GlaxoSmithKline (Australia)

Inflammatory proteases (mast cell tryptase and trypsins) cleave protease-activated receptor 2 (PAR2) on spinal afferent neurons and cause persistent inflammation and hyperalgesia by unknown mechanisms. We determined whether transient receptor potential vanilloid receptor 1 (TRPV1), a cation channel activated by capsaicin, protons, and noxious heat, mediates PAR2-induced hyperalgesia. PAR2 was coexpressed with TRPV1 in small- to medium-diameter neurons of the dorsal root ganglia (DRG), as determined by immunofluorescence. PAR2 agonists increased intracellular [Ca2+] ([Ca2+]i) in these neurons in culture, and PAR2-responsive neurons also responded to the TRPV1 agonist capsaicin, confirming coexpression of PAR2 and TRPV1. PAR2 agonists potentiated capsaicin-induced increases in [Ca2+]i in TRPV1-transfected human embryonic kidney (HEK) cells and DRG neurons and potentiated capsaicin-induced currents in DRG neurons. Inhibitors of phospholipase C and protein kinase C (PKC) suppressed PAR2-induced sensitization of TRPV1-mediated changes in [Ca2+]i and TRPV1 currents. Activation of PAR2 or PKC induced phosphorylation of TRPV1 in HEK cells, suggesting a direct regulation of the channel. Intraplantar injection of a PAR2 agonist caused persistent thermal hyperalgesia that was prevented by antagonism or deletion of TRPV1. Coinjection of nonhyperalgesic doses of PAR2 agonist and capsaicin induced hyperalgesia that was inhibited by deletion of TRPV1 or antagonism of PKC. PAR2 activation also potentiated capsaicin-induced release of substance P and calcitonin gene-related peptide from superfused segments of the dorsal horn of the spinal cord, where they mediate hyperalgesia. We have identified a novel mechanism by which proteases that activate PAR2 sensitize TRPV1 through PKC. Antagonism of PAR2, TRPV1, or PKC may abrogate protease-induced thermal hyperalgesia.

Noscapine is an antitumor alkaloid from opium poppy that binds tubulin, arrests metaphase, and induces apoptosis in dividing human cells. Elucidation of the biosynthetic pathway will enable improvement in the commercial production of noscapine and related bioactive molecules. Transcriptomic analysis revealed the exclusive expression of 10 genes encoding five distinct enzyme classes in a high noscapine-producing poppy variety, HN1. Analysis of an F(2) mapping population indicated that these genes are tightly linked in HN1, and bacterial artificial chromosome sequencing confirmed that they exist as a complex gene cluster for plant alkaloids. Virus-induced gene silencing resulted in accumulation of pathway intermediates, allowing gene function to be linked to noscapine synthesis and a novel biosynthetic pathway to be proposed.

The metabolic syndrome is characterized by insulin resistance and abnormal apolipoprotein AI (apoAI) and apolipoprotein B-100 (apoB) metabolism that may collectively accelerate atherosclerosis. The effects of atorvastatin (40 mg/day) and micronised fenofibrate (200 mg/day) on the kinetics of apoAI and apoB were investigated in a controlled cross-over trial of 11 dyslipidemic men with the metabolic syndrome. ApoAI and apoB kinetics were studied following intravenous d(3)-leucine administration using gas-chromatography mass spectrometry with data analyzed by compartmental modeling. Compared with placebo, atorvastatin significantly decreased (P < 0.001) plasma concentrations of cholesterol, triglyceride, LDL cholesterol, VLDL apoB, intermediate-density lipoprotein (IDL) apoB, and LDL apoB. Fenofibrate significantly decreased (P < 0.001) plasma triglyceride and VLDL apoB and elevated HDL(2) cholesterol (P < 0.001), HDL(3) cholesterol (P < 0.01), apoAI (P = 0.01), and apoAII (P < 0.001) concentrations, but it did not significantly alter LDL cholesterol. Atorvastatin significantly increased (P < 0.002) the fractional catabolic rate (FCR) of VLDL apoB, IDL apoB, and LDL apoB but did not affect the production of apoB in any lipoprotein fraction or in the turnover of apoAI. Fenofibrate significantly increased (P < 0.01) the FCR of VLDL, IDL, and LDL apoB but did not affect the production of VLDL apoB. Relative to placebo and atorvastatin, fenofibrate significantly increased the production (P < 0.001) and FCR (P = 0.016) of apoAI. Both agents significantly lowered plasma triglycerides and apoCIII concentrations, but only atorvastatin significantly lowered (P < 0.001) plasma cholesteryl ester transfer protein activity. Neither treatment altered insulin resistance. In conclusion, these differential effects of atorvastatin and fenofibrate on apoAI and apoB kinetics support the use of combination therapy for optimally regulating dyslipoproteinemia in the metabolic syndrome.

