Genetic analysis reveals conspecificity of two nominal species of Anaphes fairyflies (Hymenoptera: Mymaridae), egg parasitoids of Oulema leaf beetle (Coleoptera: Chrysomelidae) pests of cereal crops in Europe and of rice in East Asia.

Anaphes (Anaphes) flavipes (Foerster), a fairyfly (Hymenoptera: Mymaridae) native of Europe, is an economically important egg parasitoid for the natural control of Oulema spp. leaf beetle (Coleoptera: Chrysomelidae) pests of cereal crops such as barley, oats, rye, and wheat in Europe, and for the classical biological control of the invasive Oulema melanopus (L.) in North America. A morphologically very similar Anaphes (Anaphes) nipponicus Kuwayama, known from mainland China, Japan, Republic of Korea, Far East of Russia and Taiwan, is an egg parasitoid of Oulema oryzae (Kuwayama), a pest of rice mainly in temperate parts of East Asia. The nuclear 28S-D2 and ITS2 and the mitochondrial COI genes were used as markers to compare specimens of A. (Anaphes) flavipes reared from eggs of an Oulema sp. on barley in Germany with those of A. (Anaphes) nipponicus reared from eggs of O. oryzae on rice in Honshu Island, Japan. Because the resulting sequences are practically identical, within an expected intraspecific genetic variability, conspecificity of these two nominal species has been confirmed, and consequently A. (Anaphes) nipponicus Kuwayama, 1932, syn. n. is synonymized with A. (Anaphes) flavipes (Foerster, 1841). Taxonomic notes and illustrations are provided for the specimens of both sexes of A. (Anaphes) flavipes from Japan to facilitate their recognition.

Endogenous Probes for Drug Transporters: Balancing Vision With Reality.

Various endogenous probes have been identified for a number of hepatic and renal drug transporters and available clinical data indicate that they could be leveraged in phase I trials to facilitate subject phenotyping and drug-drug interaction (DDI) assessment. Despite the progress, however, it is recognized that the menu of probes needs expanding, that existing probes need further characterization and validation, and that compound files need to be built in support of probe absorption-metabolism-distribution-excretion-DDI modeling exercises.

Quantitative Analyses of Hepatic OATP-Mediated Interactions Between Statins and Inhibitors Using PBPK Modeling With a Parameter Optimization Method.

This study aimed to construct a widely applicable method for quantitative analyses of drug-drug interactions (DDIs) caused by the inhibition of hepatic organic anion transporting polypeptides (OATPs) using physiologically based pharmacokinetic (PBPK) modeling. Models were constructed for pitavastatin, fluvastatin, and pravastatin as substrates and cyclosporin A (CsA) and rifampicin (RIF) as inhibitors, where enterohepatic circulations (EHC) of statins were incorporated. By fitting to clinical data, parameters that described absorption, hepatic elimination, and EHC processes were optimized, and the extent of these DDIs was explained satisfactorily. Similar in vivo inhibition constant (Ki ) values of each inhibitor against OATPs were obtained, regardless of the substrates. Estimated Ki values of CsA were comparable to reported in vitro values with the preincubation of CsA, while those of RIF were smaller than reported in vitro values (coincubation). In conclusion, this study proposes a method to optimize in vivo PBPK parameters in hepatic uptake transporter-mediated DDIs.

N-methylnicotinamide is an endogenous probe for evaluation of drug-drug interactions involving multidrug and toxin extrusions (MATE1 and MATE2-K).

Multidrug and toxin extrusion 1 (MATE1) and MATE2-K are H(+)/organic cation exchangers mediating the efflux of cationic drugs into the urine. N-methylnicotinamide (NMN) was found to be an endogenous substrate of MATE1 (Michaelis constant (K(m)) 301 ± 18 µmol/l) and MATE2-K (K(m) 422 ± 63 µmol/l) as well as a basolateral influx transporter, organic cation transporter 2 (K(m) 318 ± 29 µmol/l). A potent MATE inhibitor, pyrimethamine, competitively inhibited the uptake by MATE1 and MATE2-K with inhibition constant (K(i)) values of 83 ± 15 and 56 ± 11 nmol/l, respectively. The uptake of NMN by human kidney brush border membrane vesicles with a H(+) gradient was saturable (K(m) 360 ± 55 µmol/l) and completely inhibited by pyrimethamine. The renal clearance of endogenous NMN was 403 ± 61 in healthy male subjects, and it was significantly decreased to 119 ± 16 ml/min/kg by an oral dose of pyrimethamine (50 mg). These results support the utility of NMN as an endogenous in vivo probe for investigating MATE1 and MATE2-K in humans.

