatial pharmacometabolomics method was proposed based on ambient mass spectrometry imaging and was applied to evaluate the microregional effect of olanzapine (OLZ) on brain tissue and demonstrate its effectiveness in characterizing the microregional pharmacokinetics and pharmacodynamics of OLZ for improved understanding of the molecular mechanism of drugs acting on the microregions of the brain. It accurately and simultaneously illustrated the levels dynamics and microregional distribution of various substances, including exogenous drugs and its metabolites, as well as endogenous functional metabolites from complicated brain tissue. The targeted imaging analysis of the prototype drug and its metabolites presented the absorption, distribution, metabolism, and excretion characteristics of the drug itself. Moreover, the endogenous functional metabolites were identified along with the associated therapeutic and adverse effects of the drug, which can reflect the pharmacodynamics effect on the microregional brain. Therefore, this method is significant in elucidating and understanding the molecular mechanism of central nervous system drugs at the temporo and spatial metabolic level of system biology.

Keywords: Antipsychotic drug; Mass spectrometry imaging; Pharmacodynamics; Pharmacokinetics; Pharmacometabolomics.

Graphical abstract

Scheme 1

Strategy and workflow of the proposed temporo-spatial pharmacometabolomics method to characterize the microregional pharmacokinetics and pharmacodynamics of CNS drugs in the brain.

Figure 1

Imaging results for exogenous drug and endogenous metabolites obtained using AFADESI-MSI from brain tissue section. The representative mass spectra acquired using AFADESI-MSI from treatment (A) and control groups (B) brain tissue section in positive ion mode. C1 and D1 are experimental mass spectra of olanzapine (OLZ) and its metabolite, 2-hydroxymethyl OLZ, respectively. C2 and D2 are the calculated isotopic peaks of OLZ and 2-hydroxymethyl OLZ, respectively. (E) represents the hematoxylin-eosin (H&E) staining of sections of the brain. Scale bar = 2 mm. (F1)–(F5) show ion images of OLZ, γ-aminobutyric acid (GABA), Creatine, LysoPC (16:0), PC (32:0), respectively.

Figure 2

Temporo-spatial alteration of OLZ and 2-hydroxymethyl OLZ in brain microregions. (A) MS images of OLZ and 2-hydroxymethyl OLZ in brain tissue section acquired using AFADESI-MSI. (B1, B2) The percentage of AUC in OLZ and 2-hydroxymethyl OLZ in different brain microregions. (C1, C2) The relative intensity changes of OLZ and 2-hydroxymethyl OLZ in the different brain microregions with the time. Data are presented as means ± standard deviation (SD), n = 3. CP: caudate putamen, CTX: cerebral cortex, HP: hippocampus, HY: hypothalamus, TH: thalamus, CBC: cerebellar cortex, CM: cerebellar medulla, MD: medulla, PN: pons, CA: cerebral aqueduct, MB: middle brain, FN: fornix, PC: piriform cortex, OB: olfactory bulb, and CC: corpus callosum.

Figure 3

Drug effect on the distribution and AUC change ratio for NTs in brain. (A) represents the images of H&E-stained brain sections followed by the distribution of NTs in the brain acquired by AFADESI-MSI. (B1–B7) show the AUC change ratio of NTs. AUC change ratio = (AUC after drug‒AUC before drug)/AUC before drug. AUC change ratio >0 indicates an up-regulated status. AUC change ratio <0 indicates a down-regulated status. The larger absolute values of the AUC change ratio represent greater up-regulation or down-regulation with the drug action or effect.

Figure 4

Hierarchical cluster analysis (HCA) of identified differential metabolites in control and treatment groups. The row on the right lists the metabolites, the column on the top indicates class (green: treatment groups and red: control groups), and the column on the bottom indicates sample ID (T: treatment groups sample and C: control groups sample). The metabolites were clustered, and shades of red and blue represent high expression levels and low expression levels, respectively.

Figure 5

Temporo-spatial distribution of metabolites of the alanine, aspartate, and glutamate metabolism pathway. (A) Spatial distribution and changes of metabolites involved in the alanine, aspartate, and glutamate metabolism pathway in the treatment (50 min after dosing) and control groups. (B) MS images of metabolites of the alanine, aspartate, and glutamate metabolism pathways in brain tissue section acquired using AFADESI-MSI. Statistical analysis of the metabolome data extracted from control and treatment groups (50 min) in different brain microregions. (means ± SD), n = 3. ∗P < 0.05, ∗∗P < 0.01 and ∗∗∗P < 0.001. The red circle in the pie chart represents the conversion of log2 (1) equal to 0 when the fold change of metabolite before and after administration is 1 (FC = 1). The sector outside the red circle represents the fold change greater than 1 (FC > 1) and indicates this metabolite with up-regulation after drug intervention in this microregion, while the sector inside the red circle represents the fold change less than 1 (FC < 1) and indicates this metabolite with down-regulation after drug intervention in this microregion. The distance of the radius of the sector from the red circle represents the degree of fold change. Outside the red circle, a larger radius of the sector represents more pronounced up-regulation, and within the red circle, a smaller radius of the sector represents more significant down-regulation. The size of the rounding angle represents the relative abundance in the microregion; the larger the rounding angle, the greater the relative abundance.

Figure 6

Temporo-spatial distribution of metabolites of the glycerophospholipid metabolism pathway. (A) Spatial distribution and changes of metabolites involved in the glycerophospholipid metabolism pathway in the treatment (50 min after dosing) and control groups. (B) MS images of metabolites of the glycerophospholipid metabolism pathway in brain tissue section acquired using AFADESI-MSI. Statistical analysis was performed using Student's t-test. ∗P < 0.05, ∗∗P < 0.01 and ∗∗∗P < 0.001.