Morphinan alkaloids from the opium poppy are used for pain relief. The direction of metabolites to morphinan biosynthesis requires isomerization of (S)- to (R)-reticuline. Characterization of high-reticuline poppy mutants revealed a genetic locus, designated STORR [(S)- to (R)-reticuline] that encodes both cytochrome P450 and oxidoreductase modules, the latter belonging to the aldo-keto reductase family. Metabolite analysis of mutant alleles and heterologous expression demonstrate that the P450 module is responsible for the conversion of (S)-reticuline to 1,2-dehydroreticuline, whereas the oxidoreductase module converts 1,2-dehydroreticuline to (R)-reticuline rather than functioning as a P450 redox partner. Proteomic analysis confirmed that these two modules are contained on a single polypeptide in vivo. This modular assembly implies a selection pressure favoring substrate channeling. The fusion protein STORR may enable microbial-based morphinan production.

OBJECTIVES: This study examines awareness of the potential risks associated with over-the-counter (OTC) use of paracetamol and non-steroidal anti-inflammatory drugs (NSAIDs) among Australian consumers to better understand patterns of usage of these products. METHODS: We employed two self-reported cross-sectional surveys (conducted in 2001 and 2009) using computer-aided telephone interviewing. Both survey samples were weighted to match national population proportions; data were collected for 3702 respondents (study 1, 2001, n=1901; study 2, 2009, n=1801). The inclusion criteria were age over 18 years and willingness to participate in the survey. KEY FINDINGS: Self-reported regular use (once or more per month) of OTC analgesics declined between 2001 (67.5%) and 2009 (55.0%; P<0.05). In 2009 42.0% of regular OTC analgesic users were purchasing NSAIDs outside the pharmacy setting (compared with none in 2001). Stated awareness of potential risks has increased slightly among regular paracetamol users (from 49.0% in 2001 to 52.0% in 2009) and regular NSAID users (from 25.0% in 2001 to 41.0% in 2009). Regular OTC analgesic users were considered to be using the product appropriately if there were no contraindications, warnings, precautions or potential drug interactions to the analgesic that they had used. In 2001, significantly more people were using paracetamol appropriately than were using NSAIDs appropriately (98.3 compared with 79.3%; P<0.05). Corresponding figures for 2009 were 96.4 and 69.1% (P<0.5). CONCLUSIONS: Increasing consumer awareness of the need to consider potential risks prior to taking OTC analgesics is a positive sign. However, this has not translated to an increase in appropriate use of OTC NSAIDs; since ibuprofen has become available outside the pharmacy setting in Australia fewer people are using NSAIDs appropriately according to the label. The quality use of medicines, in particular OTC NSAIDs, is becoming increasingly reliant on product labelling and the ability of consumers to understand and self-assess risk.

OBJECTIVE: HIV-1 protease inhibitors (versus no protease inhibitors) and stavudine (versus zidovudine) are independently associated with a higher risk of lipoatrophy in HIV-infected patients. We sought to determine whether the revision of stavudine and/or protease inhibitor-containing regimens to combivir/abacavir would result in prevention and/or reversibility of lipoatrophy in HIV-1-infected patients. DESIGN: The investigation was a prospective, randomized, controlled, open-label study. SUBJECTS: The subjects included 37 HIV-1-infected individuals with stable undetectable HIV-1 loads who were taking a regimen containing either stavudine or zidovudine with lamivudine and a protease inhibitor. INTERVENTION: Subjects were randomized to continue therapy or switch stavudine to zidovudine and protease inhibitor to abacavir, such that the universal switch regimen was combivir (zidovudine/lamivudine) and abacavir. MAIN OUTCOME MEASURES: Total body, leg, and arm fat mass was measured at baseline, 24 weeks, and 48 weeks using whole-body dual-energy x-ray absorptiometry. Single-cut L4 computed tomography and assays of multiple metabolic parameters were also performed. RESULTS: There was an average gain in fat mass of 0.009 kg/(leg.mo) in switch patients versus a loss of 0.010 kg/(leg.mo) in controls (p =.04, on-treatment analysis) over 48 weeks. Significant arm fat restoration was observed in patients who switched regimens, with an average gain of 0.014 kg/(arm.mo) (p =.004), whereas controls did not have a significant change from baseline. Analyses of percentage changes in arm and leg fat masses showed similar findings. No significant effects on intraabdominal fat, blood lipid levels, glycemic indices, and lactate levels were detected, although most baseline mean values were normal in study subjects. Combivir/abacavir maintained virological control in all but one case, and three (13.6%) of 22 individuals had adverse reactions to abacavir therapy. CONCLUSIONS: A switch to combivir/abacavir therapy was associated with objective evidence of limb fat-sparing and fat restoration compared with continued treatment with stavudine and/or protease inhibitor.