Effects of a MATE protein inhibitor, pyrimethamine, on the renal elimination of metformin at oral microdose and at therapeutic dose in healthy subjects.

A microdose study of metformin was conducted to investigate the predictability of drug-drug interactions at the therapeutic dose (ThD). Healthy subjects received a microdose (100 µg) or ThD (250 mg) of metformin orally, with or without a potent and competitive multidrug and toxin extrusion (MATE) inhibitor, pyrimethamine (50 mg, p.o.), in a crossover fashion. Pyrimethamine significantly reduced the renal clearance of metformin by 23 and 35% at the microdose and ThD, respectively. At ThD, but not at microdose, it caused significant increases in the maximum concentration (C(max)) and area under the plasma concentration-time curve (AUC) of metformin (142 and 139% of control values, respectively). Human canalicular membrane vesicles showed pyrimethamine-inhibitable metformin uptake. Pyrimethamine did not affect plasma lactate/pyruvate after ThD of metformin but significantly reduced the renal clearance of creatinine, thereby causing elevation of plasma creatinine level. This microdose study quantitatively predicted a drug-drug interaction involving the renal clearance of metformin at ThD by pyrimethamine. Pyrimethamine is a useful in vivo inhibitor of MATE proteins.

Prediction of fluoroquinolone-induced elevation in serum creatinine levels: a case of drug-endogenous substance interaction involving the inhibition of renal secretion.

The aim of this study was to examine the mechanism underlying the elevation in serum creatinine levels caused by a novel des-fluoro(6)-quinolone antibacterial agent, DX-619, in healthy subjects. hOCT2 showed a prominent uptake of creatinine (K(m) = 56.4 mmol/l) among renal organic ion transporters. DX-619 is a potent inhibitor of hOCT2 (K(i) = 0.94 micromol/l), hMATE1 (0.82 µmol/l), and hMATE2-K (0.10 micromol/l). The pharmacokinetic model involving the inhibition of hOCT2 (model 1), hOCT2, and MATE1 or MATE2-K (model 2) could predict the elevation in serum creatinine levels in individual subjects receiving DX-619. This assumes that a significant contribution of tubular secretion (59, 38, and 31%) and reabsorption ranged from 3-50, 4-30, and 5-21% in model 1, -2a (hOCT2/hMATE1), and -2b (hOCT2/hMATE2-K), respectively, for creatinine. In conclusion, DX-619, at its therapeutic dose, is able to inhibit hOCT2, hMATE1, and hMATE2-K, leading to a significant inhibition of tubular secretion of creatinine and consequently to elevation of serum creatinine levels.

Microdose study of 14C-acetaminophen with accelerator mass spectrometry to examine pharmacokinetics of parent drug and metabolites in healthy subjects.

A study of the pharmacokinetics of (14)C-labeled acetaminophen (AAP) was performed in healthy Japanese subjects receiving an oral microdose of the drug. After separation by high-performance liquid chromatography (HPLC), the levels of AAP and its metabolites in the pooled plasma specimens were quantified using accelerator mass spectrometry (AMS). The total body clearance (CL(tot))/bioavailability (F) of AAP was within the variation in the reported values at therapeutic doses, indicating the linearity of AAP pharmacokinetics. AAP-glucuronide (Glu) and AAP-4-O-sulfate satisfied the criteria of safety testing of drug metabolites. AMS could detect AAP-Cys, the active metabolite of AAP conjugated with cysteine, in the urine. Probenecid prolonged the systemic elimination of total radioactivity and caused a marked decrease in AAP-Glu levels in plasma. Probenecid likely inhibited the glucuronidation of AAP and the renal elimination of AAP-4-O-sulfate. Microdosing of (14)C-labeled drug followed by AMS is a powerful tool that can be used in the early phase of drug development for pharmacokinetic analysis of drugs and their metabolites and for detecting the formation of active metabolites in humans.

