Search

In search of novel therapeutic options to treat influenza virus (IV) infections, we previously identified a series of inhibitors that act by disrupting the interactions between the PA and PB1 subunits of the viral RNA polymerase. These compounds showed broad-spectrum antiviral activity against human influenza A and B viruses and a high barrier to the induction of drug resistance in vitro. In this short communication, we investigated the effects of combinations of the PA-PB1 interaction inhibitor 54 with oseltamivir carboxylate (OSC), zanamivir (ZA), favipiravir (FPV), and baloxavir marboxil (BXM) on the inhibition of influenza A and B virus replication in vitro. We observed a synergistic effect of the 54/OSC and 54/ZA combinations and an antagonistic effect when 54 was combined with either FPV or BXM. Moreover, we demonstrated the efficacy of 54 against highly pathogenic avian influenza viruses (HPAIVs) both in cell culture and in the embryonated chicken eggs model. Finally, we observed that 54 enhances OSC protective effect against HPAIV replication in the embryonated eggs model. Our findings represent an advance in the development of alternative therapeutic strategies against both human and avian IV infections., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

A green and simple UPLC method was developed and optimized, adopting a factorial design for simultaneous determination of oseltamivir phosphate and remdesivir with dexamethasone as a co-administered drug in human plasma and using daclatasvir dihydrochloride as an internal standard within 5 min. The separation was established on UPLC column BEH C 18 1.7 μm (2.1 × 100.0 mm) connected to UPLC pre-column BEH 1.7 μm (2.1 × 5.0 mm) at 50 °C with an injection volume of 10 μL. The photodiode array detector (PDA) was set at three wavelengths of 220, 315, and 245 nm for oseltamivir phosphate, the internal standard, and both dexamethasone and remdesivir, respectively. The mobile phase consisted of methanol and ammonium acetate solution (40 mM) adjusted to pH 4 in a ratio of 61.5:38.5 (v/v) with a flow rate of 0.25 mL min -1 . The calibration curves were linear over 500.0-5000.0 ng mL -1 for oseltamivir phosphate, over 10.0-500.0 ng mL -1 and 500.0-5000.0 ng mL -1 for dexamethasone, and over 20.0-500 ng mL -1 and 500.0-5000.0 ng mL -1 for remdesivir. The Gibbs free energy and Van't Hoff plots were used to investigate the effect of column oven temperatures on retention times. Fluoride-EDTA anticoagulant showed inhibition activity on the esterase enzyme in plasma. The proposed method was validated according to the M10 ICH, FDA, and EMA's bioanalytical guidelines. According to Eco-score, GAPI, and AGREE criteria, the proposed method was considered acceptable green., (© 2024. The Author(s).)

Inspired by our earlier findings regarding neuraminidase (NA) inhibitors interacting with 150-cavity or 430-cavity of NA, sixteen novel polyheterocyclic NA inhibitors with 1,3,4-oxadiazole thioetheramide as core backbone were designed and synthesized based on the lead compound ZINC13401480. Of the synthesized compounds, compound N5 targeting 150-cavity exerts the best inhibitory activity against the wild-type H5N1 NA, with IC 50 value of 0.14 μM, which is superior to oseltamivir carboxylate (OSC) (IC 50  = 0.31 μM). Compound N10 targeting 430-cavity exhibits the best activity against the H5N1-H274Y mutant NA. Although the activity of N10 is comparable to that of OSC for wild-type H5N1 inhibition, it is approximately 60-fold more potent than OSC against the H274Y mutant, suggesting that it is not easy for the virus to develop drug resistance and is attractive for drug development. N10 (EC 50  = 0.11 μM) also exhibits excellent antiviral activity against H5N1, which is superior to the positive control OSC (EC 50  = 1.47 μM). Molecular docking study shows that the occupation of aromatic fused rings and oxadiazole moiety at the active site and the extension of the substituted phenyl to the 150-cavity or 430-cavity make great contributions to the good potency of this series of polyheterocyclic NA inhibitors. Some advancements in the discovery of effective target-specific NA inhibitors in this study may offer some assistance in the development of more potent anti-influenza drugs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

To address drug resistance to influenza virus neuraminidase inhibitors (NAIs), a series of novel boron-containing N -substituted oseltamivir derivatives were designed and synthesized to target the 150-cavity of neuraminidase (NA). In NA inhibitory assays, it was found that most of the new compounds exhibited moderate inhibitory potency against the wild-type NAs. Among them, compound 2c bearing 4-(3-boronic acid benzyloxy)benzyl group displayed weaker or slightly improved activities against group-1 NAs (H1N1, H5N1, H5N8 and H5N1-H274Y) compared to that of oseltamivir carboxylate ( OSC ). Encouragingly, 2c showed 4.6 times greater activity than OSC toward H5N1-H274Y NA. Moreover, 2c exerted equivalent or more potent antiviral activities than OSC against H1N1, H5N1 and H5N8. Additionally, 2c demonstrated low cytotoxicity in vitro and no acute toxicity at the dose of 1000 mg/kg in mice. Molecular docking of 2c was employed to provide a possible explanation for the improved anti-H274Y NA activity, which may be due to the formation of key additional hydrogen bonds with surrounding amino acid residues, such as Arg152, Gln136 and Val149. Taken together, 2c appeared to be a promising lead compound for further optimization.

Encouraged by our earlier discovery of N1-selective inhibitors, the 150-cavity of influenza virus neuraminidases (NAs) could be further exploited to yield more potent oseltamivir derivatives. Herein, we report the design, synthesis and biological evaluation of a series of novel oseltamivir derivatives via the structural modifications at C 5 -NH 2 of oseltamivir targeting 150-cavity. Among them, compound 5c bearing 4-(3-methoxybenzyloxy)benzyl group exhibited the most potent activity, which was lower or modestly improved activities than oseltamivir carboxylate (OSC) against N1 (H1N1), N1 (H5N1) and N1 (H5N1-H274Y). Specifically, there was 30-fold loss of activity against the wild-type strain H1N1. However, 5c displayed 4.85-fold more potent activity than OSC against H5N1-H274Y NA. Also, 5c demonstrated low cytotoxicity in vitro and no acute toxicity in mice. Molecular docking studies provided insights into the high potency of 5c against N1 and N1-H274Y mutant NAs. Besides, the in silico prediction of physicochemical properties and CYP enzymatic inhibitory ability of representative compounds were conducted to evaluate their drug-like properties., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier Masson SAS. All rights reserved.)

MHAA4549A is a human anti-influenza A monoclonal antibody developed to treat influenza A. We report MHAA4549A serum, nasopharyngeal, and tracheal aspirate pharmacokinetics from a phase 2b study in hospitalized patients with severe influenza A. Patients were randomized 1:1:1 into 3 groups receiving single intravenous doses of 3600 mg (n = 55) or 8400 mg (n = 47) MHAA4549A or placebo (n = 56). Patients also received oral oseltamivir twice daily for ≥5 days. Serum, nasopharyngeal, and tracheal aspirate pharmacokinetic samples were collected on days 1-60 from MHAA4549A-treated groups. Day 5 plasma samples from all groups were collected for assessing the pharmacokinetics of oseltamivir and its active metabolite, oseltamivir carboxylate. Noncompartmental pharmacokinetic analysis was performed using Phoenix WinNonlin. Data were collected during a preplanned interim analysis that became final when the trial terminated because of a lack of efficacy. Serum MHAA4549A concentrations were dose-proportional and biphasic. Mean MHAA4549A clearance was 288-350 mL/day, and mean half-life was 17.8-19.0 days. Nasopharyngeal MHAA4549A concentrations were non-dose-proportional. We detected MHAA4549A in tracheal aspirate samples, but intersubject variability was high. MHAA4549A serum and nasopharyngeal exposures were confirmed in all MHAA4549A-treated patients. Serum MHAA4549A had faster clearance and a shorter half-life in influenza A-infected patients compared with healthy subjects. MHAA4549A detection in tracheal aspirate samples indicated exposure in the lower respiratory tract. Oseltamivir and oseltamivir carboxylate exposures were similar between MHAA4549A-treated and placebo groups, suggesting a lack of MHAA4549A interference with oseltamivir pharmacokinetics., (© 2020 Genentech, Inc. The Journal of Clinical Pharmacology published by Wiley Periodicals LLC on behalf of American College of Clinical Pharmacology.)