5021^ Background: Pazopanib, an oral multikinase angiogenesis inhibitor, has shown clinical efficacy in patients (pts) with advanced RCC. In this study (VEG105192), the efficacy and safety of pazopanib was compared with placebo in advanced RCC. Methods: Pts (N = 400 planned) with clear cell advanced RCC and measurable disease with no prior treatment or 1 prior cytokine-based treatment, were stratified and randomized (2:1) to pazopanib 800 mg QD or placebo. The primary endpoint was progression-free survival (PFS). Secondary endpoints included overall survival (OS), response rate (RR), and safety. The study had ≥ 90% power to detect an 80% improvement in PFS and a 50% improvement in OS, by stratified log-rank tests with α = 0.025 one-sided. Pts received continuous treatment until disease progression (PD), death or unacceptable toxicity. Upon PD, placebo pts could receive pazopanib via an extension study. Final PFS, RR and safety results are reported here. Results: A total of 233 treatment-naïve and 202 cytokine-pretreated pts were enrolled (290 pazopanib; 145 placebo). Pt characteristics were balanced between the 2 arms. ECOG 0/1 was 42%/58% and 41%/59% for pazopanib and placebo pts, respectively. PFS was significantly prolonged with pazopanib in the overall study population (9.2 vs 4.2 mos; HR: 0.46; 95% CI: 0.34, 0.62; p &lt; 0.0000001), in treatment naïve pts (11.1 vs 2.8 mos; HR: 0.40; 95% CI: 0.27, 0.60; p &lt; 0.0000001), and in cytokine-pretreated pts (7.4 vs 4.2 mos; HR: 0.54; 95% CI: 0.35, 0.84; p &lt; 0.001). RR was 30% with pazopanib (vs 3% with placebo) and median duration of response was 58.7 wks. Median duration of exposure was 7.4 mos (pazopanib) and 3.8 mos (placebo). The majority of adverse events (AEs) were grade 1 or 2. Most common AEs in pazopanib-treated pts were diarrhea (52%; 4% Gr 3/4), hypertension (40%; 4% Gr 3/4), hair color change (38%; &lt;1% Gr 3/4), nausea (26%; &lt;1% Gr 3/4), anorexia (22%; 2% Gr 3/4), and vomiting (21%; 2% Gr 3/4). The most common laboratory abnormality was ALT elevation (53%; 10% Gr 3; 2% Gr 4). Conclusions: Pazopanib monotherapy was well tolerated and demonstrated a significant improvement in PFS and RR compared to placebo. Final OS results are awaited. [Table: see text] ASCO Conflict of Interest Policy and Exceptions In compliance with the guidelines established by the ASCO Conflict of Interest Policy (J Clin Oncol. 2006 Jan 20;24[3]:519–521) and the Accreditation Council for Continuing Medical Education (ACCME), ASCO strives to promote balance, independence, objectivity, and scientific rigor through disclosure of financial and other interests, and identification and management of potential conflicts. According to the ASCO Conflict of Interest Policy, the following financial and other relationships must be disclosed: employment or leadership position, consultant or advisory role, stock ownership, honoraria, research funding, expert testimony, and other remuneration (J Clin Oncol. 2006 Jan 20;24[3]:520). The ASCO Conflict of Interest Policy disclosure requirements apply to all authors who submit abstracts to the Annual Meeting. For clinical trials that began accrual on or after April 29, 2004, ASCO's Policy places some restrictions on the financial relationships of principal investigators (J Clin Oncol. 2006 Jan 20;24[3]:521). If a principal investigator holds any restricted relationships, his or her abstract will be ineligible for placement in the 2009 Annual Meeting unless the ASCO Ethics Committee grants an exception. Among the circumstances that might justify an exception are that the principal investigator (1) is a widely acknowledged expert in a particular therapeutic area; (2) is the inventor of a unique technology or treatment being evaluated in the clinical trial; or (3) is involved in international clinical oncology research and has acted consistently with recognized international standards of ethics in the conduct of clinical research. NIH-sponsored trials are exempt from the Policy restrictions. Abstracts for which authors requested and have been granted an exception in accordance with ASCO's Policy are designated with a caret symbol (^) in the Annual Meeting Proceedings. For more information about the ASCO Conflict of Interest Policy and the exceptions process, please visit www.asco.org/conflictofinterest .

