Bench to Bed Evidences for Pharmacokinetic and Pharmacodynamic Interactions Involving Oseltamivir and Chinese Medicine

Oseltamivir (OA), an ethyl ester prodrug of oseltamivir carboxylate (OC), is clinically used as a potent and selective inhibitor of neuraminidase. Chinese medicines have been advocated to combine with conventional drug for avian influenza. The current study aims to investigate the potential pharmacokinetic and pharmacodynamic interactions of a Chinese medicine formula, namely, Yin Qiao San and Sang Ju Yin (CMF1), commonly used for anti-influenza in combination with OA in both rat and human, and to reveal the underlined mechanisms. It was found that although Cmax, AUC and urinary recovery of OC, as well as metabolic ratio (AUCOC/AUCOA), were significantly decreased in a dose-dependent manner following combination use of CMF1 and OA in rat studies (P<0.01), such coadministration in 14 healthy volunteers only resulted in a trend of minor decrease in the related parameters. Further mechanistic studies found that although CMF1 could reduce absorption and metabolism of OA, it appears to enhance viral inhibition of OA (P<0.01). In summary, although there was potential interaction between OA and CMF1 found in rat studies, its clinical impact was expected to be minimal. The coadministration of OA and CMF1 at the clinical recommended dosages is, therefore, considered to be safe

Cytokine Response Patterns in Severe Pandemic 2009 H1N1 and Seasonal Influenza among Hospitalized Adults

BACKGROUND: Studying cytokine/chemokine responses in severe influenza infections caused by different virus subtypes may improve understanding on pathogenesis. METHODS: Adults hospitalized for laboratory-confirmed seasonal and pandemic 2009 A/H1N1 (pH1N1) influenza were studied. Plasma concentrations of 13 cytokines/chemokines were measured at presentation and then serially, using cytometric-bead-array with flow-cytometry and ELISA. PBMCs from influenza patients were studied for cytokine/chemokine expression using ex-vivo culture (Whole Blood Assay,±PHA/LPS stimulation). Clinical variables were prospectively recorded and analyzed. RESULTS: 63 pH1N1 and 53 seasonal influenza patients were studied. pH1N1 patients were younger (mean±S.D. 42.8±19.2 vs 70.5±16.7 years), and fewer had comorbidities. Respiratory/cardiovascular complications were common in both groups (71.4% vs 81.1%), although severe pneumonia with hypoxemia (54.0% vs 28.3%) and ICU admissions (25.4% vs 1.9%) were more frequent with pH1N1. Hyperactivation of the proinflammatory cytokines IL-6, CXCL8/IL-8, CCL2/MCP-1 and sTNFR-1 was found in pH1N1 pneumonia (2-15 times normal) and in complicated seasonal influenza, but not in milder pH1N1 infections. The adaptive-immunity (Th1/Th17)-related CXCL10/IP-10, CXCL9/MIG and IL-17A however, were markedly suppressed in severe pH1N1 pneumonia (2-27 times lower than seasonal influenza; P-values<0.01). This pattern was further confirmed with serial measurements. Hypercytokinemia tended to be sustained in pH1N1 pneumonia, associated with a slower viral clearance [PCR-negativity: day 3-4, 55% vs 85%; day 6-7, 67% vs 100%]. Elevated proinflammatory cytokines, particularly IL-6, predicted ICU admission (adjusted OR 12.6, 95%CI 2.6-61.5, per log(10)unit increase; P = 0.002), and correlated with fever, tachypnoea, deoxygenation, and length-of-stay (Spearman's rho, P-values<0.01) in influenza infections. PBMCs in seasonal influenza patients were activated and expressed cytokines ex vivo (e.g. IL-6, CXCL8/IL-8, CCL2/MCP-1, CXCL10/IP-10, CXCL9/MIG); their 'responsiveness' to stimuli was shown to change dynamically during the illness course. CONCLUSIONS: A hyperactivated proinflammatory, but suppressed adaptive-immunity (Th1/Th17)-related cytokine response pattern was found in severe pH1N1 pneumonia, different from seasonal influenza. Cytokine/immune-dysregulation may be important in its pathogenesis

Seasonality of Influenza A(H3N2) Virus: A Hong Kong Perspective (1997–2006)

