The role of Abcb5 alleles in susceptibility to haloperidol-induced toxicity in mice and humans

Abstract

Background: We know very little about the genetic factors affecting susceptibility to drug-induced central nervous system (CNS) toxicities, and this has limited our ability to optimally utilize existing drugs or to develop new drugs for CNS disorders. For example, haloperidol is a potent dopamine antagonist that is used to treat psychotic disorders, but 50% of treated patients develop characteristic extrapyramidal symptoms caused by haloperidol-induced toxicity (HIT), which limits its clinical utility. We do not have any information about the genetic factors affecting this drug-induced toxicity. HIT in humans is directly mirrored in a murine genetic model, where inbred mouse strains are differentially susceptible to HIT. Therefore, we genetically analyzed this murine model and performed a translational human genetic association study.

Methods and findings: A whole genome SNP database and computational genetic mapping were used to analyze the murine genetic model of HIT. Guided by the mouse genetic analysis, we demonstrate that genetic variation within an ABC-drug efflux transporter (Abcb5) affected susceptibility to HIT. In situ hybridization results reveal that Abcb5 is expressed in brain capillaries, and by cerebellar Purkinje cells. We also analyzed chromosome substitution strains, imaged haloperidol abundance in brain tissue sections and directly measured haloperidol (and its metabolite) levels in brain, and characterized Abcb5 knockout mice. Our results demonstrate that Abcb5 is part of the blood-brain barrier; it affects susceptibility to HIT by altering the brain concentration of haloperidol. Moreover, a genetic association study in a haloperidol-treated human cohort indicates that human ABCB5 alleles had a time-dependent effect on susceptibility to individual and combined measures of HIT. Abcb5 alleles are pharmacogenetic factors that affect susceptibility to HIT, but it is likely that additional pharmacogenetic susceptibility factors will be discovered.

Conclusions: ABCB5 alleles alter susceptibility to HIT in mouse and humans. This discovery leads to a new model that (at least in part) explains inter-individual differences in susceptibility to a drug-induced CNS toxicity.

Fig 1. A pharmacogenetic factor for HIT.

(A) The average latency measured after 30 days of haloperidol administration to 17 inbred strains is shown in the top panel. The bottom panel shows ten genes whose genetic pattern was most highly correlated with the latency data by the HBCGM program. The genes within correlated haplotype blocks are indicated by their symbol; an orange, yellow, or white background indicates whether SNPs causing a significant, minor, or no amino acid change are present, respectively. (A blue background indicates a SNP that affects a potential splice site alteration is present.) The haplotypic pattern is shown as colored rectangles that are arranged in the same order as the input data. Strains with the same colored rectangle have the same haplotype within the block. The p-values and genetic effect size were calculated as previously described [14]. (B) Missense SNPs in Abcb5. The 1,255 amino acids in Abcb5 contain two transmembrane (shaded blocks) and two ATP binding transporter domains (solid blocks). The alleles in eight missense SNPs in the 17 analyzed strains are shown, and the alleles that differ from the C57BL/6 reference strain are highlighted. The Gly170Cys is within the first transmembrane domain, and Gly491Ser and Lys493Glu are within the first transporter domain. These three SNPs divide the strains into three haplotypic groupings (each indicated by a different color) that correspond with the haloperidol-induced latency.

Fig 2. (A) HIT in C57BL/6J, A/J, and four chromosome substitution strains.

The time (latency) required for a mouse to make a coordinated movement on a vertical mesh screen after treatment with haloperidol (3 mg/kg/day IP) for the indicated number of days is shown. Each bar represents the average ± SEM for three to four mice per strain. Only CSS12 mice (which have A/J alleles for every gene on Chromosome 12) had a significantly increased latency (p < 0.005) after 5 and 10 days of haloperidol treatment. (B) The amount of haloperidol and its metabolite (HPP⁺) in brain tissue obtained from A/J and C57BL/6 mice after 4 days of treatment with haloperidol (10 mg/kg/day IP) was assessed in two independent experiments. Each bar represents the average ± SEM of the LC/MS determined abundance (molecules per unit mass of brain tissue) for n = 4 mice per strain. (C) The brain level of the oxidative metabolite of haloperidol (HPP⁺) was measured after 10 days of haloperidol administration (3 mg/kg/day) to A/J (n = 6), C57BL/6 (n = 6), and Chromosome 12 substitution strain (CSS12) (n = 8) mice. Each bar shows the average ± standard error for the six to eight measurements for each strain, and the p-value is calculated using the Student’s t test on log2-transformed relative abundance data. Of note, CSS12 mice have a significantly higher (1.3-fold, p = 0.004) brain HPP⁺ level than C57BL/6J mice, but this level is below that in A/J mice.

Fig 3. DESI-MSI of haloperidol in brain tissue.

