Members of the organic anion transporting polypeptide (OATP) family are responsible for the cellular uptake of a broad range of endogenous compounds and xenobiotics in multiple tissues. This review ...focuses on OATP1B1 and -1B3, which are specifically expressed in the liver and considered to be of particular importance for hepatic drug elimination and drug pharmacokinetics. Recent literature has indicated that inhibition of these transporters may result in drug-drug interactions. Furthermore, genetic polymorphisms in the genes encoding OATP1B1 and -1B3 have been described that increase or decrease transport in vitro and in vivo. Alteration of transporter function by either of these mechanisms may contribute to interindividual variability in drug disposition and response. In this review an update of this rapidly emerging field is provided.
Deletion of exon 9 from Cullin‐3 (CUL3, residues 403–459: CUL3Δ403–459) causes pseudohypoaldosteronism type IIE (PHA2E), a severe form of familial hyperkalaemia and hypertension (FHHt). CUL3 binds ...the RING protein RBX1 and various substrate adaptors to form Cullin‐RING‐ubiquitin‐ligase complexes. Bound to KLHL3, CUL3‐RBX1 ubiquitylates WNK kinases, promoting their ubiquitin‐mediated proteasomal degradation. Since WNK kinases activate Na/Cl co‐transporters to promote salt retention, CUL3 regulates blood pressure. Mutations in both KLHL3 and WNK kinases cause PHA2 by disrupting Cullin‐RING‐ligase formation. We report here that the PHA2E mutant, CUL3Δ403–459, is severely compromised in its ability to ubiquitylate WNKs, possibly due to altered structural flexibility. Instead, CUL3Δ403–459 auto‐ubiquitylates and loses interaction with two important Cullin regulators: the COP9‐signalosome and CAND1. A novel knock‐in mouse model of CUL3WT/Δ403–459 closely recapitulates the human PHA2E phenotype. These mice also show changes in the arterial pulse waveform, suggesting a vascular contribution to their hypertension not reported in previous FHHt models. These findings may explain the severity of the FHHt phenotype caused by CUL3 mutations compared to those reported in KLHL3 or WNK kinases.
Synopsis
Molecular defects observed in a mutant form of the ubiquitin E3 protein CUL3 causing a severe form of familial hypertension (PHA2E) result in E3 ligase loss of function. A novel mouse model with the same CUL3 disease mutation was generated, and it closely recapitulates the human PHA2E phenotype.
Mutations in CUL3 that cause hereditary hypertension prevent the ubiquitylation and degradation of WNK kinases probably due to increased flexibility of the cullin backbone.
The mutant CUL3Δ403–459 protein shows marked auto‐ubiquitylation and loss of binding to the critical cullin regulators, CSN and CAND1.
The phenotype of knock‐in mice carrying the CUL3Δ403–459 mutation closely recapitulates PHA2E; kidney DCTs contain striking accumulation of WNK and SPAK proteins.
The mice expressing CUL3Δ403–459 have a vascular phenotype suggesting increased vascular tone that may contribute to the severity of the hypertension seen in PHA2E compared to other forms (PHA2A‐D).
Molecular defects observed in a mutant form of the ubiquitin E3 protein CUL3 causing a severe form of familial hypertension (PHA2E) result in E3 ligase loss of function. A novel mouse model with the same CUL3 disease mutation was generated, and it closely recapitulates the human PHA2E phenotype.
Abstract Objective Although the taxanes represent the most active agents for the first-line treatment of metastatic hormone-refractory prostate cancer (HRPC), most patients eventually progress while ...receiving taxane-based treatments. No agents are approved for second-line therapy in HRPC, but common standard practice for the oncologists is to treat patients also after docetaxel failure. Methods Twenty highly pretreated patients with HRPC received bevacizumab (10 mg/kg) and docetaxel (60 mg/m2 ) every 3 wk. All patients had bone metastases and eight had measurable lesions. Results Eleven patients (55%) had major prostate-specific antigen (PSA) responses, and 3 (37.5%) had objective responses. Seven major PSA responses were recorded in the same patients who had reported a >50% PSA decrease after first-line docetaxel. However, four major PSA responses were observed in patients previously nonresponsive to docetaxel alone. The treatment was well tolerated. Conclusions Our results show that the combination of bevacizumab and docetaxel is active and well tolerated. Continued investigation of bevacizumab with cytotoxic chemotherapy is warranted in HRPC.