BACKGROUND: The underlying basis for the seasonality of influenza A viruses is still uncertain. Phylogenetic studies investigated this phenomenon but have lacked sequences from more subtropical and tropical regions, particularly from Southeast Asia. METHODOLOGY/PRINCIPAL FINDINGS: 281 complete hemagglutinin (HA) and neuraminidase (NA) sequences were obtained from influenza A(H3N2) viruses, collected over 10 years (1997-2006) from Hong Kong. These dated sequences were analyzed with influenza A(H3N2) vaccine strain sequences (Syd/5/97, Mos/10/99, Fuj/411/02, Cal/7/04) and 315 other publicly available dated sequences from elsewhere, worldwide. In addition, the NA sequence alignment was inspected for the presence of any naturally occurring, known, neuraminidase inhibitor (NAI) resistance-associated amino acid mutations (R292K and E119V). Before 2001, the Hong Kong HA and NA sequences clustered more closely with the older vaccine sequences (Syd/5/97, Mos/10/99) than did sequences from elsewhere. After 2001, this trend reversed with significant clusters containing HA and NA sequences from different locations, isolated at different times, suggesting that viral migration may account for much of the influenza A(H3N2) seasonality during this 10-year period. However, at least one example from Hong Kong was found suggesting that in some years, influenza A(H3N2) viruses may persist in the same location, perhaps continuing to circulate, sub-clinically, at low levels between seasons, to re-emerge in the influenza season the following year, relatively unchanged. None of these Hong Kong influenza A(H3N2) NA sequences contained any of the known NAI-resistance associated mutations. CONCLUSIONS/SIGNIFICANCE: The seasonality of influenza A(H3N2) may be largely due to global migration, with similar viruses appearing in different countries at different times. However, occasionally, some viruses may remain within a single location and continue to circulate within that population, to re-emerge during the next influenza season, with relatively little genetic change. Naturally occurring NAI resistance mutations were absent or, at least, very rare in this population

Replication and transcription activities of ribonucleoprotein complexes reconstituted from avian H5N1, H1N1pdm09 and H3N2 influenza A viruses.

Avian influenza viruses pose a serious pandemic threat to humans. Better knowledge on cross-species adaptation is important. This study examined the replication and transcription efficiency of ribonucleoprotein complexes reconstituted by plasmid co-transfection between H5N1, H1N1pdm09 and H3N2 influenza A viruses, and to identify mutations in the RNA polymerase subunit that affect human adaptation. Viral RNA polymerase subunits PB1, PB2, PA and NP derived from influenza viruses were co-expressed with pPolI-vNP-Luc in human cells, and with its function evaluated by luciferase reporter assay. A quantitative RT-PCR was used to measure vRNA, cRNA, and mRNA levels for assessing the replication and transcription efficiency. Mutations in polymerase subunit were created to identify signature of increased human adaptability. H5N1 ribonucleoprotein complexes incorporated with PB2 derived from H1N1pdm09 and H3N2 viruses increased the polymerase activity in human cells. Furthermore, single amino acid substitutions at PB2 of H5N1 could affect polymerase activity in a temperature-dependent manner. By using a highly sensitive quantitative reverse transcription-polymerase chain reaction, an obvious enhancement in replication and transcription activities of ribonucleoproteins was observed by the introduction of lysine at residue 627 in the H5N1 PB2 subunit. Although less strongly in polymerase activity, E158G mutation appeared to alter the accumulation of H5N1 RNA levels in a temperature-dependent manner, suggesting a temperature-dependent mechanism in regulating transcription and replication exists. H5N1 viruses can adapt to humans either by acquisition of PB2 from circulating human-adapted viruses through reassortment, or by mutations at critical sites in PB2. This information may help to predict the pandemic potential of newly emerged influenza strains, and provide a scientific basis for stepping up surveillance measures and vaccine production

Comparison of <i>in vitro</i> polymerase activity of H1N1pdm09, seasonal H3N2, avian H5N1, and WSN H1N1.

<p>293T cells were co-transfected with expression plasmids of NP, PA, PB1 and PB2 together with pPolI-vNP-Luc and a reporter plasmid pGL4.73 [hRluc/SV40], encoding a <i>Renilla</i> luciferase gene. Cells were incubated at (A) 33^o and (B) 37°C. Polymerase activity was normalized with the expression of a reporter plasmid. Relative polymerase activity (%) was expressed as relative activity to WSN H1N1 in percentage. Results shown are means with standard deviations from three independent assays. *indicates <i>P</i><0.05 when compared to WSN H1N1, and ***indicates <i>P</i><0.001 when compared to H5N1.</p

Polymerase activity of H5N1 RNP complexes containing mutations E158G, T271A and E627K in PB2.