Influenza A and B viruses cause seasonal worldwide influenza epidemics each winter, and are a major public health concern and cause of morbidity and mortality. A substantial reduction in influenza-related deaths can be attributed to both vaccination and administration of oseltamivir (OS), which is approved for oral administration and inhibits viral neuraminidase (NA), a transmembrane protein. OS carboxylate (OSC), the active form of OS, is formed by the action of endogenous esterase, which targets NA and is shown to significantly reduce influenza-related deaths. However, the development of resistance in various viral variants, including H3N2 and H5N1, has raised concern about the effectiveness of OS. This comprehensive review covers a range of OS analogs shown to be effective against influenza virus, comparing different types of substituent group that contribute to the activity and bioavailability of these compounds., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

A mechanistic population-pharmacokinetic model was developed to predict oseltamivir exposures in neonates and infants accounting for physiological changes during the first 2 years of life. The model included data from 13 studies, comprising 436 subjects with normal renal function (317 pediatric subjects (≥ 38 weeks postmenstrual age (PMA), ≥ 13 days old) and 119 adult subjects < 40 years). Concentration-time profiles of oseltamivir and its active metabolite, oseltamivir carboxylate (OC), were characterized by a four-compartment model, with absorption described by three additional compartments. Renal maturational changes were implemented by description of OC clearance with allometric function of weight and Hill function of PMA. Clearance of OC increased with weight up to 43 kg (allometric coefficient 0.75). Half the adult OC clearance was reached at a PMA of 45.6 weeks (95% confidence interval (CI) 41.6-49.6) with a Hill coefficient of 2.35 (95% CI 1.67-3.04). The model supports the European Union/United States-approved 3 mg/kg twice-daily oseltamivir dose for infants < 1 year (PMA ≥ 38 weeks) and allows prediction of exposures in preterm neonates., (© 2020 The Authors. Clinical Pharmacology & Therapeutics published by Wiley Periodicals, Inc. on behalf of American Society for Clinical Pharmacology and Therapeutics.)

Among any drugs, no comparative pharmacological study on how prodrug and its active metabolite behave in animal bodies is available. Immunohistochemistry (IHCs) using newly prepared two monoclonal antibodies, AOS-96 and AOC-160, monospecific for oseltamivir (OS) and its metabolite oseltamivir carboxylate (OC) were developed, simultaneously detecting the uptake or excretion of OS and OC in the intestine, liver, and kidney of rats to which OS was orally administered. In the intestine, IHC for OS revealed OS highly distributed to the absorptive epithelia with heavily stained cytoplasmic small granules (CSGs). IHC for OC showed that OC also distributed highly in the epithelia, but without CSGs, suggesting that OS was partly converted to OC in the cells. In the liver, OS distributed in the hepatocytes and on their bile capillaries, as well as on the lumina from the bile capillaries to the interlobular bile ducts. OC distributed in the whole cell of the hepatocytes, but without CSGs nor on any lumina through the interlobular bile ducts. In the kidney, a few levels of OS distributed in the cytoplasm of almost all the renal tubule cells, but they contained numerous CSGs. In contrast, OC distributed highly in the proximal tubules, but very slightly in the lower renal tubules of the nephrons. Thus, it was concluded that the two drugs behave in completely different ways in rat bodies. This paper also discusses a possibility of the correlation of OS or OC levels in tissue cells with their known transporters., (© 2020 The Authors. Pharmacology Research & Perspectives published by John Wiley & Sons Ltd, British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics.)

Oseltamivir is an antiviral drug approved to treat influenza in humans. Although the dosing regimen of this drug is well established for non-pregnant patients, it is not clear if the significant physiological alterations associated with pregnancy affect the pharmacokinetics of oseltamivir and, thus, warrant different dosing regimens to assure efficacy. In this study, we investigated the suitability of rhesus macaques as an animal model for studying oseltamivir pharmacokinetics during all trimesters of pregnancy in comparison to pre-pregnant conditions. Specifically, we compared the pharmacokinetics of oseltamivir and its pharmacologically active metabolite oseltamivir carboxylate in rhesus monkeys after intravenous and nasogastric administration of 2.5 mg oseltamivir phosphate/kg body weight given prior to and during the first, second, and third trimesters of pregnancy. Pregnancy had only a modest effect upon the pharmacokinetic parameters of oseltamivir and oseltamivir carboxylate. Monkeys treated intravenously in the third trimester had a reduction in Vd and CL, compared to non-pregnant monkeys. These changes did not occur in the other two trimesters. Pregnant monkeys treated intravenously had 20-25% decrease in AUC 0-∞ of oseltamivir carboxylate and a corresponding increase in Vd and CL. Pregnant monkeys treated nasogastrically with oseltamivir phosphate demonstrated a pattern that recapitulated intravenous dosing. Taken together these data indicate that rhesus monkeys are an acceptable model for studying drug-pregnancy interactions., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier Inc.)

Glycosphingolipids (GSLs) hexosylceramides and lactosylceramides are elevated in lupus mice and human patients with nephritis. Whereas other renal diseases characterized by increased GSL levels are thought to be a result of upregulated GSL synthesis, our results suggest elevated hexosylceramides and lactosylceramides in lupus nephritis is a result of increased catabolism of ganglioside GM3 due to significantly increased neuraminidase (NEU) activity. Thus, we hypothesized GM3 would be decreased in lupus nephritis kidneys and blocking NEU activity would reduce GSLs and improve disease in lupus mice. Female MRL/lpr lupus mice were treated with water or the NEU inhibitor oseltamivir phosphate at the onset of proteinuria to block GSL catabolism. Age-matched (non-nephritic) female MRL/MpJ lupus mice served as controls. Renal GM3 levels were significantly higher in the nephritic MRL/lpr water-treated mice compared to non-nephritic MRL/MpJ mice, despite significantly increased renal NEU activity. Blocking GSL catabolism increased, rather than decreased, renal and urine GSL levels and disease was not significantly impacted. A pilot study treating MRL/lpr females with GlcCer synthase inhibitor Genz-667161 to block GSL synthesis resulted in a strong significant negative correlation between Genz-667161 dose and renal GSL hexosylceramide and GM3 levels. Splenomegaly was negatively correlated and serum IgG levels were marginally correlated with increasing Genz-667161 dose. These results suggest accumulation of renal GM3 may be due to dysregulation of one or more of the GSL ganglioside pathways and inhibiting GSL synthesis, but not catabolism, may be a therapeutic approach for treating lupus nephritis., Competing Interests: The authors have no financial or commercial conflicts of interest with regards to the presented studies. Sanofi Genzyme Corporation (Waltham, MA) provided the glucosylceramide synthase inhibitor Genz-667161. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Oseltamivir is a neuraminidase inhibitor widely used to treat and prevent influenza A and B infections, although its safety and pharmacokinetics have not been evaluated in patients with severe hepatic impairment. A physiologically based pharmacokinetic (PBPK) model of the prodrug oseltamivir and its active metabolite, oseltamivir carboxylate (OC), was established and validated to simulate their disposition in adults and predict the exposure in patients with Child-Pugh C cirrhosis (CP-C). The simulated results from PBPK modeling and the observed data after oral administration of various oseltamivir regimens were consistent according to the fold error values of less than 2. Furthermore, the clinical observations published in the literature were comparable with our pharmacokinetic predictions. In patients with CP-C, the oseltamivir C max was approximately 2-fold increased, and its AUC was approximately 6-fold higher compared with those in normal subjects. In contrast, the AUC of OC in CP-C patients did not differ significantly from that in normal subjects, whereas its C max was reduced by approximately 30% in the patients. Examination of drug exposure in different health conditions indicated that the oseltamivir exposure was significantly increased in conditions with elevated cirrhosis severity, which might be associated with a higher risk of adverse drug effects, e.g., neuropsychiatric adverse events (NPAEs). In conclusion, the pharmacokinetics of oseltamivir and OC were correctly predicted by PBPK modeling. The model further predicted that the pharmacokinetics of oseltamivir might be altered in liver cirrhosis, depending on the degree of severity.

