Igor Mosyagin

MDR1-P-Glycoprotein (ABCB1) Mediates Transport of Alzheimer’s Amyloid-β Peptides—Implications for the Mechanisms of Aβ Clearance at the Blood–Brain Barrier

Amyloid‐β (Aβ) is the major component of the insoluble amyloid plaques that accumulate intracerebrally in patients with Alzheimer’s disease (AD). It has been suggested that MDR1‐P‐glycoprotein (ABCB1, P‐gp) plays a substantial role in the elimination of Aβ from the brain. In the present study, MDR1‐transfected LLC cells growing in a polarized cell layer were used to characterize the interaction of Aβ1‐40/1‐42 with P‐gp. In this system, P‐gp‐mediated transport can be followed by the efflux of the fluorescent dye rhodamine‐123, or of Aβ itself from the cells into the apical extracellular space. Aβ significantly decreased the apical efflux of rhodamine‐123, and the transcellular transport of Aβ1‐40 and Aβ1‐42 into the apical chamber could be demonstrated using both ELISA and fluorescence (FITC)‐labeled peptides. This transport was inhibited by a P‐gp modulator. Furthermore, ATP‐dependent, P‐gp‐mediated transport of the fluorescence‐labeled peptides could be demonstrated in isolated, inside‐out membrane vesicles. Our data support the concept that P‐gp is important for the clearance of Aβ from brain, and thus may represent a target protein for the prevention and/or treatment of neurodegenerative disorders such as AD.

Candidate Gene Analysis Identifies a Polymorphism in HLA-DQB1 Associated With Clozapine-Induced Agranulocytosis

OBJECTIVE Clozapine is considered to be the most efficacious drug to treat schizophrenia, although it is underutilized, partially due to a side effect of agranulocytosis. This analysis of 74 candidate genes was designed to identify an association between sequence variants and clozapine-induced agranulocytosis (CIA). METHOD Blood and medical history were collected for 33 CIA cases and 54 clozapine-treated controls enrolled between April 2002 and December 2003. Significant markers from 4 genes were then assessed in an independently collected case-control cohort (49 CIA cases, 78 controls). RESULTS Sequence variants in 5 genes were found to be associated with CIA in the first cohort: HLA-DQB1, HLA-C, DRD1, NTSR1, and CSF2RB. Sequence variants in HLA-DQB1 were also found to be associated with CIA in the second cohort. After refinement analyses of sequence variants in HLA-DQB1, a single SNP (single nucleotide polymorphism), 6672G>C, was found to be associated with risk for CIA; the odds of CIA are 16.9 times greater in patients who carry this marker compared to those who do not. CONCLUSIONS A sequence variant (6672G>C) in HLA-DQB1 is associated with increased risk for CIA. This marker identifies a subset of patients with an exceptionally high risk of CIA, 1,175% higher than the overall clozapine-treated population under the current blood-monitoring system. Assessing risk for CIA by testing for this and other genetic variants yet to be determined may be clinically useful when deciding whether to begin or continue treatment with clozapine.

The ATP-binding cassette transporter ABCG2 (BCRP), a marker for side population stem cells, is expressed in human heart

Efforts to improve severely impaired myocardial function include transplantation of autologous hematopoietic side population (SP) stem cells. The transmembrane ABC-type (ATP binding cassette) half-transporter ABCG2 (BCRP) serves as a marker protein for SP cell selection. We have recently shown that other ABC transport proteins such as ABCB1 and ABCC5 are differentially expressed in normal and diseased human heart. Here we investigated localization and individual ABCG2 expression in 15 ventricular (including 10 cardiomyopathic) and 51 auricular heart tissue samples using immunohistochemistry, confocal laser scanning fluorescence microscopy, and real-time RT-PCR. Individual genotypes were assigned using PCR–restriction fragment length polymorphism (RFLP) analysis and subsequently correlated to ABCG2 mRNA levels. ABCG2 was localized in endothelial cells of capillaries and arterioles of all samples. Ventricular samples from cardiomyopathic hearts exhibited significantly increased levels of ABCG2 mRNA (ABCG2/18S rRNA: 1.08 ± 0.30 × 10−7; p = 0.028 (dilative cardiomyopathy) and 1.16 ± 0.46 × 10−7; p = 0.009 (ischemic cardiomyopathy) compared with 0.44 ± 0.26 × 10−7 in nonfailing hearts). The individual haplotypes were not associated with altered mRNA expression. ABCG2 is variably expressed in endothelial cells of human heart, where it may function as a protective barrier against cardiotoxic drugs such as anthracyclines or mitoxantrone. ABCG2 expression is induced in dilative and ischemic cardiomyopathies.