<p>293T cells were co-transfected with expression plasmids of NP, PA, PB1 and either wild type (WT) or PB2 mutants with the indicated amino acid substitution of E158G, T271A or E627K, together with pPolI-vNP-Luc and a reporter plasmid pGL4.73. Cells were incubated at (A) 33°C, (B) 37°C. Polymerase activity was normalized with the expression of a reporter plasmid. Relative polymerase activity (%) was expressed as relative activity to WT. Results shown are means with standard deviations from three independent assays. *indicates <i>P</i><0.05 when compared to WT.</p

Quantitation of viral RNA levels of H5N1 RNP complexes containing PB2 mutations.

<p>293T cells were co-transfected with expression plasmids of NP, PA, PB1 and either wild type (WT) of indicated PB2 mutants with amino acid substitution of E158G, T271A or E627K, together with pPolI-NA plasmid. Total cellular RNA was isolated after 48 hours post-transfection and subjected to quantitative RT-PCR for segment 6 (NA genes) transcripts. Cells were incubated at (A) 33°C, (B) 37°C. RNA levels were expressed as relative activity to wild-type. Results shown are means with standard deviations from three independent assays. *indicates <i>P</i><0.05 when compared to wild-type.</p

Comparison of <i>in vitro</i> polymerase activity of reconstituted RNP complexes.

<p>Activities of polymerase complexes with a single gene replacement following expression in human 293T cells. (A) H5N1 polymerase complexes substituted with one H3N2 gene, (B) H3N2 polymerase complexes substituted with one H5N1 gene, (C) H5N1 polymerase complexes substituted with one H1N1pdm09 gene, and (D) H1N1pdm09 polymerase complexes substituted with one H5N1 gene, were analyzed in 293T cells transfected with the indicated plasmids of NP, PA, PB1 and PB2 together with pPolI-vNP-Luc and a reporter plasmid pGL4.73. Cells were incubated at 33°C and 37°C. Polymerase activity was normalized with the expression of a reporter plasmid. Relative polymerase activity (%) was expressed as relative activity to the corresponding parental vRNPs. Results shown are means with standard deviations from three independent assays.</p

Knowledge, attitude, practice and barriers on vaccination against human papillomavirus infection: a cross-sectional study among primary care physicians in Hong Kong.

This study explored the knowledge, attitude, practice and barriers to prescribe human papillomavirus (HPV) vaccines among private primary care physicians in Hong Kong. A self-administered questionnaire survey was conducted by sending letters to doctors who had joined a vaccination program for school girls. From 720 surveys sent, 444 (61.7%) completed questionnaires were returned and analyzed. For knowledge, few responded to questions accurately on the prevalence of cervical HPV (27.9%) and genital wart infection (13.1%) among sexually active young women in Hong Kong, and only 44.4% correctly answered the percentage of cervical cancers caused by HPV. For attitude, most agreed that HPV vaccination should be fully paid by the Government (68.3%) as an important public health strategy. Vaccination against HPV was perceived as more important than those for genital herpes (52.2%) and Chlamydia (50.1%) for adolescent health, and the majority selected adolescents aged 12-14 years as the ideal group for vaccination. Gardasil(®) (30.9%) and Cervarix(®) (28.0%) were almost equally preferred. For practice, the factors influencing the choice of vaccine included strength of vaccine protection (61.1%), long-lasting immunity (56.8%) and good antibody response (55.6%). The most significant barriers to prescribe HPV vaccines consisted of parental refusal due to safety concerns (48.2%), and their practice of advising vaccination was mostly affected by local Governmental recommendations (78.7%). A substantial proportion of physicians had recommended HPV vaccines for their female clients/patients aged 18-26 years for protection of cervical cancer (83.8%) or both cervical cancer and genital warts (85.5%). The knowledge on HPV infection was low among physicians in Hong Kong. Prescription of HPV vaccine was hindered by the perceived parental concerns and was mostly relied on Governmental recommendations. Educational initiatives should be targeted towards both physicians and parents, and the Government should consider full subsidy to enhance vaccine uptake rate

Knowledge on human papillomavirus (HPV) infection (N = 444).

<p>Knowledge on human papillomavirus (HPV) infection (N = 444).</p