Encouraged by our earlier discovery of neuraminidase inhibitors targeting 150-cavity or 430-cavity, herein, to yield more potent inhibitors, we designed, synthesized, and biologically evaluated a series of novel oseltamivir derivatives via modification of C-1 and C5-NH 2 of oseltamivir by exploiting 150-cavity and/or 430-cavity. Among the synthesized compounds, compound 15e, the most potent N1-selective inhibitor targeting 150-cavity, showed 1.5 and 1.8 times greater activity than oseltamivir carboxylate (OSC) against N1 (H5N1) and N1 (H5N1-H274Y). In cellular assays, 15e also exhibited greater potency than OSC against H5N1 with EC 50 of 0.66 μM. In addition, 15e demonstrated low cytotoxicity in vitro and low acute toxicity in mice. Molecular docking studies provided insights into the high potency of 15e against N1 and N1-H274Y mutant NA. Overall, we envisioned that the significant breakthrough in the discovery of potent group-1-specific neuraminidase inhibitors may lead to further investigation of more potent anti-influenza agents., (Copyright © 2019 Elsevier Masson SAS. All rights reserved.)

A series of NH 2 -sulfonyl oseltamivir analogues were designed, synthesized, and their inhibitory activities against neuraminidase from H5N1 subtype evaluated. The results indicated that the IC 50 value of compound 4a , an oseltamivir analogue via methyl sulfonylation of C5- NH 2 , was 3.50 μM. Molecular docking simulations suggested that 4a retained most of the interactions formed by oseltamivir carboxylate moieties and formed an additional hydrogen bond with the methylsulfonyl group. Meanwhile, 4a showed high stability towards human liver microsomes. More importantly, 4a without basic moieties is not a zwitterion as reported on the general structure of neuraminidase inhibitors. This research will provide valuable reference for the research of new types of neuraminidase inhibitors.

The aim of this study was to investigate the pharmacokinetics of oseltamivir phosphate, a prodrug, and its active moiety in plasma and lung after its nebulization and intravenous administration in rats. Only 2% of prodrug was converted into active moiety presystematically, attesting to a low advantage of oseltamivir phosphate nebulization, suggesting that oseltamivir phosphate nebulization is not a good option to obtain a high exposure of the active moiety at the infection site within lung., (Copyright © 2019 American Society for Microbiology.)

We present a rapid Raman assay for on-site analysis of stockpiled drugs in aqueous solution. This approach was tested on Tamiflu (oseltamivir phosphate). Tamiflu is a drug approved by the FDA for treatment of influenza and is the most common antiviral included in stockpiles for use in the event of a national emergency. Rapid assays were performed on three concentrations (30, 45, and 75 mg) of oseltamivir using three different portable & handheld Raman instruments. PLS regression models were developed to establish a calibration curve and applied to the Tamiflu samples. Raman assay values were compared against the standard HPLC assay to demonstrate the viability of this approach, yielding an average assay value within 0.3% of that obtained from the HPLC analysis for the 35 different capsules analyzed. The Raman method demonstrates the potential for rapid screening of stockpiled pharmaceuticals on-site using portable Raman instrumentation and readily available consumables for sample preparation. In addition to routine screening to ensure product quality past the expiration date, this approach could also be used to assist in rapid deployment of such medications in the case of a national emergency., (Published by Elsevier B.V.)

Influenza neuraminidase is responsible for the escape of new viral particles from the infected cell surface. Several neuraminidase inhibitors are used clinically to treat patients or stockpiled for emergencies. However, the increasing development of viral resistance against approved inhibitors has underscored the need for the development of new antivirals effective against resistant influenza strains. A facile, sensitive, and inexpensive screening method would help achieve this goal. Recently, we described a multiwell plate-based DNA-linked inhibitor antibody assay (DIANA). This highly sensitive method can quantify femtomolar concentrations of enzymes. DIANA also has been applied to high-throughput enzyme inhibitor screening, allowing the evaluation of inhibition constants from a single inhibitor concentration. Here, we report the design, synthesis, and structural characterization of a tamiphosphor derivative linked to a reporter DNA oligonucleotide for the development of a DIANA-type assay to screen potential influenza neuraminidase inhibitors. The neuraminidase is first captured by an immobilized antibody, and the test compound competes for binding to the enzyme with the oligo-linked detection probe, which is then quantified by qPCR. We validated this novel assay by comparing it with the standard fluorometric assay and demonstrated its usefulness for sensitive neuraminidase detection as well as high-throughput screening of potential new neuraminidase inhibitors., (© 2018 The Author(s).)

In this study, a series of carboxyl-modified oseltamivir analogs with improved lipophilicity were designed and synthesized, and their inhibitory activities against neuraminidase from influenza A virus H5N1 subtype were evaluated. The results demonstrated that compound 5m exhibited potent inhibitory activity (IC 50  = 1.30 ± 0.23 μM), and it targeted the recently discovered 430-cavity. Compound 5m (LogD = -0.12) is more lipophilic than oseltamivir carboxylate (LogD = -1.69) at pH 7.4, which is potentially propitious to improved membrane permeability and oral drug absorption. Meanwhile, 5m showed high stability in human liver microsomes. The findings of this study can be valuable in identifying neuraminidase inhibitors with optimal lipophilicity and in the exploration of 430-cavity., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Aims: The aim of this study was to evaluate the drug-drug interaction between pimodivir, a novel, non-nucleoside polymerase basic protein 2 (PB2) subunit inhibitor of the influenza A virus polymerase complex, and oseltamivir, to assess the feasibility of this combination therapy. Furthermore, single- and multiple-dose pharmacokinetics and safety of pimodivir in healthy volunteers were assessed., Methods: In Part 1 of this open-label Phase 1 study, healthy volunteers (n = 18) were randomized to one of six cross-over treatment sequences, each comprising administration of oseltamivir 75 mg or pimodivir 600 mg or combination thereof twice daily on Days 1-4, followed by a single morning dose on Day 5. Between each treatment session, there was a minimum 5-day washout period. In Part 2, healthy volunteers (n = 16) randomly received pimodivir 600 mg or placebo (3:1) twice daily on Days 1-9, followed by a single morning dose on Day 10. Pharmacokinetics of pimodivir, oseltamivir and oseltamivir carboxylate, and safety were assessed., Results: In Part 1, co-administration of pimodivir with oseltamivir increased the C max of pimodivir by 31% (90% CI: 0.92-1.85) with no change in C min or AUC 12h . Pimodivir had no effect on oseltamivir or oseltamivir carboxylate pharmacokinetics. In Part 2, after single- and multiple-dose administration of pimodivir, there was a 1.2- and 1.8-fold increase in C max and AUC 12h , respectively, between Day 1 and Day 10. The most frequently reported treatment-emergent adverse event was diarrhoea (n = 7 each in Part 1 and 2)., Conclusion: Combination treatment with pimodivir and oseltamivir in healthy volunteers showed no clinically relevant drug-drug interactions. No safety concerns were identified with pimodivir 600 mg twice daily alone or in combination with oseltamivir 75 mg twice daily., (© 2018 The British Pharmacological Society.)

We report a turn-on tetravalent sialic acid-coated tetraphenylethene luminogen (TPE4S) with excellent hydrophilicity, good stability, high sensitivity and unique selectivity towards sialidases, and the maximum fluorescence enhancement was ∼40 fold. More importantly, TPE4S was successfully utilized for the screening of sialidase inhibitors and diagnosis of bacterial vaginosis.

