Current status and future perspective on preclinical pharmacokinetic and pharmacodynamic (PK/PD) analysis: Survey in Japan pharmaceutical manufacturers association (JPMA).

Preclinical pharmacokinetic/pharmacodynamic (PK/PD) analysis is an efficient tool for the translational research and proof of mechanism/concept in animals. The questionnaire survey on the practice of preclinical PK/PD analysis was conducted in the member companies of the Japan Pharmaceutical Manufacturers Association (JPMA). According to the survey, 60% of companies conducted preclinical PK/PD analysis and its impact for drug development was different between each of the companies. The frequently analyzed therapeutic areas of preclinical PK/PD analysis were neurology, inflammation and metabolic disease, and those are different from the therapeutic area (infectious disease and oncology) in which PK/PD analysis was considered as effective by the present survey. Many companies which have used preclinical PK/PD analysis for the translation to human PK/PD and for the prediction of dose/regimen had good communication with other research & development (R&D) departments (e.g. pharmacology/clinical pharmacology). The increase in resources for preclinical PK/PD analysis including education was highly demanded. As a future perspective, the closer collaboration between pharmacokinetics scientists, pharmacologists, toxicologists and clinical pharmacologists and the increase in resources including upskilling and the comprehension of preclinical PK/PD analysis by the project team are considered to lead to efficient contributions to improve the success ratio of drug discovery and development.

Alterations in the reduced pteridine contents in the cerebrospinal fluids of LRRK2 mutation carriers and patients with Parkinson's disease.

Tetrahydrobiopterin (BH4) is a cofactor for tyrosine hydroxylase that is essential for the biosynthesis of dopamine. Parkinson's disease (PD) is characterized by a progressive degeneration of nigrostriatal dopaminergic neurons, and biomarkers reflecting the degree of neurodegeneration are important not only for basic research but also for clinical diagnosis and the treatment of the disease. Although the total neopterin and biopterin levels in the cerebrospinal fluids (CSF) of the patients with PD were reported, alterations in the composition of reduced and oxidized forms of pteridine compounds have not been examined. In this study, we first examined the time-dependent alterations in BH4 and other reduced pteridine compounds in the CSF of an MPTP-treated monkey as a primate PD model. We found that the CSF levels of BH4 and dihydroneopterin, an intermittent metabolite of BH4-biosynthesis, altered inversely with progression of neurodegeneration, whereas those of dihydrobiopterin and neopterin were relatively low and constant. Next, we assayed the amounts of reduced pteridine compounds in the CSF of 36 pre-symptomatic LRRK2-mutation (N1437H or G2019S) carriers (LRRK2-carrier), 13 patients with PD symptoms (LRRK2-PD), 46 patients with sporadic PD (sPD), and 26 non-PD individuals. The BH4 levels were significantly lower in both the LRRK2-PD and sPD patients, and the LRRK2-carriers exhibited higher BH4 levels compared with the sPD patients. The total neopterin levels in the CSF of the LRRK2-PD were significantly higher than those in the sPD and non-PD individuals, which indicated greater inflammatory responses in the brains of LRRK2-PD patients. The present results suggest that detailed analyses of pteridine levels in the CSF might be useful for understanding the pathophysiology of familial PD and for monitoring PD progression.

Biopterin levels in the cerebrospinal fluid of patients with PARK8 (I2020T).

PARK8 is the most common form of familial Parkinson's disease (PD). We measured biopterin and monoamine metabolite levels in the cerebrospinal fluids of 7 PARK8 patients (I2020T mutation in leucine-rich repeat kinase 2), 2 asymptomatic mutation carriers, and 21 sporadic PD patients. The biopterin levels in PARK8 patients were significantly higher than those in sporadic PD patients, although the symptoms were comparable in both groups, suggesting that PARK8 patients exhibit parkinsonian symptoms with higher biopterin levels than sporadic PD patients.