Differentiation of feline immunodeficiency virus vaccination, infection, or vaccination and infection in cats.

BACKGROUND: Serodiagnosis of feline immunodeficiency virus (FIV) is complicated by the use of a formalin-inactivated whole-virus FIV vaccine. Cats respond to immunization with antibodies indistinguishable from those produced during natural infection by currently available diagnostic tests, which are unable to distinguish cats that are vaccinated against FIV, infected with FIV, or both. HYPOTHESIS: An enzyme-linked immunosorbent assay (ELISA) detecting antibodies against formalin-treated FIV whole virus and untreated transmembrane peptide will distinguish uninfected from infected cats, regardless of vaccination status. ANIMALS: Blood samples were evaluated from uninfected unvaccinated cats (n = 73 samples), uninfected FIV-vaccinated cats (n = 89), and FIV-infected cats (n = 102, including 3 from cats that were also vaccinated). METHODS: The true status of each sample was determined by virus isolation. Plasma samples were tested for FIV antibodies by a commercial FIV diagnostic assay and an experimental discriminant ELISA. RESULTS: All samples from uninfected cats were correctly identified by the discriminant ELISA (specificity 100%). Of the samples collected from FIV-infected cats, 99 were correctly identified as FIV-infected (sensitivity 97.1%). CONCLUSIONS AND CLINICAL IMPORTANCE: With the exception of viral isolation, the discriminant ELISA is the most reliable assay for diagnosis of FIV. A practical strategy for the diagnosis of FIV infection would be to use existing commercial FIV antibody assays as screening tests. Negative results with commercial assays are highly reliable predictors for lack of infection. Positive results can be confirmed with the discriminant ELISA. If the discriminant ELISA is negative, the cat is probably vaccinated against FIV but not infected. Positive results are likely to represent infection.

Pharmacogenetic characterization of sulfasalazine disposition based on NAT2 and ABCG2 (BCRP) gene polymorphisms in humans.

The role of breast cancer resistance protein (BCRP), an efflux ABC transporter, in the pharmacokinetics of substrate drugs in humans is unknown. We investigated the impact of genetic polymorphisms of ABCG2 (421C>A) and NAT2 on the pharmacokinetics of sulfasalazine (SASP), a dual substrate, in 37 healthy volunteers, taking 2,000 mg of conventional SASP tablets. In ABCG2, SASP AUC(0-48) of C/C, C/A, and A/A subjects was 171 +/- 85, 330 +/- 194, and 592 +/- 275 microg h/ml, respectively, with significant differences among groups. In contrast, AUC(0-48) of sulfapyridine (SP) tended to be lower in subjects with the ABCG2-A allele as homozygosity. In NAT2, AUC(AcSP)/AUC(SP) was significantly higher in rapid than in intermediate and slow acetylator (SA) genotypes. We successfully described the pharmacokinetics of SASP, SP, and N -acetylsulfapyridine (AcSP) simultaneously by nonlinear mixed-effects modeling (NONMEM) analysis with regard to both gene polymorphisms. The data indicate that SASP is a candidate probe of BCRP, particularly in its role in intestinal absorption.

SLCO1B1 (OATP1B1, an uptake transporter) and ABCG2 (BCRP, an efflux transporter) variant alleles and pharmacokinetics of pitavastatin in healthy volunteers.