The synthesis, antiviral and pharmacokinetic properties of zanamivir (ZMV) dimers 8 and 13 are described. The compounds are highly potent neuraminidase (NA) inhibitors which, along with dimer 3, are being investigated as potential second generation inhaled therapies both for the treatment of influenza and for prophylactic use. They show outstanding activity in a 1 week mouse influenza prophylaxis assay, and compared with ZMV, high concentrations of 8 and 13 are found in rat lung tissue after 1 week. Retention of compounds in rat lung tissue correlated both with molecular weight (excluding 3 and 15) and with a capacity factor K' derived from immobilized artificial membrane (IAM) chromatography (including 3 and 15). Pharmacokinetic parameters for 3, 8 and 13 in rats show the compounds have short to moderate plasma half-lives, low clearances and low volumes of distribution. Dimer 3 shows NA inhibitory activity against N1 viruses including the recent highly pathogenic H5N1 A/Chicken/Vietnam/8/2004. In plaque reduction assays, 3, 8 and 13 show good to outstanding potency against a panel of nine flu A and B virus strains. Consistent with its shorter and more rigid linking group, dimer 8 has been successfully crystallized.

BACKGROUND: Estimates of the disease burden from childhood pneumonia are available for most developed countries, but they are based mainly on models. Measured country-specific pneumonia burden data are limited to a few nations and differ in case definitions and case ascertainment methods. This review describes pneumonia disease burden in developed countries. METHODS: We reviewed studies describing childhood pneumonia incidence in North America, Europe, Australia, New Zealand and Japan. Available estimates suggest that each year in developed countries there are up to 2.6 million cases of pneumonia, including 1.5 million hospitalized cases and around 3000 pneumonia deaths (compared with approximately 640 annual deaths from meningitis) in children <5 years of age. RESULTS: Data to inform policy decisions would be improved by information on burden and etiology of severe pneumonia, population-based incidence of ambulatory visits and hospitalizations and prevalence of complications and sequelae.

OBJECTIVE: To examine the evidence that dopamine (DA) dysfunction contributes to melancholic depression. METHOD: Database (EMBASE, PsychLit and MEDLINE) searches using relevant key words were conducted and citations were scrutinized. RESULTS: In this paper, we assume that the definition of melancholia is contingent upon the presence of psychomotor disturbance (PMD). In melancholic depression PMD comprises both a cognitive and motor component and DA is found to be important in both. DA neurotransmission modulates cognition in particular in attention, adaptation and motivational processes and has a pivotal role in motor function. CONCLUSION: DA is a credible aetiological candidate for the PMD in melancholic depression. However, melancholia needs first to be characterized both clinically and in terms of its pathophysiology. In this regard, illnesses such as bipolar depression and Parkinson's disease warrant consideration as they provide suitable models of both the cognitive and motor aspects of PMD, and hold the necessary markers to better define melancholia.