On the basis of our earlier discovery of N1-selective inhibitors, the 150-cavity of influenza virus neuraminidases (NAs) could be further exploited to yield more potent oseltamivir derivatives. Among the synthesized compounds, 15b and 15c were exceptionally active against both group-1 and -2 NAs. Especially for 09N1, N2, N6, and N9 subtypes, they showed 6.80-12.47 and 1.20-3.94 times greater activity than oseltamivir carboxylate (OSC). They also showed greater inhibitory activity than OSC toward H274Y and E119V variant. In cellular assays, they exhibited greater potency than OSC toward H5N1, H5N2, H5N6, and H5N8 viruses. 15b demonstrated high metabolic stability, low cytotoxicity in vitro, and low acute toxicity in mice. Computational modeling and molecular dynamics studies provided insights into the role of R group of 15b in improving potency toward group-1 and -2 NAs. We believe the successful exploitation of the 150-cavity of NAs represents an important breakthrough in the development of more potent anti-influenza agents.

Oseltamivir phosphate (OP) is the first line therapy for influenza, and its primary metabolite oseltamivir carboxylate (OC) is the active agent via inhibition of neuraminidase of influenza virus. Dosages of OP and OC might affect human causing nausea and vomiting and it is therefore necessary to evaluate their toxicity and safety. The separation system: liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a powerful technique to monitor OP and OC. However, quantification of OP and OC needs isotopic analogs as internal standards to monitor the stability of the sample pretreatment procedures and instruments. In this study, we demonstrated a modified method (i.e., reductive amination) to synthesize OP and OC deuterated and hydrogenated analogs as internal standards (ISs) and for illustration of calibration curves, respectively. This modification allowed to overcome ISs selection and to enhance the signal intensities via high yield reductive amination in MS detection. We utilized the multiple reaction monitoring (MRM) mode to target m/z values of precursor and product ions. N-dimethylated OP and N-dimethylated OC showed linearity ranging from 1 to 1000 ng/mL with coefficient of determination (R 2 ) values of 0.9995 and 0.9999, respectively. Additionally, the relative standard deviations (RSD) of intra-day ranged from 0.3% to 5.2%, and the RSD of inter-day ranged from 2.0% to 18.8%, respectively. This quantitative method utilized spiked OP and OC at low (20 ng/mL), intermediate (100 ng/mL), and high (500 ng/mL) concentrations in human serum samples. The average recoveries for OP and OC were 84.6%-107.7% and 94.9%-98.5%, respectively., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Neuraminidase (NA) is one of the particular potential targets for novel antiviral therapy. In this work, a series of neuraminidase inhibitors with the cyclohexene scaffold were studied based upon the combination of 3D-QSAR, molecular docking, and molecular dynamics techniques. The results indicate that the built 3D-QSAR models yield reliable statistical information: the correlation coefficient (r 2 ) and cross-validation coefficient (q 2 ) of CoMFA (comparative molecular field analysis) are 0.992 and 0.819; the r 2 and q 2 of CoMSIA (comparative molecular similarity analysis) are 0.992 and 0.863, respectively. Molecular docking and MD simulations were conducted to confirm the detailed binding mode of enzyme-inhibitor system. The new NA inhibitors had been designed, synthesized, and their inhibitory activities against group-1 neuraminidase were determined. One agent displayed excellent neuraminidase inhibition, with IC 50 value of 39.6 μM against NA, while IC 50 value for oseltamivir is 61.1 μM. This compound may be further investigated for the treatment of infection by the new type influenza virus., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Several subtypes of avian influenza viruses (AIVs) are emerging as novel human pathogens, and the frequency of related infections has increased in recent years. Although neuraminidase (NA) inhibitors (NAIs) are the only class of antiviral drugs available for therapeutic intervention for AIV-infected patients, studies on NAI resistance among AIVs have been limited, and markers of resistance are poorly understood. Previously, we identified unique NAI resistance substitutions in AIVs of the N3, N7, and N9 NA subtypes. Here, we report profiles of NA substitutions that confer NAI resistance in AIVs of the N4, N5, N6, and N8 NA subtypes using gene-fragmented random mutagenesis. We generated libraries of mutant influenza viruses using reverse genetics (RG) and selected resistant variants in the presence of the NAIs oseltamivir carboxylate and zanamivir in MDCK cells. In addition, two substitutions, H274Y and R292K (N2 numbering), were introduced into each NA gene for comparison. We identified 37 amino acid substitutions within the NA gene, 16 of which (4 in N4, 4 in N5, 4 in N6, and 4 in N8) conferred resistance to NAIs (oseltamivir carboxylate, zanamivir, or peramivir) as determined using a fluorescence-based NA inhibition assay. Substitutions conferring NAI resistance were mainly categorized as either novel NA subtype specific (G/N147V/I, A246V, and I427L) or previously reported in other subtypes (E119A/D/V, Q136K, E276D, R292K, and R371K). Our results demonstrate that each NA subtype possesses unique NAI resistance markers, and knowledge of these substitutions in AIVs is important in facilitating antiviral susceptibility monitoring of NAI resistance in AIVs. IMPORTANCE The frequency of human infections with avian influenza viruses (AIVs) has increased in recent years. Despite the availability of vaccines, neuraminidase inhibitors (NAIs), as the only available class of drugs for AIVs in humans, have been constantly used for treatment, leading to the inevitable emergence of drug-resistant variants. To screen for substitutions conferring NAI resistance in AIVs of N4, N5, N6, and N8 NA subtypes, random mutations within the target gene were generated, and resistant viruses were selected from mutant libraries in the presence of individual drugs. We identified 16 NA substitutions conferring NAI resistance in the tested AIV subtypes; some are novel and subtype specific, and others have been previously reported in other subtypes. Our findings will contribute to an increased and more comprehensive understanding of the mechanisms of NAI-induced inhibition of influenza virus and help lead to the development of drugs that bind to alternative interaction motifs., (Copyright © 2017 American Society for Microbiology.)

Influenza viruses are major human respiratory pathogens that cause high morbidity and mortality worldwide. Currently, prophylactic vaccines and therapeutic antiviral agents are used to prevent and control influenza virus infection. Oseltamivir free base (OSV-FB), a modified generic antiviral drug of Tamiflu (oseltamivir phosphate, OSV-P), was launched in the Republic of Korea last year. Here, we examine the bioequivalence of these two compounds by assessing their antiviral efficacy in infected cells and in a mouse model. It was observed that both antivirals showed comparable efficacy against 11 different influenza A and B viruses in vitro. Moreover, in mice infected with influenza A virus (mouse-adapted A/Puerto Rico/8/34), they showed a dose-dependent therapeutic activity and alleviated infection-mediated reductions in body weight, leading to significantly better survival. There was histopathological disappearance of virus-induced inflammatory cell infiltration of the lung after oral treatment with either antiviral agent (at 10 mg/kg). Pharmacokinetic analysis also exhibited similar plasma concentrations of the active drug, oseltamivir carboxylate, metabolised from both OSV-B and OSV-P. This is the first report showing bioequivalence of OSV-FB to its phosphate salt form in the mouse system. The free base drug has some beneficial points including simple drug formulation process and reduced risk of undesirable cation-phosphate precipitation within solution. The long term stability of OSV-FB requires further monitoring when it is provided as a national stock in readiness for an influenza pandemic.