To investigate the contribution of genetic polymorphisms of SLCO1B1 and ABCG2 to the pharmacokinetics of a dual substrate, pitavastatin, 2 mg of pitavastatin was administered to 38 healthy volunteers and pharmacokinetic parameters were compared among the following groups: 421C/C(*)1b/(*)1b (group 1), 421C/C(*)1b/(*)15 (group 2), 421C/C(*)15/(*)15 and 421C/A(*)15/(*)15 (group 3), 421C/A(*)1b/(*)1b (group 4), 421A/A(*)1b/(*)1b (group 5), and 421C/A(*)1b/(*)15 (group 6). In SLCO1B1, pitavastatin area under plasma concentration-time curve from 0 to 24 h (AUC(0-24)) for groups 1, 2, and 3 was 81.1+/-18.1, 144+/-32, and 250+/-57 ng h/ml, respectively, with significant differences among all three groups. In contrast to SLCO1B1, AUC(0-24) in groups 1, 4, and 5 was 81.1+/-18.1, 96.7+/-35.4, and 78.2+/-8.2 ng h/ml, respectively. Although the SLCO1B1 polymorphism was found to have a significant effect on the pharmacokinetics of pitavastatin, a nonsynonymous ABCG2 variant, 421C>A, did not appear to be associated with the altered pharmacokinetics of pitavastatin.

An atomic seesaw switch formed by tilted asymmetric Sn-Ge dimers on a Ge (001) surface.

When tin (Sn) atoms are deposited on a clean germanium (Ge) (001) surface at room temperature, buckled dimers originating from the Sn atoms are formed at the Ge-dimer position. We identified the dimer as a heterogeneous Sn-Ge dimer by reversing its buckling orientation with a scanning tunneling microscope (STM) at 80 kelvin. An atomic seesaw switch was formed for one-dimensional electronic conduction in the Ge dimer-row direction by using the STM to reversibly flip the buckling orientation of the Sn-Ge dimer and to set up standing-wave states.

Design and assessment of a tissue-engineered model of human phalanges and a small joint.

OBJECTIVES: To develop models of human phalanges and small joints by suturing different cell-polymer constructs that are then implanted in athymic (nude) mice. DESIGN: Models consisted of bovine periosteum, cartilage, and/or tendon cells seeded onto biodegradable polymer scaffolds of either polyglycolic acid (PGA) or copolymers of PGA and poly-L-lactic acid (PLLA) or poly-epsilon-caprolactone (PCL) and PLLA. Constructs were fabricated to produce a distal phalanx, middle phalanx, or distal interphalangeal joint. SETTING AND SAMPLE POPULATION: Studies of more than 250 harvested implants were conducted at the Northeastern Ohio Universities College of Medicine. EXPERIMENTAL VARIABLE: Polymer scaffold, cell type, and implantation time were examined. OUTCOME MEASURE: Tissue-engineered specimens were characterized by histology, transmission electron microscopy, in situ hybridization, laser capture microdissection and qualitative and quantitative polymerase chain reaction analysis, magnetic resonance microscopy, and X-ray microtomography. RESULTS: Over periods to 60 weeks of implantation, constructs developed through vascularity from host mice; formed new cartilage, bone, and/or tendon; expressed characteristic genes of bovine origin, including type I, II and X collagen, osteopontin, aggrecan, biglycan, and bone sialoprotein; secreted corresponding proteins; responded to applied mechanical stimuli; and maintained shapes of human phalanges with small joints. CONCLUSION: Results give insight into construct processes of tissue regeneration and development and suggest more complete tissue-engineered cartilage, bone, and tendon models. These should have significant future scientific and clinical applications in medicine, including their use in plastic surgery, orthopaedics, craniofacial reconstruction, and teratology.

CD8+ T cells from feline immunodeficiency virus (FIV) infected cats suppress exogenous FIV replication of their peripheral blood mononuclear cells in vitro.

Feline immunodeficiency virus (FIV) isolates from domestic cats have been classified into five subtypes, designated A, B, C, D and E. Although many FIV-infected cats may have frequent contact with multiple strains of FIV, they usually become infected with a single FIV subtype. In the present study, we demonstrate that peripheral blood mononuclear cells (PBMC) of FIV infected cats were resistant to exogenous FIV (second virus) replication in vitro and that the resistance of these PBMC was mediated by CD8+ T cells. In cats with a low anti-FIV activity of CD8+ T cells, the proviral DNA of the second virus inoculated into PBMC was detected intracellularly, and both the second and the originally infecting strain (original virus) were produced in the culture supernatant. In contrast, in cats with a high anti-FIV activity of CD8+ T cells, both the proviral DNA of the second virus and the original virus were detected in PBMC intracellularly, but neither virus was produced in the culture supernatant. However, when PBMCs from these cats were depleted of CD8+ T cells, the RNA of both viruses was detected in the culture supernatant. These results suggest that CD8+ T cells inhibit the late phase of FIV replication after viral integration. Moreover, the inhibition was also effective against FIV strains of different subtypes from that of the original strain. It appears that the CD8+ T cell-mediated immune response plays important roles in the maintenance of an asymptomatic state in FIV-infected cats and their resistance to superinfection.