A/J and C57BL/6J mice (n = 3 per strain) were treated with haloperidol (10 mg/kg/day IP) for 5 days. Two sagittal sections, which were separated by a distance of 120 μm, were prepared the same brain region of haloperidol-treated mice. The amount of haloperidol was analyzed DESI-MSI, and the relative abundance of haloperidol is indicated by the color shown in the scale bar. The HE-stained brain section shown at the bottom shows the sagittal plane analyzed, and the location of the frontal cortex (FC) and cerebellum (CB) are indicated for orientation. Haloperidol was diffusely present in the brain tissue obtained from both strains. Although the amount of haloperidol in brain tissue varied among different mice, A/J brain tissue had remarkably increased amount of haloperidol relative to C57BL/6J brain tissue.

Fig 4. Abcb5 mRNA expression in mouse brain.

In situ hybridization was performed using anti-sense probes for Abcb5 and Drd2 mRNA on C57BL/6 mouse brain sections. For each anti-sense probe, negative-control hybridizations were also performed using the corresponding sense RNA probe on adjacent tissue sections. (A) Abcb5 mRNA was specifically expressed in the perivascular regions, as shown in two separate views of cortical and cerebellar regions. (B) Purkinje cells in cerebellum express Abcb5 mRNA, and these cells also express the dopamine receptor D2 (Drd2) that is bound by haloperidol. The left and right images are shown at 4× magnification, and magnified views (20×) of the boxed regions are shown in the center image.

Fig 5. (A, B) Abcb5 knockout mice have a prolonged haloperidol-induced latency.

In two independent experiments shown in (A) and (B), Abcb5 knockout mice and wild-type littermates (gender indicated by male or female) were treated with haloperidol (10 mg/kg IP) for 7 days, and the haloperidol-induced latency was measured on the indicated days. In both experiments, the latencies in Abcb5 knockout mice were significantly prolonged relative to wild-type mice after 4, 5, 6, and 7 days of haloperidol treatment. The p-values comparing the latencies measured in the Abcb5 knockout and wild-type groups are shown for each treatment day. (C) DESI-MSI of haloperidol in brain tissues of Abcb5 knockout (n = 3) and wild-type littermate (n = 3) mice. The mice were treated with haloperidol (10mg/kg/day IP) for 4 days, and brain tissues were collected 4 hours after the last treatment. The amount of haloperidol was analyzed by DESI-MSI on 20 μm brain sections, and the relative abundance of haloperidol is indicated by the color shown in the scale bar. The HE-stained brain section shown at the bottom shows the sagittal plane analyzed. Abcb5 knockout mice have a 7-fold increased brain haloperidol level relative to wild-type littermates (p = 0.02).

Fig 6. Human ABCB5 alleles and HIT.

(A) The average of three toxicity measurements (± SEM) for 85 patients determined after the indicated time of haloperidol treatment is shown. Of note, the average Dyskinesia score peaked at 3 days post treatment and then declined, while the two other toxicity measurements (akathesia and parkinsonoid) increased with time after treatment until day 14. For this analysis, the toxicity measurement data for days 0, 1, 3, 7, 14, and 21 were available for 85, 85, 85, 85, 81, and 54, respectively, of the 85 patients in this cohort. (B) The graph shows the combined toxicity measurement (y-axis) relative to the time (x-axis) for ABCB5 SNP rs17143212. The average combined toxicity measurement was calculated for each genotype at each SNP, and then plotted (± SEM) according to the colors shown in the legend. (C) The LD map for genetic variants in ABCB5, which was compiled using data obtained from the International HapMap project (http://hapmap.ncbi.nlm.nih.gov/). The three regions where the alleles have a high level of LD are enclosed in boxes. Region 2 contains a cluster of four SNPs with alleles that were associated with HIT, and arrows in the diagram at the top of the figure indicate their relative positions.

Fig 7. A diagram of the effect of Abcb5-mediated haloperidol transport on HIT.

Abcb5 is expressed in brain capillaries and by cerebellar Purkinje cells. Haloperidol will enter the brain due to its intrinsic properties, but vectorial transport by perivascular Abcb5 will reduce the brain concentration of haloperidol. An individual with alleles that reduce Abcb5 activity are more susceptible to HIT because they will have an increased brain haloperidol concentration. Cerebellar Purkinje cells and dopaminergic neurons in the substantia nigra both express the Drd2 receptor that is bound by haloperidol. In Drd2-expressing dopaminergic neurons haloperidol is converted to its oxidative metabolite (HPP⁺), which inhibits mitochondrial energy generation and causes the characteristic Parkinsonian-like symptoms to develop. Although Purkinje cells express Drd2, they also express Abcb5, which exports of haloperidol from these cells and protects them from HIT.