Neuraminidaseis a key enzyme in the life cycle of influenza viruses and is present in some bacterial pathogens. We here assess the inhibitory potency of plant tannins versus clinically used inhibitors on both a viral and a bacterial model neuraminidase by applying the 2'-(4-methylumbelliferyl)-α-d- N -acetylneuraminic acid (MUNANA)-based activity assay. A range of flavan-3-ols, ellagitannins and chemically defined proanthocyanidin fractions was evaluated in comparison to oseltamivir carboxylate and zanamivir for their inhibitory activities against viral influenza A (H1N1) and bacterial Vibrio cholerae neuraminidase (VCNA). Compared to the positive controls, all tested polyphenols displayed a weak inhibition of the viral enzyme but similar or even higher potency on the bacterial neuraminidase. Structure-activity relationship analyses revealed the presence of galloyl groups and the hydroxylation pattern of the flavan skeleton to be crucial for inhibitory activity. The combination of zanamivir and EPs ® 7630 (root extract of Pelargonium sidoides ) showed synergistic inhibitory effects on the bacterial neuraminidase. Co-crystal structures of VCNA with oseltamivir carboxylate and zanamivir provided insight into bacterial versus viral enzyme-inhibitor interactions. The current data clearly indicate that inhibitor potency strongly depends on the biological origin of the enzyme and that results are not readily transferable. The therapeutic relevance of our findings is briefly discussed., Competing Interests: The authors declare no conflict of interest.

Background: The aim of this study was to compare the bioavailability of a new generic formulation of oseltamivir 75-mg capsule (test) and a branded formulation Tamiflu ® (reference) to meet regulatory criteria for marketing the test product in healthy Chinese male volunteers., Methods: This single-dose, randomized-sequence, open-label, two-period crossover study was conducted in fasted healthy Chinese male volunteers, who first received a single oral dose of the test or reference formulation with a 7-day washout period, and then the alternative formulation. The study drug was administered after a 10-hour overnight fast. Blood samples were collected at baseline and at 0.25, 0.5, 0.75, 1.0, 1.5, 2, 4, 6, 8, 10, 12, 24, and 36 hours after administration of the study drug. Plasma concentrations of the parent oseltamivir and its metabolite oseltamivir carboxylate were determined using an LC-MS/MS method. The formulations were considered bioequivalent if the 90% confidence intervals (CIs) for the log-transformed values were within the predetermined equivalence range (70 - 143% for C max , 80 - 125% for AUC) according to the guidelines of the State Food and Drug Administration of China. Adverse events (AEs) were monitored throughout the study based on clinical parameters and patient reports., Results: Characteristics of the 20 male volunteers included were as follows: mean age 23 (± 0.7, SD) years (range 21 - 24 years); weight 69 (± 7.1) kg (range 60 - 88 kg); height 177 (± 5.9) cm (range 168 - 192 cm). All included subjects completed the study. The mean geometric ratio between the test and reference formulations of oseltamivir was 99.5% (90% CI), 86.3 - 114.8%) for C max , 104.4% (95.7 - 113.9%) for AUC 0-t , and 104.4% (95.6 - 113.9%) for AUC 0-∞. That of oseltamivir carboxylate was 103.7% (90% CI, 95.3 - 112.8%) for C max , 101.7% (96.6 - 107.1%) for AUC 0-t , and 101.4% (96.5 - 106.5%) for AUC 0-∞ . There was no significant difference in pharmacokinetic parameters between the two groups. Only 1 AE (nausea) occurred in 1 subject who received the test formulation; the AE resolved without any treatment., Conclusions: The result of this single-dose study indicated that the test formulation of oseltamivir capsule met the Chinese regulatory criteria for bioequivalence vs. the reference formulation in fasted healthy Chinese male volunteers.
.

Inhibition of influenza A virus infection by multivalent sialic acid inhibitors preventing viral hemagglutinin binding to host cells of the respiratory tract is a promising strategy. However, optimal geometry and optimal ligand presentation on multivalent scaffolds for efficient inhibition both in vitro and in vivo application are still unclear. Here, by comparing linear and dendritic polyglycerol sialosides (LPGSA and dPGSA) we identified architectural requirements and optimal ligand densities for an efficient multivalent inhibitor of influenza virus A/X31/1 (H3N2). Due to its large volume, the LPGSA at optimal ligand density sterically shielded the virus significantly better than the dendritic analog. A statistical mechanics model rationalizes the relevance of ligand density, morphology, and the size of multivalent scaffolds for the potential to inhibit virus-cell binding. Optimized LPGSA inhibited virus infection at IC 50 in the low nanomolar nanoparticle concentration range and also showed potent antiviral activity against two avian influenza strains A/Mallard/439/2004 (H3N2) and A/turkey/Italy/472/1999 (H7N1) post infection. In vivo application of inhibitors clearly confirmed the higher inhibition potential of linear multivalent scaffolds to prevent infection. The optimized LPGSA did not show any acute toxicity, and was much more potent than the neuraminidase inhibitor oseltamivir carboxylate in vivo. Combined application of the LPGSA and oseltamivir carboxylate revealed a synergistic inhibitory effect and successfully prevented influenza virus infection in mice., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Combination therapies against multiple targets are currently being developed to prevent resistance to a single chemotherapeutic agent and to extirpate pre-existing resistance in heterogeneous cancer cells in tumors due to selective pressure from the single agent. Gemcitabine (GEM), a chemotherapeutic agent, is the current standard of care for patients with pancreatic cancer. Patients with pancreatic cancer receiving GEM have a low progression-free survival. Given the poor response rate to GEM, cancer cells are known to develop rapid resistance to this drug. Metronomic chemotherapy using combinatorial and sequential delivery systems are novel developmental approaches to disrupt tumor neovascularization, reduce systemic drug toxicity, and increase the sensitivity of chemotherapeutics in cancer. Here, implantable double-layered poly(d,l-lactic-co-glycolic acid) (PLGA) cylinders were engineered to sequentially release GEM in combination with oseltamivir phosphate (OP) over an extended time. Double-layered PLGA cylindrical implants loaded with these active hydrophilic drugs were fabricated with minimal loss of drugs during the formulation, enabling extensive control of drug loading and establishing uniform drug distribution throughout the polymer matrix. OP is used in the formulation because of its anticancer drug properties targeting mammalian neuraminidase 1 (Neu1) involved in multistage tumorigenesis. OP and GEM encapsulated in inner/outer GEM in /OP out or OP in /GEM out implantable double-layered PLGA cylinders displayed sustained near linear release over 30 days. OP and GEM released from the double-layered PLGA cylinders effectively reduced cell viability in pancreatic cancer cell line PANC1 and its GEM-resistant variant for up to 15 days., Competing Interests: Disclosure The authors report no conflicts of interest in this work.

Objective: The objective of the study was to determine transplacental of oseltamivir phosphate (OP) and its active metabolite oseltamivir carboxylate (OC) using the ex vivo human placental perfusion model in Chinese Hans population., Study Design: Perfusion studies were performed on 20 placentas from healthy term pregnancies. Concentrations typical for 75 mg, twice-daily oral dose were tested (OP 65.2 ng/ml and OC 348 ng/ml), along with the positive control antipyrine 0.1 mg/ml. Each perfusion experiment was conducted for 180 min while samples were taken from both maternal and fetal compartments. Concentrations of OP and its metabolite OC were determined by ultrafast-performance liquid chromatography/tandem mass spectrometry. Integrity and viability of the placenta were determined by measuring fetal volume loss, pH, pO 2 , ΔhCG, glucose consumption and lactate production during the perfusion experiments., Results: Following 3 h of perfusion, the fetal transfer rates of OP and its metabolite OC were 12.39%±3.26%, 10.17%±2.03%, respectively. The clearance indexes of OP and OC were 0.36 ± 0.11 and 0.29 ± 0.06, respectively., Conclusion: These data suggest that OP and its metabolite OC pass through the healthy term placenta at a small amount according to the ex vivo human placental perfusion model, fetal exposure must be considered when treating pregnant women with OP.