A physiologically based pharmacokinetic analysis of capecitabine, a triple prodrug of 5-FU, in humans: the mechanism for tumor-selective accumulation of 5-FU.

PURPOSE: To identify the factors governing the dose-limiting toxicity in the gastrointestine (GI) and the antitumor activity after oral administration of capecitabine, a triple prodrug of 5-FU, in humans. METHOD: The enzyme kinetic parameters for each of the four enzymes involved in the activation of capecitabine to 5-FU and its elimination were measured experimentally in vitro to construct a physiologically based pharmacokinetic model. Sensitivity analysis for each parameter was performed to identify the parameters affecting tissue 5-FU concentrations. RESULTS: The sensitivity analysis demonstrated that (i) the dihydropyrimidine dehydrogenase (DPD) activity in the liver largely determines the 5-FU AUC in the systemic circulation, (ii) the exposure of tumor tissue to 5-FU depends mainly on the activity of both thymidine phosphorylase (dThdPase) and DPD in the tumor tissues, as well as the blood flow rate in tumor tissues with saturation of DPD activity resulting in 5-FU accumulation, and (iii) the metabolic enzyme activity in the GI and the DPD activity in liver are the major determinants influencing exposure to 5-FU in the GI. The therapeutic index of capecitabine was found to be at least 17 times greater than that of other 5-FU-related anticancer agents, including doxifluridine, the prodrug of 5-FU, and 5-FU over their respective clinical dose ranges. CONCLUSIONS: It was revealed that the most important factors that determine the selective production of 5-FU in tumor tissue after capecitabine administration are tumor-specific activation by dThdPase, the nonlinear elimination of 5-FU by DPD in tumor tissue, and the blood flow rate in tumors.

Functional characterization of rat organic anion transporter 2 in LLC-PK1 cells.

Rat organic anion transporter 2 (rOat2) is abundantly expressed in the liver and localized to the basolateral membrane. A previous study using the Xenopus laevis oocyte expression system has shown that rOat2 transports organic anions such as salicylate () and, in the present study, rOat2 was characterized using a mammalian expression system. In addition to the substrates previously shown to be transported by rOat2, three substrates, indomethacin [IDM, Michaelis-Menten constant (K(m)) of 0.37 microM] and nucleoside derivatives such as 3'-azido-3'-deoxythymidine (AZT, K(m) of 26 microM) and 2',3'-dideoxycytidine (ddC, K(m) of 3.08 mM), were also identified for the first time The rank order of rOat2-mediated transport of these substrates was IDM > salicylate > prostaglandin E(2) > AZT > ddC > p-aminohippurate (PAH). Ketoprofen, indocyanine green and glibenclamide are potent inhibitors of the uptake of [(14)C]salicylate via rOat2 (K(i) of approximately 12 microM), while diclofenac, benzoate, verapamil, ibuprofen, and tolbutamide are moderate inhibitors (K(i) of approximately 150 microM). The affinity of PAH, a common substrate for the OAT family, for rOat2 is low (K(i) > 1 mM) compared with the other members of the OAT family (rOat1 and rOat3). Salicylate and IDM are also substrates for rOat1, but their affinity for rOat2 was higher than that for rOat1. The present study shows that rOat2 is a multispecific transporter and suggests that it may be involved at least partly, in the hepatic uptake of IDM, salicylate and nucleoside derivatives.

Characterization of the efflux transport of 17beta-estradiol-D-17beta-glucuronide from the brain across the blood-brain barrier.