Non-response to antiepileptic pharmacotherapy is associated with the ABCC2 -24C>T polymorphism in young and adult patients with epilepsy.

Objective We aimed to evaluate the association of non-response to antiepileptic pharmacotherapy with the frequency of variant alleles in the drug transporter genes ABCB1 and ABCC2 or in the CYP2C locus in young patients with epilepsy and an independent cohort of adults with drug-refractory epilepsy. Methods A total of 221 pediatric or adolescent Caucasian patients with epilepsy (105 females; age: 14.5±6.54 years) were genotyped for nine putatively functionally relevant ABCB1, ABCC2, CYP2C8, CYP2C9, and CYP2C19 polymorphisms. In addition, 70 adult patients (35 females, age: 41.9±11.5 years) with drug-refractory epilepsy who had earlier undergone neurosurgical therapy were genotyped and partly (n = 22) investigated for hippocampal ABCB1 and ABCC2 mRNA expression. Finally, 242 healthy volunteers (167 females, age: 27.0±6.77 years) from the same region were included as controls. Results The young cohort consisted of 103 (46.6%) responders and 118 (53.4%) non-responders to the first-line anticonvulsant. Carriers of the putatively low-expression ABCC2 −24T variant were significantly overrepresented among non-responders [odds ratio (OR) 2.15 (1.16–3.99); P = 0.016)]. This overrepresentation was confirmed by comparing young responders with adult drug-refractory patients [OR 3.36 (1.71–6.59); P<0.001]. Conversely, ABCB1 genotype distribution did not significantly differ between young responders and non-responders or adult drug-refractory patients. Excluding patients with febrile convulsions, heterozygous CYP2C8*4 [OR 0.35 (0.13–0.95); P = 0.038] and CYP2C9*3 [OR 0.34 (0.14–0.81); P = 0.015] variant allele carriers were underrepresented among non-responders. ABCC2 −24C>T genotype did not affect hippocampal ABCC2 expression, but was associated with increased ABCB1 expression (P = 0.034). Conclusion These data suggest a higher risk of antiepileptic drug failure in ABCC2 −24T allele carriers possibly because of compensatory upregulation of ABCB1.

Impact of myeloperoxidase and NADPH-oxidase polymorphisms in drug-induced agranulocytosis.

Abstract: Therapy of the atypical antipsychotic drug clozapine is limited by a comparatively high incidence of agranulocytosis in 0.8% of patients. This severe side effect is possibly based on the clozapine-mediated stimulation of cytokines and soluble cytokine receptors release, followed by induction of granulocyte proliferation and induction of myeloperoxidase (MPO) and NADPH-oxidase. Because NADPH-oxidase/MPO may oxidize clozapine to highly reactive nitrenium ions, we investigated the role of hereditary polymorphisms in the NADPH oxidase/myeloperoxidase system in agranulocytosis patients who received clozapine (n = 49), ticlopidine (n = 11), and other drugs prior to the event. The low active MPO -436A allelic variant frequency was 22.2% in cases and 19.9% in controls, but AA carriers were overrepresented among cases compared with the sum of AG and GG-carriers (odds ratio 4.16, 95% confidence limits 0.86-20.3, P = 0.056). Particularly in clozapine-induced agranulocytosis, this finding was most pronounced (P = 0.04). In the CYBA gene, encoding the p22phox subunit of the NADPH-oxidase, 2 polymorphisms were investigated. C242T (His72Tyr) had an allele frequency of 31.9% and 32.2% (P = NS) and A640G in the 3′-UTR was less frequent in cases (48.7%) than controls (60.0%), odds ratio 0.63 (0.39-1.02), P = 0.048. CYBA 640GG-carriers were marginally less frequent in cases compared with controls (28.2% vs. 38.7%, P = 0.062). Sequencing the entire coding region of the NADPH subunit CYBB (gpS1phase) disclosed that CYBB is a highly conserved gene, which does not represent a risk factor for clozapine-induced agranulocytosis. The impact of the polymorphic myeloperoxidase, however, needs further verification to predict a patients risk to develop drug-induced agranulocytosis.