5031 Background: Pazopanib is a potent and selective multi-targeted receptor tyrosine kinase inhibitor of VEGFR-1, VEGFR-2, VEGFR-3, PDGFR-a/β, and c-kit that blocks tumor growth &amp; inhibits angiogenesis. This Phase II RDT determined effects of pazopanib on tumor growth in patients with adv/met RCC after 12 wks of treatment. Methods: Cytokine naïve and refractory (failed 1 prior cytokine or bevacizumab-containing regimen) patients with adv/met RCC, ECOG 1–2, were enrolled. Pazopanib 800 mg po qd was administered. Response (RECIST) assessed at wk 12: SD patients randomized to continue pazopanib or placebo (blinded); PR/CR patients continued pazopanib. Interim analysis (first 60 patients) endpoints: % randomized (SD) and % CR/PR at wk 12. Futility boundary was based on a randomization rate of &lt;40%. Safety was analyzed in enrolled patients (n=161) at the time of interim analysis. Results: In the first 60 patients, response at wk 12 by independent review: PR in 24 (40%); SD in 25 (42%); PD in 5 (8%); unknown response in 2 (3%); and withdrawal prior to wk 12 (reasons other than PD/AE) in 4 (7%). 27 (45%) patients were randomized based on investigator review at wk 12. Total disease control rate was 82% (PR + SD). 67% of patients were treatment naïve, 33% had failed one prior treatment regimen (23% cytokine, 8% bevacizumab, 2% both). Most common AEs/lab abnormalities in all patients (n=161; 71% M, 29% F; mean 60.3 yrs; 81% Caucasian) were ALT/AST elevations, diarrhea, fatigue, nausea, hair depigmentation, &amp; hypertension. Gr 3/4 AEs occurred in 26% of patients; most common were hypertension (8%) &amp; ALT elevation (8%). One patient had Gr 5 AE due to large intestinal perforation. AEs led to discontinuation in 5% of patients. Conclusions: Interim analysis of this Ph II study demonstrated that pazopanib treatment resulted in a PR rate at wk 12 of 40% among patients with adv/met RCC and an acceptable toxicity profile. Based on these results, the Independent Data Monitoring Committee recommended discontinuation of randomization to placebo. Patients without PD were offered continued treatment with pazopanib beyond wk 12. At the time of publication, efficacy and safety data will be available for all 225 patients. [Table: see text]

Although the number of countries participating in pivotal trials submitted to enable drug registration has nearly doubled over the past 25 years, there has not been a substantial increase in the diversity of clinical trial populations. In parallel, our understanding of factors that influence medicine response and variability has continued to evolve. The notion of intrinsic and extrinsic sources of variability has been embedded into different regulatory guidelines, including the recent guideline on the importance of enhancing the diversity of clinical trial populations. In addition to presenting the clinical and scientific reasons for ensuring that clinical trial populations represent the demographics of patient populations, this overview outlines the efforts of regulatory agencies, patient advocacy groups and clinical researchers to attain this goal through strategies to meet representation in recruitment targets and broaden eligibility criteria. Despite these efforts, challenges to participation in clinical trials remain, and certain groups continue to be underrepresented in development programmes. These challenges are amplified when the representativeness of specific groups may vary across countries and regions in a global clinical programme. Whilst enhanced trial diversity is a critical step towards ensuring that results will be representative of patient populations, a concerted effort is required to characterise further the factors influencing interindividual and regional differences in response for global populations. Quantitative clinical pharmacology principles should be applied to allow extrapolation of data across groups or regions as well as provide insight into the effect of patient-specific characteristics on a medicine's dose rationale and efficacy and safety profiles.