In search of novel anti-influenza agents with higher potency, a series of acylguanidine oseltamivir carboxylate analogues were synthesized and evaluated against influenza viruses (H1N1 and H3N2) in vitro. The representative compounds with strong inhibitory activities (IC 50 <40nM) against neuraminidase (NA) were further tested against the NA from oseltamivir-resistant strain (H259Y). Among them, compounds 9 and 17 were potent NA inhibitors that exhibited a 5 and 11-fold increase in activity comparing with oseltamivir carboxylate (2, OC) against the H259Y mutant, respectively. Furthermore, the effect against influenza virus H259Y mutant (H1N1) replication and cytotoxicity assays indicated that compounds 9 and 17 exhibited a 20 and 6-fold increase than the parent compound 2, and had no obvious cytotoxicity in vitro. Moreover, the molecular docking studies revealed that the docking modes of compounds 9 and 17 were different from that of oseltamivir, and the new hydrogen bonds and hydrophobic interaction were formed in this case. This work provided unique insights in the discovery of potent inhibitors against NAs from wild-type and oseltamivir-resistant strains., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Background: Human carboxylesterase 1 (CES1) is a serine esterase that hydrolyses various exogenous and endogenous compounds including oseltamivir, a prodrug used to treat influenza. A novel CES1 c.662A>G single nucleotide polymorphism (SNP) was predicted to decrease CES1 enzymatic activity in an in silico analysis. This study evaluated the effect of the c.662A>G SNP on the pharmacokinetics (PK) of oseltamivir in humans., Methods: A single oral dose of oseltamivir at 75 mg was administered to 20 healthy subjects, 8 heterozygous c.662A>G carriers (c.662AG) and 12 non-carriers (c.662AA). The concentrations of oseltamivir and its active metabolite, oseltamivir carboxylate, were measured in plasma and urine using a validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. The PK parameters were calculated using a noncompartmental method. The geometric mean ratios (GMR, c.662AG to c.662AA) of the PK parameters and their 90% confidence intervals (CI) were calculated., Results: The systemic exposure to oseltamivir, as assessed by the AUC0-48h of oseltamivir, was increased by 10% in c.662AG subjects, whereas the AUC0-48h of oseltamivir carboxylate was 5% lower in c.662AG subjects. The GMR and 90% CI of the metabolic ratio (AUC0-48h, Oseltamivir carboxylate/AUC0-48h, Oseltamivir) was 0.87 (0.66-1.14). The amount of unchanged oseltamivir excreted in the urine was increased by 15% in subjects with the c.662AG genotype., Conclusions: This result suggests that CES1 enzymatic activity may be decreased in these heterozygous allele carriers, although further studies are warranted to investigate the clinical implications of this genetic variation on CES1 substrate drugs., Trial Registration: ClinicalTtrials.gov NCT01902342.

Influenza virus causes severe respiratory infections that are responsible for up to half a million deaths worldwide each year. Two inhibitors targeting viral neuraminidase have been approved to date (oseltamivir, zanamivir). However, the rapid development of antiviral drug resistance and the efficient transmission of resistant viruses among humans represent serious threats to public health. The approved influenza neuraminidase inhibitors have (oxa)cyclohexene scaffolds designed to mimic the oxonium transition state during enzymatic cleavage of sialic acid. Their active forms contain a carboxylate that interacts with three arginine residues in the enzyme active site. Recently, the phosphonate group was successfully used as an isostere of the carboxylate in oseltamivir, and the resulting compound, tamiphosphor, was identified as a highly active neuraminidase inhibitor. However, the structure of the complex of this promising inhibitor with neuraminidase has not yet been reported. Here, we analyzed the interaction of a set of oseltamivir and tamiphosphor derivatives with neuraminidase from the A/California/07/2009 (H1N1) influenza virus. We thermodynamically characterized the binding of oseltamivir carboxylate or tamiphosphor to the neuraminidase catalytic domain by protein microcalorimetry, and we determined crystal structure of the catalytic domain in complex with tamiphosphor at 1.8 Å resolution. This structural information should aid rational design of the next generation of neuraminidase inhibitors., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.)

The aim of this study was to determine the biopharmaceutical characteristics of oseltamivir carboxylate (OC) after pulmonary delivery. After OC bolus and intratracheal nebulization (NEB) in rats, blood was collected and bronchoalveolar lavages (BALs) were performed. Epithelial lining fluid (ELF) concentrations were estimated from BAL fluid. The area under the curve (AUC) ratio for ELF to plasma was 842 times higher after NEB than after intravenous (i.v.) administration, indicating that OC nebulization offers a biopharmaceutical advantage over i.v. administration., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Aims: The aims of the present study were to compare the pharmacokinetics of oseltamivir and its active antiviral metabolite oseltamivir carboxylate in obese and non-obese individuals and to determine the effect of obesity on the pharmacokinetic properties of oseltamivir and oseltamivir carboxylate., Methods: The population pharmacokinetic properties of oseltamivir and oseltamivir carboxylate were evaluated in 12 obese [body mass index (BMI) ≥30 kg m(-2) ) and 12 non-obese (BMI <30 kg m(-2) ) Thai adult volunteers receiving a standard dose of 75 mg and a double dose of 150 mg in a randomized sequence. Concentration-time data were collected and analysed using nonlinear mixed-effects modelling., Results: The pharmacokinetics of oseltamivir and oseltamivir carboxylate were described simultaneously by first-order absorption, with a one-compartment disposition model for oseltamivir, followed by a metabolism compartment and a one-compartment disposition model for oseltamivir carboxylate. Creatinine clearance was a significant predictor of oseltamivir carboxylate clearance {3.84% increase for each 10 ml min(-1) increase in creatinine clearance [95% confidence interval (CI) 0.178%, 8.02%]}. Obese individuals had an approximately 25% (95% CI 24%, 28%) higher oseltamivir clearance, 20% higher oseltamivir volume of distribution (95% CI 19%, 23%) and 10% higher oseltamivir carboxylate clearance (95% CI 9%, 11%) compared with non-obese individuals. However, these altered pharmacokinetic properties were small and did not change the overall exposure to oseltamivir carboxylate., Conclusions: The results confirmed that a dose adjustment for oseltamivir in obese individuals is not necessary on the basis of its pharmacokinetics., (© 2016 The British Pharmacological Society.)

A target-free and cell-based approach was applied to evaluate the anti-influenza properties of six newly synthesized benzoic acid derivatives. SA-2, the ethyl 4-(2-hydroxymethyl-5-oxopyrrolidin-1-yl)-3-[3-(3-methylbenzoyl)-thioureido] benzoate (compound 2) was screened as a potential drug candidate. In a cytopathic effect assay, SA-2 dose dependently inhibited H1N1, H3N2 and the oseltamivir-resistant mutant H1N1-H275Y influenza viruses in both virus-infected MDCK and A549 cells, with 50% effective concentrations (EC50) in MDCK cells of 9.6, 19.2 and 19.8 μM respectively, and 50% cytotoxic concentration (CC50) of 444.5 μM, showing competitive antiviral activity with oseltamivir in vitro. Orally administered SA-2 effectively protected mice infected with lethal doses of H1N1 or oseltamivir-resistant strain H1N1-H275Y, conferring 70% or 50% survival at a dosage of 100 mg/kg/d, reducing body weight loss, alleviating the influenza-induced acute lung injury, and reducing lung virus titer. Mechanistic studies showed that SA-2 efficiently inhibited the activity of RNA polymerase and suppressed NP and M1 levels during viral biosynthesis by interfering with gene transcription without having an obvious influence on virus entry and release. Based on these favourable findings, SA-2, a novel anti-influenza agent, with its potent anti-influenza activity in vitro and in vivo, could be a promising antiviral for the treatment of infection of influenza A viruses, including oseltamivir-resistant mutants., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The goal of this study was to improve the intestinal mucosal cell membrane permeability of the poorly absorbed guanidino analogue of a neuraminidase inhibitor, oseltamivir carboxylate (GOC) using a carrier-mediated strategy. Valyl amino acid prodrug of GOC with isopropyl-methylene-dioxy linker (GOC-ISP-Val) was evaluated as the potential substrate for intestinal oligopeptide transporter, hPEPT1 in Xenopus laevis oocytes heterologously expressing hPEPT1, and an intestinal mouse perfusion system. The diastereomers of GOC-ISP-Val were assessed for chemical and metabolic stability. Permeability of GOC-ISP-Val was determined in Caco-2 cells and mice. Diastereomer 2 was about 2 times more stable than diastereomer 1 in simulated intestinal fluid and rapidly hydrolyzed to the parent drug in cell homogenates. The prodrug had a 9 times-enhanced apparent permeability (P(app)) in Caco-2 cells compared with the parent drug. Both diastereomer exhibited high effective permeability (P(eff)) in mice, 6.32 ± 3.12 and 5.20 ± 2.81 × 10(-5) cm/s for diastereomer 1 and 2, respectively. GOC-ISP-Val was found to be a substrate of hPEPT1. Overall, this study indicates that the prodrug, GOC-ISP-Val, seems to be a promising oral anti-influenza agent that has sufficient stability at physiologically relevant pHs before absorption, significantly improved permeability via hPEPT1 and potentially rapid activation in the intestinal cells., (Copyright © 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