Production of cells with targeted integration of gene variants of human ABC transporter for stable and regulated expression using the Flp recombinase system.

The vector-mediated introduction of cDNA into mammalian cells by calcium phosphate co-precipitation or permeation with lipofectamine is widely used for the integration of cDNA into genomic DNA. Such integration, however, of cDNA occurs randomly at unpredictable sites in the host's chromosomal DNA, and the number of integrated recombinant DNAs is not controllable. To overcome this problem, we developed the Flp-In method to integrate one single copy of cDNA encoding the human ABC transporter ABCG2 into FRT-tagged genomic DNA. Examination of more than 20 metaphase spreads for both fluorescence in situ hybridization (FISH) mapping and multicolor-FISH analysis revealed that ABCG2 cDNA was incorporated into the telomeric region of the short arm on one of chromosomes 12 in Flp-In-293 cells. By using those cells, we investigated the effect of genetic polymorphisms and post-translational modifications of human ABC transporter ABCG2 on the protein expression and degradation. On the basis of our experience, it has been concluded that the Flp recombinase system provides a useful tool to quantitatively analyze the protein stability and endoplasmic reticulum (ER)-associated degradation of proteins like the ABC transporter. This system is also applicable for similar studies of the biogenesis of other proteins using the secretory pathway as well as proteins with other cellular localizations.

Disruption of N-linked glycosylation enhances ubiquitin-mediated proteasomal degradation of the human ATP-binding cassette transporter ABCG2.

The human ATP-binding cassette (ABC) transporter, ABCG2 (BCRP/MXR/ABCP), is a plasma membrane protein containing intramolecular and intermolecular disulfide bonds and an N-linked glycan at Asn596. We have recently reported that the intramolecular disulfide bond is a critical checkpoint for determining the degradation fates of ABCG2. In the present study, we aimed to analyze quantitatively the impact of the N-linked glycan on the protein stability of ABCG2. For this purpose, we incorporated one single copy of ABCG2 cDNA into a designated site of genomic DNA in Flp-In-293 cells to stably express ABCG2 or its variant proteins. When ABCG2 wild type-expressing cells were incubated with various N-linked glycosylation inhibitors, tunicamycin profoundly suppressed the protein expression level of ABCG2 and, accordingly, reduced the ABCG2-mediated cellular resistance to the cancer chemotherapeutic SN-38. When Asn596 was converted to Gln596, the resulting variant protein was not glycosylated, and its protein level was about one-third of the wild type level in Flp-In-293 cells. Treatment with MG132, a proteasome inhibitor, increased the level of the variant protein. Immunoblotting with anti-ubiquitin IgG1k after immunoprecipitation of ABCG2 revealed that the N596Q protein was ubiquitinated at levels that were significantly enhanced by treatment with MG132. Immunofluorescence microscopy demonstrated that treatment with MG132 increased the level of ABCG2 N596Q protein both in intracellular compartments and in the plasma membrane. In conclusion, we propose that the N-linked glycan at Asn596 is important for stabilizing de novo-synthesized ABCG2 and that disruption of this linkage results in protein destabilization and enhanced ubiquitin-mediated proteasomal degradation.

In vitro evaluation of photosensitivity risk related to genetic polymorphisms of human ABC transporter ABCG2 and inhibition by drugs.

Since porphyrins are regarded as endogenous substrates for the ATP-binding cassette (ABC) transporter ABCG2, it is hypothesized that functional impairment owing to genetic polymorphisms or inhibition of ABCG2 by drugs may result in a disruption of cellular porphyrin homeostasis. In the present study, we expressed ABCG2 genetic variants, i.e., V12M, Q141K, S441N, and F489L, as well as the wild type (WT) in Flp-In-293 cells to examine the hypothesis. Cells expressing S441N and F489L variants exhibited high levels of both cellularly accumulated pheophorbide a and photosensitivity, when those cells were incubated with pheophorbide a and irradiated with visible light. To further elucidate the significance of ABCG2 in cellular porphyrin homeostasis, we observed cellular accumulation and compartmentation of porphyrin and pheophorbide a by means of a new fluorescence microscopy technology, and found that accumulation of porphyrin and pheophorbide a in the cytoplasm compartment was maintained at low levels in Flp-In-293 cells expressing ABCG2 WT, V12M, or Q141K. When ABCG2 was inhibited by imatinib or novobiocin, however, those cells became sensitive to light. Based on these results, it is strongly suggested that certain genetic polymorphisms and/or inhibition of ABCG2 by drugs can enhance the potential risk of photosensitivity.