5561 Background: Pazopanib is a potent and selective multi-targeted receptor tyrosine kinase inhibitor of VEGFR-1, VEGFR-2, VEGFR-3, PDGFR-a/β, and c-kit that blocks tumor growth &amp; inhibits angiogenesis. Clinical studies have demonstrated activity of anti-angiogenesis agents in ovarian carcinoma. Methods: Pts with epithelial ovarian, fallopian tube or primary peritoneal carcinoma; ECOG PS 0–1 with complete CA-125 response to initial platinum-based chemotherapy with subsequent rise to = 42 U/mL; no disease on CT; or non-bulky disease (masses ≤ 4 cm) were eligible. All pts must have received 1–2 prior regimens. Treatment consisted of pazopanib 800mg QD until PD, withdrawal due to AEs, or withdrawal of consent. Two-stage Green-Dahlberg design was employed requiring 2 CA-125 responses in Stage I (20 pts) to proceed to Stage II (15 pts). Primary endpoint was CA-125 response (= 50% decrease from 2 baseline samples, confirmed ≥ 21 d after initial response sample). Results: Data were available from 15/17 pts enrolled. Median age was 60 yrs (46–79). Majority of pts had platinum-sensitive disease to first line therapy (74%) and had 1 prior line of therapy (60%). 40% of pts relapsed &lt; 6 mos, 27% relapsed 6–12 mos; 27% relapsed &gt; 12 mos. CA-125 responses were seen in 7 pts (47%) with a median time to response of 29 d (5 pts have continuing response of 56–140 d and 2 pts had response duration of 56 &amp; 112 d). SD was observed in 4 pts (27%) and PD in 4 pts (27%). Most common AEs were fatigue, diarrhea, nausea, vomiting, and headache. Most common Gr 3/4 AEs were diarrhea (n=2) and ALT elevation (n=2). Five pts (33%) withdrew due to an AE; 1pt (7%) withdrew due to a potentially disease-related AE (Gr 3 ascites) and 4 pts (26%) withdrew due to toxicity (Gr 3 fatigue, Gr 2 vomiting, Gr 3 double vision, Gr 3 AST/ALT elevations). Conclusions: Preliminary review of Stage I suggests that pazopanib monotherapy demonstrates biologic activity in pts with ovarian cancer with biochemical relapse following prior platinum chemotherapy. The study has met the response criteria to proceed to stage II of enrollment. No significant financial relationships to disclose.

OBJECTIVES: To investigate the utility of metrics of CYP1A2 activity using caffeine as a probe, and saliva and plasma sampling with or without a 24-h caffeine abstinence. METHODS: This was a cross-over pharmacokinetic study in 30 healthy male subjects who received a single oral 100mg caffeine dose after 24-h caffeine abstinence or after maintaining their regular caffeine intake (no caffeine abstinence). Serial blood and saliva samples were collected simultaneously over 24h. Caffeine and paraxanthine concentrations were measured using a validated HPLC assay. KEY FINDINGS: There was a strong correlation between the paraxanthine/caffeine AUC(0-24) ratio (reference metric) and the paraxanthine/caffeine concentration (C(t) ) ratio at 4h (C(4) ) in both saliva and plasma (r≥0.75). The paraxanthine/caffeine AUC(0-24) ratio in plasma and saliva did not differ between the 24-h caffeine abstinence and the no abstinence period (P>0.05). The optimal paraxanthine/caffeine C(t) that correlated with the plasma paraxanthine/caffeine AUC(0-24) ratio in the 24-h abstinence period was 2 and 4h (r=0.88) in plasma, and 4 and 6h in saliva (r=0.70), while it was the saliva 4h time-point in the no abstinence period (r=0.78). CONCLUSIONS: The saliva paraxanthine/caffeine concentration ratio at 4h was a suitable metric to assess CYP1A2 activity after oral administration of caffeine without the need for 24-h caffeine abstinence.

The drug-metabolizing enzyme CYP1A2 contributes to the metabolism of a number of commonly used medicines and displays wide interindividual variability. The aim of this study was to investigate CYP1A2 activity in a population of South Asian ancestry and compare it with a population of European ancestry. CYP1A2 activity was determined using the 4 h paraxanthine/caffeine saliva concentration ratio following a 100-mg oral dose of caffeine in healthy individuals of South Asian (n = 166) and European (n = 166) ancestry. Participants were surveyed for extrinsic ethnic factors and genotyped for polymorphisms in CYP1A2 and related genes. Significantly lower CYP1A2 activity was observed in South Asian participants (median: 0.42; range: 0.10-1.06) as compared with European participants (0.54; 0.12-1.64) (P < 0.01). Multiple linear regression indicated that 41% of the variability in CYP1A2 activity could be explained by the diet, lifestyle, and genetic factors studied.

Meningococcal conjugate vaccines induce herd protection by preventing nasopharyngeal meningococcal acquisition, which is a prerequisite for invasive disease. Thus, meningococcal carriage epidemiology is important in understanding relationships between carriage and disease. A literature search traced information on meningococcal carriage in 27 EU countries. Meningococcal carriage prevalence differed within and between countries, varying across age groups, serogroup distribution and over time. Carriage prevalence increased during childhood, peaking in 15-24-year-olds. While serogroup B was usually the dominant serogroupable carried serogroup, serogroups C, W-135 and Y were also frequently carried. Current carriage studies in Europe are limited. New studies using standardized methods are needed to improve our understanding of meningococcal disease etiology and transmission, and to monitor the impact of meningococcal conjugate vaccines in populations.

