Influence of peptide transporter 2 (PEPT2) on the distribution of cefadroxil in mouse brain: A microdialysis study

Abstract

Peptide transporter 2 (PEPT2) is a high-affinity low-capacity transporter belonging to the proton-coupled oligopeptide transporter family. Although many aspects of PEPT2 structure-function are known, including its localization in choroid plexus and neurons, its regional activity in brain, especially extracellular fluid (ECF), is uncertain. In this study, the pharmacokinetics and regional brain distribution of cefadroxil, a β-lactam antibiotic and PEPT2 substrate, were investigated in wildtype and Pept2 null mice using in vivo intracerebral microdialysis. Cefadroxil was infused intravenously over 4h at 0.15mg/min/kg, and samples obtained from plasma, brain ECF, cerebrospinal fluid (CSF) and brain tissue. A permeability-surface area experiment was also performed in which 0.15mg/min/kg cefadroxil was infused intravenously for 10min, and samples obtained from plasma and brain tissues. Our results showed that PEPT2 ablation significantly increased the brain ECF and CSF levels of cefadroxil (2- to 2.5-fold). In contrast, there were no significant differences between wildtype and Pept2 null mice in the amount of cefadroxil in brain cells. The unbound volume of distribution of cefadroxil in brain was 60% lower in Pept2 null mice indicating an uptake function for PEPT2 in brain cells. Finally, PEPT2 did not affect the influx clearance of cefadroxil, thereby, ruling out differences between the two genotypes in drug entry across the blood-brain barriers. These findings demonstrate, for the first time, the impact of PEPT2 on brain ECF as well as the known role of PEPT2 in removing peptide-like drugs, such as cefadroxil, from the CSF to blood.

Keywords: Blood-cerebrospinal fluid barrier; Brain extracellular fluid; Cefadroxil; Microdialysis; Peptide transporter 2.

Fig. 1

Concentration-time profiles of cefadroxil in the plasma (total drug, C_p) and brain extracellular fluid (unbound drug, C_u,ECF) during a 4-hr intravenous infusion of 0.15 mg/min/kg cefadroxil in wildtype and Pept2 null mice. Data are expressed as mean ± SEM (n=10–12). ^***p<0.001 when comparing C_p or C_u,ECF between the two genotypes, as indicated by two-way ANOVA with Bonferroni correction for multiple comparisons.

Fig. 2

Concentrations of cefadroxil in the plasma (total drug, C_p,240), brain extracellular fluid (unbound drug, C_u,ECF or C_{u,ECF,220-240}) and cerebrospinal fluid (C_csf), as well as amount of cefadroxil in the brain parenchyma (A_brain) and brain cells (A_cell) of wildtype and Pept2 null mice at the end of the 4-hr intravenous infusion of 0.15 mg/min/kg cefadroxil. Data are expressed as mean ± SEM (n=6–12). *p<0.05 and **p<0.001 when comparing a parameter between two genotypes, as indicated by Welch’s t-test (for unequal variance) and by student’s t-test (for equal variance).

Fig. 3

Ratio of unbound concentration in brain ECF to total plasma concentration (C_u,ECF/C_p) as a function of time during a 4-hr constant intravenous infusion of 0.15 mg/min/kg cefadroxil in wildtype and Pept2 null mice. Data are expressed as mean ± SEM (n=10–12). *p<0.05 and ***p<0.001 when comparing a ratio between two genotypes, as indicated by two-way ANOVA with Bonferroni correction for multiple comparisons.

Fig. 4

Unbound volume of distribution (V_u,brain) of cefadroxil in different brain regions (A) and whole brain (averaged from the five regions) (B) of wildtype and Pept2 null mice at the end of a 4-hr intravenous infusion of 0.15 mg/min/kg cefadroxil. Data are expressed as mean ± SEM (n=11–12). Two-way ANOVA indicated that genotype and not brain region was an influencing factor for V_u,brain. Welch’s t-test indicated a significant differences in V_u,brain between the two genotypes.

Fig. 5

Permeability-surface area (PS) product of cefadroxil in different brain regions based on the study design using a 10-min intravenous infusion of 0.15 mg/min/kg cefadroxil in wildtype and Pept2 null mice. Data are expressed as mean ± SEM (n=3). Two-way ANOVA indicated that brain region but not genotype was an influencing factor for PS product (i.e., specific to cerebellum as compared to the other brain regions).

Fig. 6

A schematic illustration showing the dual role of PEPT2 in affecting the disposition of cefadroxil (CEF) in brain. Apical membrane (AP), basolateral membrane (BL), blood-brain barrier (BBB), blood-cerebrospinal fluid barrier (BCSFB), cerebrospinal fluid (CSF), choroid plexus (CP), ependyma (EP), extracellular fluid (ECF), wildtype (WT) and Pept2 null (KO) mice.