Cardiac remodeling and hypertrophy are the pathological consequences of cardiovascular disease and are correlated with its associated mortality. Activity of the transcription factor NF-κB is ...increased in the diseased heart; however, our present understanding of how the individual subunits contribute to cardiovascular disease is limited. We assign a new role for the c-Rel subunit as a stimulator of cardiac hypertrophy and fibrosis. We discovered that c-Rel-deficient mice have smaller hearts at birth, as well as during adulthood, and are protected from developing cardiac hypertrophy and fibrosis after chronic angiotensin infusion. Results of both gene expression and cross-linked chromatin immunoprecipitation assay analyses identified transcriptional activators of hypertrophy, myocyte enhancer family, Gata4, and Tbx proteins as Rel gene targets. We suggest that the p50 subunit could limit the prohypertrophic actions of c-Rel in the normal heart, because p50 overexpression in H9c2 cells repressed c-Rel levels and the absence of cardiac p50 was associated with increases in both c-Rel levels and cardiac hypertrophy. We report for the first time that c-Rel is highly expressed and confined to the nuclei of diseased adult human hearts but is restricted to the cytoplasm of normal cardiac tissues. We conclude that c-Rel-dependent signaling is critical for both cardiac remodeling and hypertrophy. Targeting its activities could offer a novel therapeutic strategy to limit the effects of cardiac disease.
Imatinib, a tyrosine kinase inhibitor currently approved for treatment of several malignancies, has been shown to be a substrate for multiple efflux-transporter proteins, including ABCB1 ...(P-glycoprotein) and ABCG2 (BCRP). The effect of inhibiting these transporters on tissue exposure to imatinib remains unclear.
To assess the role of these transporters on drug disposition, 50 mg/kg imatinib was administered to Balb/C mice, 30 minutes after receiving tariquidar (10 mg/kg), an inhibitor of both ABCB1 and ABCG2, or vehicle, via oral gavage.
Quantitative determination of imatinib in mouse plasma, liver and brain was performed using a newly-developed and validated liquid-chromatography-mass spectrometric method.
Exposure to imatinib was 2.2-fold higher in plasma, liver and brain in mice that received tariquidar, as compared to those that received the vehicle (P = 0.001). The peak plasma concentration did not increase substantially, suggesting that tariquidar is affecting the distribution, metabolism and/or excretion of imatinib, rather than absorption. Though tariquidar increased the absolute exposure of imatinib, the brain-to-plasma ratio of imatinib was unaffected.
This study suggests that intentional inhibition of ABCB1 and ABCG2 function at the blood-brain barrier is unlikely to significantly improve clinical outcome of imatinib with currently used dosing regimens.
Purpose: To evaluate the effect of naturally occurring variants in genes encoding the cytochrome P 450 (CYP) isoforms CYP3A4 and CYP3A5 in patients with cancer receiving midazolam as a phenotyping ...probe.
Experimental Design: Five variants in CYP3A4 and CYP3A5 were evaluated in 58 patients (21 women and 37 men) receiving a short i.v. bolus of midazolam (dose, 0.0145 or 0.025 mg/kg).
Midazolam concentrations in plasma were determined using liquid chromatography-mass spectrometry, and pharmacokinetic variables
were calculated using noncompartmental analysis. Genomic DNA was characterized for the variants by PCR-RFLP, and all genotypes
were confirmed by direct nucleotide sequencing.
Results: The mean clearance of midazolam was 24.4 ± 9.12 L/h, and phenotypic CYP3A activity varied about 4-fold in this population
(range, 10.8-44.3 L/h). There were six carriers of the CYP3A4*1B allele (allele frequency, 0.061). No variant alleles for CYP3A4*17, CYP3A4*18A , or CYP3A5*6 were identified. Forty-eight of the 58 patients were homozygous variant for CYP3A5*3C , eight were heterozygous, and two were homozygous wild type (allele frequency, 0.897). No associations were noted between
any of the studied genotypes and the phenotypic measures ( P ≥ 0.16). Likewise, a common variant in exon 26 in the gene encoding P-glycoprotein i.e., ABCB1 ( MDR1 ) 3435C>T that was previously reported to be linked to CYP3A4 mRNA levels was unrelated to any of the studied phenotypic measures ( P ≥ 0.49).
Conclusions. The studied genetic variants in CYP3A4 and CYP3A5 are unlikely to have an important functional significance to phenotypic CYP3A activity in patients with cancer.