What is already known about this subject • Gliclazide is a widely used oral hypoglycaemic agent. • The major metabolites of gliclazide formed in vivo have been identified. • However, the cytochrome P450 enzymes catalysing the rate‐limiting pathways of gliclazide elimination are unknown. What this study adds • CYP2C9 is the major enzyme involved in the various hydroxylation pathways of gliclazide, although a contribution of CYP2C19 to tolymethylhydroxylation, the major metabolic route, cannot be discounted. • Factors known to influence CYP2C9 activity will provide the main source of variability in gliclazide pharmacokinetics. Aims To identify the human cytochrome P450 (CYP) enzymes responsible for the formation of the 6β‐hydroxy (6β‐OHGz), 7β‐hydroxy (7β‐OHGz) and hydroxymethyl (MeOH‐Gz) metabolites of gliclizide (Gz). Methods 6β‐OHGz, 7β‐OHGz and MeOH‐Gz formation by human liver microsomes and a panel of recombinant human P450s was measured using a high‐performance liquid chromatography procedure, and the kinetics of metabolite formation was determined for each pathway. Effects of prototypic CYP enzyme selective inhibitors were characterized for each of the microsomal metabolic pathways. Results Microsomes from six human livers converted Gz to its 6β‐OHGz, 7β‐OHGz, and MeOH‐Gz metabolites, with respective mean (± SD) K m values of 461 ± 139, 404 ± 143 and 334 ± 75 µ m and mean V max values of 130 ± 55, 82 ± 31 and 268 ± 115 pmol min −1 mg −1 , respectively. V max / K m ratios for the microsomal reactions parallelled relative metabolite formation in vivo . Sulfaphenazole inhibited microsomal 6β‐OHGz, 7β‐OHGz and MeOH‐Gz formation by 87, 83 and 64%, respectively, whereas S‐mephenytoin caused significant inhibition (48%) of only MeOH‐Gz formation. Recombinant CYP2C9, CYP2C18 and CYP2C19 catalysed all hydroxylation pathways, whereas CYP2C8 formed only 6β‐OHGz and 7β‐OHGz. Conclusion Taken together, the results indicate that CYP2C9 is the major contributor to Gz metabolic clearance, although CYP2C19 may also be involved in MeOH‐Gz formation (the major metabolic pathway). Factors known to influence CYP2C9 activity will provide the main source of variability in Gz pharmacokinetics.

CONTEXT: Reduced high density lipoprotein (HDL) concentration in the metabolic syndrome (MetS) is associated with increased risk of diabetes and cardiovascular disease and is related to defects in the kinetics of HDL apolipoprotein (apo) A-I and A-II. OBJECTIVE: The objective of the study was to investigate HDL apoA-I and apoA-II kinetics in nondiabetic men with MetS and lean controls by developing a model that describes the kinetics of lipoprotein (Lp)A-I and LpA-I:A-II particles. DESIGN: Twenty-three MetS men and 10 age-matched lean controls were investigated. ApoA-I and apoA-II tracer/tracee ratios were studied after iv d3-leucine administration using gas chromatography mass spectrometry. RESULTS: Compared with lean subjects, MetS subjects had accelerated catabolism of LpA-I (P < 0.001), LpA-I:A-II (P = 0.005), and apoA-II (P = 0.005); the production rate of LpA-I was also significantly elevated in MetS, so that the dominant changes in plasma concentrations were reduction in LpA-I:A-II (P < 0.001) and apoA-II (P < 0.05). Increased catabolism of LpA-I and LpA-I:A-II was directly related to increased waist circumference, hypertriglyceridemia, low HDL-cholesterol, small HDL particle size, hyperinsulinemia, and low phospholipid transfer protein (PLTP) activity; overproduction of LpA-I was significantly associated with increased waist circumference, insulin resistance, and low PLTP activity. CONCLUSIONS: MetS men exhibit hypercatabolism of the two major HDL lipoprotein particles, LpA-I and LpA-I:A-II, but selective overproduction of LpA-I maintains a normal plasma concentration of LpA-I. These kinetic perturbations are probably related to central obesity, insulin resistance, hypertriglyceridemia, and low plasma PLTP activity.