Aims: Physiological changes in pregnancy are expected to alter the pharmacokinetics of various drugs. The objective of this study was to evaluate systematically the pharmacokinetics of oseltamivir (OS), a drug used in the treatment of influenza during pregnancy., Methods: A multicentre steady-state pharmacokinetic study of OS was performed in 35 non-pregnant and 29 pregnant women. Plasma concentration-time profiles were analyzed using both non-compartmental and population pharmacokinetic modelling (pop PK) and simulation approaches. A one compartment population pharmacokinetic model with first order absorption and elimination adequately described the pharmacokinetics of OS., Results: The systemic exposure of oseltamivir carboxylate (OC, active metabolite of OS) was reduced approximately 30 (19-36)% (P < 0.001) in pregnant women. Pregnancy significantly (P < 0.001) influenced the clearance (CL/F) and volume of distribution (V/F) of OC. Both non-compartmental and population pharmacokinetic approaches documented approximately 45 (23-62)% increase in clearance (CL/F) of OC during pregnancy., Conclusion: Based on the decrease in exposure of the active metabolite, the currently recommended doses of OS may need to be increased modestly in pregnant women in order to achieve comparable exposure with that of non-pregnant women., (© 2015 The British Pharmacological Society.)

End-stage renal disease (ESRD) patients receiving hemodialysis (HD) are at heightened risk for influenza, but the optimal oseltamivir dosage regimen for treating or preventing influenza in this high-risk population is still uncertain. Pharmacokinetic data for 24 adults with ESRD were pooled from a single-dose and a multiple-dose study to develop a population pharmacokinetic model using nonlinear mixed-effects modeling. The final model comprised five compartments, two each to describe the systemic pharmacokinetics of oseltamivir phosphate and its metabolite, oseltamivir carboxylate (OC), and a delay compartment to describe oseltamivir metabolism. Estimated OC clearance in the model was markedly faster during HD sessions (7.43 liters/min) than at other times (0.19 liter/min). Model simulations showed that 30 mg oseltamivir given after every HD session is the most suitable regimen for influenza treatment, producing trough OC concentrations above the median value achieved with the 75-mg twice-daily regimen in patients with normal renal function and peak concentrations below the highest oseltamivir exposures known to be well tolerated (median exposures after twice-daily dosing of 450 mg). Administration of the first dose following diagnosis of influenza need not wait until after the next HD session: addition of a single 30-mg dose during the 12 h before the next HD session raises OC exposures quickly without posing any safety risk. Further simulation showed that 30 mg oseltamivir given after every other HD session is the most suitable regimen for influenza prophylaxis., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Avian influenza (AI) H9N2 viruses are endemic in many bird species, and human infections of H9N2 viruses have been reported. Oseltamivir phosphate (Tamiflu(®)) is the available antiviral drug for the treatment and prophylaxis of influenza. There are no reports of use of embryonated chicken egg as a model to study susceptibility of AI viruses to oseltamivir carboxylate (OC), the active metabolite. The present study was undertaken to explore the use of embryonated chicken eggs as a model for testing OC against the AI H9N2 viruses. A total of three AI H9N2 viruses, isolated in poultry in India, were used. Various virus dilutions were tested against 14μg/ml of OC. Three methods, namely (1) the in vitro virus-drug treatment, (2) drug delivery and virus challenge by allantoic route, and (3) drug delivery by albumen route and virus challenge by allantoic route were explored. The viruses were also tested using the fluorescence-based neuraminidase inhibitor (NAI) assay. There was significant inhibition (p<0.05) of the H9N2 viruses in presence of OC. The infectious virus titers as well as hemagglutination titers were significantly lower in presence of OC as compared to controls. The in vitro treatment of virus and drug; and drug and virus delivery at the same time by allantoic route showed significantly higher inhibition (p<0.05) of virus growth than that by the albumen route. In the NAI assay, the half maximal inhibitory concentration (IC50) values of the H9N2 viruses were within the standard range for known susceptible reference virus. In conclusion, the H9N2 viruses used in the study were susceptible to OC. Embryonated chicken egg could be used as a model to study susceptibility of AI viruses to antiviral drugs., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The ferret is a suitable small animal model for preclinical evaluation of efficacy of antiviral drugs against various influenza strains, including highly pathogenic H5N1 viruses. Rigorous pharmacokinetics/pharmacodynamics (PK/PD) assessment of ferret data has not been conducted, perhaps due to insufficient information on oseltamivir PK. Here, based on PK data from several studies on both uninfected and influenza-infected groups (i.e., with influenza A viruses of H5N1 and H3N2 subtypes and an influenza B virus) and several types of anesthesia we developed a population PK model for the active compound oseltamivir carboxylate (OC) in the ferret. The ferret OC population PK model incorporated delayed first-order input, two-compartment distribution, and first-order elimination to successfully describe OC PK. Influenza infection did not affect model parameters, but anesthesia did. The conclusion that OC PK was not influenced by influenza infection must be viewed with caution because the influenza infections in the studies included here resulted in mild clinical symptoms in terms of temperature, body weight, and activity scores. Monte Carlo simulations were used to determine that administration of a 5.08 mg/kg dose of oseltamivir phosphate to ferret every 12 h for 5 days results in the same median OC area under the plasma concentration-time curve 0-12 h (i.e., 3220 mg h/mL) as that observed in humans during steady state at the approved dose of 75 mg twice daily for 5 days. Modeling indicated that PK variability for OC in the ferret model is high, and can be affected by anesthesia. Therefore, for proper interpretation of PK/PD data, sparse PK sampling to allow the OC PK determination in individual animals is important. Another consideration in appropriate design of PK/PD studies is achieving an influenza infection with pronounced clinical symptoms and efficient virus replication, which will allow adequate evaluation of drug effects.

This investigation evaluated the adsorption behavior of the antiviral drugs of oseltamivir (OE) and its metabolites (i.e., oseltamivir carboxylate (OC)) on three types of carbon nanotubes (CNTs) including single-walled CNT (SWCNT), multi-walled CNT (MWCNT), and carboxylated SWCNT (SWCNT-COOH). CNTs can efficiently remove more than 90% of the OE and OC from aqueous solution when the initial concentration was lower than 10(-4) mmol/L. The Polanyi-Manes model depicted the adsorption isotherms of OE and OC on CNTs better than the Langmuir and Freundlich models. The properties of OE/OC and the characteristics of CNTs, particularly the oxygen functional groups (e.g., SWCNT-COOH) played important roles during the adsorption processes. OE showed a higher adsorption affinity than OC. By comparing the different adsorbates adsorption on each CNT and each adsorbate adsorption on different CNTs, the adsorption mechanisms of hydrophobic interaction, electrostatic interaction, van der Waals force, and H-bonding were proposed as the contributing factors for OE and OC adsorption on CNTs. Particularly, for verifying the contribution of electrostatic interaction, the changes of adsorption partition efficiency (Kd) of OE and OC on CNTs were evaluated by varying pH from 2 to 11 and the importance of isoelectric point (pHIEP) of CNTs on OE and OC adsorption was addressed., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Availability of lower-dose oseltamivir capsules, an increased pharmacokinetic database, and a desire to align drug exposure across the spectrum of renal function prompted reassessment of oral dosing in patients with renal impairment. The data set comprised 128 subjects (71 with varying degrees of renal impairment) from 8 studies, which included single and multiple doses of 20-1000 mg. Pharmacokinetic profiles of oseltamivir phosphate (OP) and oseltamivir carboxylate (OC) were modeled simultaneously in NONMEM. Exposure metrics of OP and OC (AUC48  h , Cmax , Cmin ) after administration of various dosing regimens were simulated for renal impairment subgroups and compared with exposures in patients with normal renal function receiving approved regimens. For influenza treatment, 30 mg once-daily and twice-daily regimens were selected for severe and moderate impairment, respectively. These regimens provided OC exposures similar or above those of the approved 75-mg twice-daily treatment regimen in subjects with normal renal function. For influenza prophylaxis, 30 mg once every other day and once-daily regimens were selected for severe and moderate impairment, respectively. No dosing adjustments were required for mild impairment. This analysis supported revised labeling in the United States and Europe for oral oseltamivir dosing in patients with moderate and severe renal impairment., (© 2015, The American College of Clinical Pharmacology.)