Vascular smooth muscle cell (VSMC) apoptosis occurs in advanced atherosclerotic plaques where it may contribute to plaque instability. VSMCs express the death receptor Fas but are relatively ...resistant to Fas-induced apoptosis due in part to the intracellular sequestration of Fas. Although inflammatory cytokines such as interferon (IFN)-γ present in plaques can prime VSMCs to FasL-induced death, the mechanism of this effect is unclear. We examined Fas expression and FasL-induced apoptosis in human VSMCs in response to IFN-γ. IFN-γ induced Fas trafficking to the cell surface within 24 hours, an effect that required Jak2/Stat1 activity. IFN-γ also stimulated Akt activity, and both Fas trafficking and Stat1 activation were inhibited by blocking PI3K, Akt, or Jak-2. IFN-γ increased Fas-induced apoptosis
in vitro by 46 ± 8% (mean ± SEM,
P = 0.04), an event that could be abrogated by inhibition of PI3K, Akt, or Jak-2. IFN-γ also increased Fas-induced apoptosis
in vivo 7.5- to 15-fold (
P < 0.05) in human arteries transplanted into immunodeficient mice, accompanied by increased Fas and phospho-Ser
727-Stat1. We conclude that IFN-γ primes VSMCs to Fas-induced apoptosis, in part by relocation of Fas to the cell surface, a process that involves PI3K, Akt, and Jak-2/Stat1. IFN-γ present in plaques may co-operate with FasL to induce VSMC apoptosis in atherosclerosis.
Background:
Several microtubule targeting agents are capable of inducing CYP3A4 via activation of the pregnane X receptor (PXR; NR1I2).
Objective:
To evaluate the CYP3A4 induction potential of ...vinblastine both clinically and in vitro and determine the involvement of the nuclear receptors NR1I2 and the constitutive androstane receptor (NR1I3).
Methods:
Midazolam pharmacokinetics were evaluated in 6 patients who were enrolled in a Phase 1/2 study of infusional vinblastine given in combination with the ABCB1 (P-glycoprotein) antagonist valspodar (PSC 833) and received the CYP3A4 phenotyping probo midazolam on more than 1 occasion. Genotyping was conducted in CYP3A4, CYP3A5, and ABCB1 to rule out potential pharmacogenetic influences. Clinical data were followed-up by Western blotting and reporter assays in HepG2 and NIH3T3 cells treated with vinbfastine over a dose range of 150-4800 ng/mL for 48 hours.
Results:
In 6 patients with cancer, vinblastine increased the median (95% CI) clearance of the CYP3A4 phenotyping probe midazolam from 21.7 L/h (12.6 to 28.1) to 32.3 L/h (17.3 to 53.9) (p = 0.0156, Wilcoxon signed-rank test). No obvious effect of polymorphisms in CYP3A4, CYP3A5, and ABCB1 on midazolam clearance was observed. In vitro, vinblastine induced CYP3A4 protein. Furthermore, cell-based reporter gene assays using transiently transfected HepG2 and NIH3T3 cells indicated that vinblastine (150-4800 ng/mL) weakly activated human and mouse full-length NR1I2, but had no influence on NR1I3.
Conclusions:
Collectively, these findings suggest that vinblastine is able to induce CYP3A4, at least in part, via an NR1I2-dependent mechanism, and thus has the potential to facilitate its own elimination and cause interactions with other CYP3A4 substrates.
Abstract Objectives Combining antineoplastic agents is the key to improving the treatment options for men with hormone refractory prostate cancer (HRPC). The current study investigated the ...combination of docetaxel, vinorelbine, and zoledronic acid as a first-line treatment for HRPC. Methods Patients were treated repeatedly with docetaxel (25 mg/mq) and vinorelbine (10 mg/mq) intravenously for three consecutive weeks followed by a 1-wk rest until disease progression or side effects. Zoledronic acid was administered every 4 wk. Changes in prostate-specific antigen (PSA) levels and objective responses were evaluated after two and three cycles, respectively. Toxicity and pain evaluation, based on pain intensity reduction and analgesic drug reduction, were assessed every cycle. Results Forty men with HRPC (median age: 65 yr) were treated. Among 38 evaluable patients, complete and major PSA responses were observed in seven (18%) and 12 (32%), respectively; a partial objective response was observed in six of 15 (40%) patients with measurable disease. Neutropenia (25%) was the most important grade 3 haematologic toxicity observed. Only three patients (7.5%) reported grade 4 neutropenia. Nineteen patients (47.5%) achieved a reduction of pain intensity and analgesic drug use after two cycles. Median progression-free survival was 7 mo (95% CI: 2–10 mo), with a median overall survival of 17 mo (95% CI: 6–22 mo). Conclusions The combination of docetaxel, vinorelbine, and zoledronic acid is associated with improvement in biochemical, objective, and pain responses and is well tolerated as a first-line treatment for HRPC.
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