Caffeine has been extensively used as a probe to measure CYP1A2 activity in humans with caffeine clearance or the paraxanthine (major metabolite of caffeine) to caffeine concentration ratio being regarded as the preferred metric. A simple reverse-phased C(18) HPLC assay using ethyl acetate liquid-liquid extraction was developed to quantitate caffeine and paraxanthine concentrations in saliva and plasma. The mobile phase consisted of acetonitrile-acetic acid-H(2)O (100:1:899) and analytes were quantitated with UV detection at 280 nm. The extraction recovery for paraxanthine and caffeine was approximately 70% in both saliva and plasma. The assay was linear over the concentration ranges 0.05-2.50 and 0.05-5.00 µg/mL, for paraxanthine and caffeine, respectively, in saliva. In plasma the assay was linear over the ranges 0.025-2.50 and 0.025-5.00 µg/mL for paraxanthine and caffeine, respectively. Intra- and inter-assay precision and accuracy were less than 15%. Detection limits were 0.015 µg/mL for paraxanthine and caffeine in saliva, while it was 0.005 µg/mL for paraxanthine and caffeine in plasma. Utility was established in samples collected from two healthy volunteers who abstained from caffeine for 24 h and received a single 100 mg oral dose of caffeine. The assay developed is a robust, simple and precise technique to measure caffeine and paraxanthine in saliva and plasma of healthy volunteers after a single oral dose of caffeine.

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT • An increasing number of drugs on the market or under development have been identified as substrates of the ATP‐binding cassette drug efflux transporter breast cancer resistance protein (BCRP; ABCG2), which can affect the pharmacokinetics of drugs by reducing absorption and/or increasing biliary elimination. • ABCG2 C421A, a single nucleotide polymorphism associated with decreased protein expression/transport activity in vitro and higher anti‐cancer drug concentrations in carriers of the C421A polymorphism, may contribute to the intersubject pharmacokinetic variability of BCRP substrates. • Predicting the potential influence of BCRP on drug disposition or drug interactions is challenging because of the lack of a well‐characterized, BCRP‐selective clinical probe substrate. • Nitrofurantoin is potentially a suitable clinical BCRP probe substrate based on preclinical and clinical information available (e.g. in vitro transport studies, Bcrp knockout mouse studies, inhibition studies in rats, and milk secretion studies in rats and humans). WHAT THIS STUDY ADDS • The ABCG2 C421A SNP had no effect on oral nitrofurantoin plasma and urine pharmacokinetic parameters in healthy male Chinese subjects. • Nitrofurantoin does not appear to be a useful clinical BCRP probe. AIMS A number of drugs are substrates or inhibitors of the efflux transporter breast cancer resistance protein (BCRP; ABCG2), which can limit systemic exposure by reducing absorption and/or increasing biliary elimination. The identification of a BCRP‐selective clinical probe drug would provide a useful tool to understand the effect of genetic polymorphisms and transporter‐based drug interactions on drug pharmacokinetics. The aim of this study was to assess the utility of nitrofurantoin as a clinical probe substrate for BCRP activity by evaluating the impact of genetic variation on nitrofurantoin pharmacokinetics. METHODS Nitrofurantoin pharmacokinetics were studied in an open‐label, single‐oral dose (100 mg) study in 36 male Chinese subjects who were pre‐screened for ABCG2 421 CC, CA and AA genotypes ( n = 12 each). Plasma and urine concentrations of nitrofurantoin were determined by LC/MS/MS and LC/UV respectively. anova was used to compare pharmacokinetic parameters among genotypes. RESULTS There were no significant differences in nitrofurantoin pharmacokinetics among the genotypic cohorts. The geometric mean nitrofurantoin plasma AUC (0‐∞) (95% confidence interval) values were 2.21 (2.00, 2.45), 2.42 (2.11, 2.78) and 2.32 (1.99, 2.70) µg h ml −1 and half‐life values were 0.79 (0.59, 1.0), 0.76 (0.64, 0.89) and 0.72 (0.62, 0.84) h for ABCG2 421 genotypes CC, CA and AA, respectively. The percentage of dose excreted unchanged in the urine was 43, 44 and 39%, respectively. CONCLUSIONS The ABCG2 C421A polymorphism had no effect on nitrofurantoin plasma and urine pharmacokinetic parameters in healthy Chinese subjects. These results indicate that nitrofurantoin is not a suitable clinical probe substrate for assessing BCRP activity.