Development of polyclonal antibodies specific to ATP-binding cassette transporters human ABCG4 and mouse Abcg4: site-specific expression of mouse Abcg4 in brain.

In our recent study on seeking new mouse ATP-binding cassette (ABC) transporters of the G subfamily, we succeeded in cloning mouse Abcg4 from a cDNA library of mouse brain, and we characterized the tissue-specific expression and chromosomal localization of the mouse Abcg4 gene. To further characterize the physiological function of mouse Abcg4 protein and to compare its function with that of ABCG2, in the present study, we developed polyclonal antibodies against mouse Abcg4 and established the Abcg4-expression system. To raise antibodies, we selected three different epitope peptides that correspond to the amino acid residues of 46-60, 465-479, and 600-613 in mouse Abcg4 protein. The antibody raised against the epitope encoding the amino acids 46-60 was found to be specific to mouse Abcg4, exhibiting a band with molecular weight of 63,000 on immunoblotting, whereas this band was dose-dependently diminished by adding the corresponding epitope peptide into the immunoblot medium. Use of the antibody for immunoblot detection in mouse normal tissues revealed that the Abcg4 protein is expressed in brain, spleen, and testis. Immunohistochemical studies showed that mouse Abcg4 is site-specifically expressed in the cerebral cortex and medulla of mouse brain. These results suggest that mouse Abcg4 plays a certain physiological role in the brain. It is of importance to note that the sequence of amino acids 46-60 is completely identical between mouse Abcg4 and human ABCG4. Thus, this antibody is applicable to the detection of human ABCG4 as well as mouse Abcg4.

Intramolecular disulfide bond is a critical check point determining degradative fates of ATP-binding cassette (ABC) transporter ABCG2 protein.

Human ABCG2 belongs to the ATP-binding cassette (ABC) transporter family and plays an important role in various biological reactions, such as xenobiotic elimination and homeostasis of protoporphyrin. We previously reported that ABCG2 exists in the plasma membrane as a homodimer bound via a disulfide bond at Cys-603. In the present study, we examined the importance of an intramolecular disulfide bond for stability of the ABCG2 protein. Substitution of either Cys-592 or Cys-608 located in the extracellular loop to glycine resulted in a significant decrease in protein levels of ABCG2 when expressed in Flp-In-293 cells. Interestingly, the protein levels of those ABCG2 variants were remarkably enhanced by treatment with the proteasome inhibitor MG132. Concomitantly, increases in ubiquitinated forms of those variant proteins were detected by immunoprecipitation. In contrast, neither the protein level nor the ubiquitinated state of the ABCG2 wild-type (WT) was affected by MG132 treatment. Ubiquitin-mediated protein degradation is suggested to be involved in degradation of misfolded ABCG2 proteins lacking the intramolecular disulfide bond. On the other hand, the protein level of ABCG2 WT increased more than 4-fold when cells were treated with bafilomycin A(1), which inhibits lysosomal degradation, whereas the C592G or C608G variant was little affected by the same treatment. These results strongly suggest that two distinct pathways exist for protein degradation of ABCG2 WT and mutants lacking the intramolecular disulfide bond. Namely, the WT ABCG2 is degraded in lysosomes, and the misfolded ABCG2 lacking intramolecular disulfide bond undergoes ubiquitin-mediated protein degradation in proteasomes.