The contribution of organic anion transporters to the total efflux of 17beta-estradiol-D-17beta-glucuronide (E(2)17betaG) through the blood-brain barrier (BBB) was investigated using the Brain Efflux Index method by examining the inhibitory effects of probenecid, taurocholate (TCA), p-aminohippurate (PAH), and digoxin. E(2)17betaG was eliminated through the BBB with a rate constant of 0.037 min(-1) after the microinjection into the brain. Probenecid and TCA inhibited this elimination with an IC50 value of 34 and 1.8 nmol/0.5 microl of injectate, respectively, whereas PAH and digoxin reduced the total efflux to about 80 and 60% of the control value, respectively. The selectivity of these inhibitors was confirmed by examining their inhibitory effects on the transport via organic anion transporting polypeptide 1 (Oatp1), Oatp2, organic anion transporter 1 (Oat1), and Oat3 transfectants using LLC-PK1 cells as hosts. Digoxin specifically inhibited the transport via Oatp2 (K(i) = 0.037 microM). The K(i) values of TCA for Oatp1 and Oatp2 (11 and 39 microM, respectively) were about 20 times lower than those for Oat1 and Oat3 (2.8 and 0.8 mM, respectively). PAH did not affect the transport via the Oatp family, but had a similar affinity for Oat1 and Oat3 (85 and 300 microM, respectively). Probenecid had a similar affinity for these transporters (Oatp1, Oatp2, Oat1, and Oat3) examined in this study. Taking the selectivity of these inhibitors into consideration, the maximum contribution made by the Oatp2 and Oat family to the total efflux of E(2)17betaG from the brain appears to be about 40 and 20%, respectively.

Efflux transport systems for drugs at the blood-brain barrier and blood-cerebrospinal fluid barrier (Part 2).

Penetration of the blood-brain barrier or blood-cerebrospinal fluid barrier is necessary if a drug is to achieve the required concentration for a desired pharmacological effect. Efflux transport systems at such barriers provide protection for the CNS by removing drugs from the brain or cerebrospinal fluid, and transferring them to the systemic circulation. In Part 2 of this review, in vivo and in vitro studies of efflux transport via these barriers are discussed, with reference to the transporters previously described in Part 1(1).

Efflux transport systems for drugs at the blood-brain barrier and blood-cerebrospinal fluid barrier (Part 1).

Penetration through the blood-brain barrier (BBB) and blood-cerebrospinal fluid barrier (BCSFB) is necessary if a drug is to achieve the required concentration for a desired pharmacological effect. Efflux transport systems at the BBB and BCSFB provide a protective barrier function by removing drugs from the brain or cerebrospinal fluid and transferring them to the systemic circulation, respectively; several transporters at the BBB and BCSFB have been identified. Efflux transport should be taken into consideration during drug development to improve brain penetration and to avoid drug-drug interactions involving these transporters and subsequent side effects.

Y-24180, an antagonist of platelet-activating factor, suppresses interleukin 5 production in cultured murine th(2)cells and human peripheral blood mononuclear cells.

Interleukin (IL)-5 has been shown to play an essential role in the pathogenesis of airway inflammation. We investigated the effect of 4-(2-chlorophenyl)-2-[2-(4-isobutylphenyl)ethyl]-6, 9-dimethyl-6 H -thieno[3,2- f ][1,2,4]triazolo[4,3- a][1,4]diazepine (Y-24180), an antagonist of platelet-activating factor (PAF), on the production of IL-5 in cultured D10.G4.1 cells, a murine Th(2)clone, and human peripheral blood mononuclear cells (PBMC). As a result, Y-24180 was found to suppress both the mRNA expression of IL-5 and the subsequent secretion of this cytokine in antigen-stimulated D10.G4.1 cells. Y-24180 also suppressed the production of IL-4, another Th(2)type cytokine, at the level of mRNA expression, however, it hardly affected the mRNA expression for IL-6 or IL-10, thus indicating it to have a selective action against IL-5 and IL-4. The suppressive effect of Y-24180 on the secretion of IL-5 by human PBMC was more potent than that of WEB2086, which is another PAF-antagonist. These results suggest that Y-24180 suppresses IL-5 production through a common pathway which also affects the production of IL-4, even though the mechanism remains to be elucidated as to whether the PAF-antagonistic actions are involved or not.