Association of ABCB1 genetic variants 3435C>T and 2677G>T to ABCB1 mRNA and protein expression in brain tissue from refractory epilepsy patients

Purpose: There is evidence from studies in rodents that P‐glycoprotein (P‐gp) overexpression is implicated in the causation of refractory epilepsy. Genetic variants in the human ABCB1 (MDR1) gene were shown to affect the expression levels of the transporter in various tissues and to be associated with refractory epilepsy. However, the effect of the genetic variants on the P‐gp level in epileptogenic brain tissue is poorly investigated. In the present study, we examined the impact of putatively functional polymorphisms 3435C>T and 2677G>T in the ABCB1 gene on the ABCB1 mRNA expression and P‐gp content in human brain tissue from epileptogenic foci of the patients with refractory epilepsy.

Targeting CNS Transporters for Treatment of Neurodegenerative Diseases

Molecular transporters that are expressed in brain, especially at the blood-brain barrier (BBB), are increasingly recognized as possible therapeutic targets in the treatment of neurodegenerative disorders such as Alzheimers disease and Parkinsons disease. Some ATP-binding cassette (ABC) transporters, particularly P-glycoprotein (ABCB1), MRP1 (ABCC1) and BCRP (ABCG2), have been implicated in the clearance of neurotoxic polypeptides that characteristically accumulate in the brain, such as amyloid-β (Aβ) peptides in Alzheimers disease. Several lines of evidence also implicate lipid transporters of the A-branch of ABC transporters in pathogenesis. Induction of transporters via the activation of specific nuclear receptors may represent a novel approach to restoring diminished BBB function. On the other hand, transporters in the brain capillary endothelium regulate the permeation of therapeutic compounds into the brain. In addition to the export pumps that limit brain entry of exogenous substances, SLC-type uptake transporters, especially of the OCT (SLC22A) family, are of potential relevance in that they mediate not only the uptake of several drugs used for the treatment of neurodegenerative diseases, but also of certain neurotoxins. Here, we summarize recent findings and novel strategies targeting transporters to reduce brain pathology or to improve drug therapy.

Drug-induced agranulocytosis: impact of different fcgamma receptor polymorphisms?

Abstract: Drug-induced agranulocytosis is a rare but life-threatening side effect which is possibly based on immunogenetic mechanisms. Some studies regarding agranulocytosis induced by the atypical antipsychotic clozapine dealing with HLA subtyping and enzyme polymorphisms have been performed to elucidate its genetic background. To further screen possibly genetically based pathways of developing agranulocytosis, we assessed clinically relevant polymorphisms of immunoglobulin G or Fcγ receptors in patients with clozapine-induced (n = 48), ticlopidine-induced (n = 11), thyroid inhibitors-induced agranulocytosis (n = 8), and controls (n = 75). We found significant age-related effects in each of the drug-induced agranulocytoses but no further associations that underline an effect of polymorphisms in FcγRIIa, FcγRIIIa, and FcγRIIIb genes on drug-induced agranulocytosis. Thus, Fcγ receptors may not serve as a genetic marker to identify patients at risk for this life-threatening side effect.