Influenza A virus (IAV) has its natural reservoir in wild waterfowl, and new human IAVs often contain gene segments originating from avian IAVs. Treatment options for severe human influenza are principally restricted to neuraminidase inhibitors (NAIs), among which oseltamivir is stockpiled in preparedness for influenza pandemics. There is evolutionary pressure in the environment for resistance development to oseltamivir in avian IAVs, as the active metabolite oseltamivir carboxylate (OC) passes largely undegraded through sewage treatment to river water where waterfowl reside. In an in vivo mallard (Anas platyrhynchos) model, we tested if low-pathogenic avian influenza A(H7N9) virus might become resistant if the host was exposed to low levels of OC. Ducks were experimentally infected, and OC was added to their water, after which infection and transmission were maintained by successive introductions of uninfected birds. Daily fecal samples were tested for IAV excretion, genotype, and phenotype. Following mallard exposure to 2.5 μg/liter OC, the resistance-related neuraminidase (NA) I222T substitution, was detected within 2 days during the first passage and was found in all viruses sequenced from subsequently introduced ducks. The substitution generated 8-fold and 2.4-fold increases in the 50% inhibitory concentration (IC50) for OC (P < 0.001) and zanamivir (P = 0.016), respectively. We conclude that OC exposure of IAV hosts, in the same concentration magnitude as found in the environment, may result in amino acid substitutions, leading to changed antiviral sensitivity in an IAV subtype that can be highly pathogenic to humans. Prudent use of oseltamivir and resistance surveillance of IAVs in wild birds are warranted., (Copyright © 2015, Gillman et al.)

Abnormal behaviors and death associated with the use of oseltamivir (Tamiflu(®)) have emerged as a major issue in influenza patients. We have previously reported that the mechanisms underlying the effects of caffeine, a non-selective adenosine A1/A2 receptor antagonist, combined with oseltamivir. Oseltamivir is rapidly hydrolyzed to its active form (oseltamivir carboxylate, OCB). In this study, we investigated the effects of an adenosine system and OCB on the action of oseltamivir on mice behavior. Oseltamivir for 1 day (150 mg/kg, intraperitoneally (i.p.)) alone did not affect ambulation at 2 h post-injection. However, caffeine (10 mg/kg, i.p.) in combination with oseltamivir for 1 day increased ambulation. Moreover, caffeine (30 mg/kg, i.p.) in combination with oseltamivir for 3 days increased ambulation, but caffeine (10 mg/kg, i.p.) in combination with oseltamivir for 3 days did not increase. These enhancements were inhibited by an adenosine A2 receptor agonist, CGS21680 (0.2 mg/kg, subcutaneously (s.c.)). Furthermore, an adenosine A2 receptor antagonist, SCH58261 (1 and 3 mg/kg, i.p.) in combination with oseltamivir for 1 day increased ambulation. Moreover, SCH58261 (3 mg/kg, i.p.) in combination with oseltamivir for 3 days increased ambulation, but SCH58261 (1 mg/kg, i.p.) in combination with oseltamivir for 3 days did not. Conversely, in phenobarbital (PB)-treated mice, caffeine (3 mg/kg, i.p.) in combination with oseltamivir for 1 day increased ambulation. Moreover, OCB for 1 day (0.3 μg/mouse intracerebroventricular (i.c.v.)) alone increased ambulation. These findings suggest that the actions of oseltamivir may involve the adenosine systems and its metabolism. Our findings suggest an interaction between the central blockade of adenosine A2 receptors by caffeine and OCB-induced behavioral changes., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

This is the first report of the detection of two new anti-influenza drugs, peramivir (PER) and laninamivir (LAN), in Japanese sewage effluent and river waters. Over about 1 year from October 2013 to July 2014, including the influenza prevalence season in January and February 2014, we monitored for five anti-influenza drugs-oseltamivir (OS), oseltamivir carboxylate (OC), zanamivir (ZAN), PER, and LAN-in river waters and in sewage effluent flowing into urban rivers of the Yodo River system in Japan. The dynamic profiles of these anti-influenza drugs were synchronized well with that of the numbers of influenza patients treated with the drugs. The highest levels in sewage effluents and river waters were, respectively, 82 and 41 ng/L (OS), 347 and 125 ng/L (OC), 110 and 35 ng/L (ZAN), 64 and 11 ng/L (PER), and 21 and 9 ng/L (LAN). However, application of ozone treatment before discharge from sewage treatment plants was effective in reducing the levels of these anti-influenza drugs in effluent. The effectiveness of the ozone treatment and the drug dependent difference in susceptibility against ozone were further evidenced by ozonation of a STP effluent in a batch reactor. These findings should help to promote further environmental risk assessment of the generation of drug-resistant influenza viruses in aquatic environments.

This work characterizes the pharmacokinetics (PK) of oseltamivir phosphate (OP) and its active metabolite, oseltamivir carboxylate (OC), and investigates oseltamivir i.v. dosing regimens for treatment of influenza in patients with normal renal function and with various degrees of renal impairment. Initially, data collected from 149 subjects with normal renal function and mild to severe renal impairment who were administered 40-200 mg oseltamivir i.v. were described by a four-compartment model. Two compartments described OP, one compartment described OC and one compartment described OP to OC metabolism. Then, data of 128 subjects administered 20-1,000 mg oseltamivir orally were added. The absorption model included three first-order processes with direct (via first-pass) input in the OC compartment and two (direct and delayed) inputs in the OP compartment. Simulations and PK bridging were used to recommend i.v. dosing regimens. The analysis demonstrated that renal function had a major effect on OC clearance (CL M ) and exposure. CL M for subjects with mild, moderate and severe renal impairment was 18, 50, and 84 % lower than for subjects with normal renal function. Simulations were used to select i.v. dosing regimens that provide OC Cmin coverage and exposures comparable to those achieved in subjects with normal renal function administered 75 mg b.i.d. orally. The oseltamivir dose depended on the degree of renal impairment and was independent of route of administration. Specifically, 75 mg b.i.d. is recommended for subjects with normal renal function or mild renal impairment, 30 mg b.i.d. for subjects with moderate renal impairment, and 30 mg q.d. for subjects with severe renal impairment. Recommended i.v. doses were the same as those recommended for oral administration in corresponding renal impairment groups.

Background: Tamiflu (oseltamivir phosphate ester, OE) is a widely used antiviral active against influenza A virus. Its active metabolite, oseltamivir carboxylate (OC), is chemically stable and secreted into wastewater treatment plants. OC contamination of natural habitats of waterfowl might induce OC resistance in influenza viruses persistently infecting waterfowl, and lead to transfer of OC-resistance from avian to human influenza. The aim of this study was to evaluate whether such has occurred., Methods: A genomics approach including phylogenetic analysis and probability calculations for homologous recombination was applied on altogether 19,755 neuraminidase (N1 and N2) genes from virus sampled in humans and birds, with and without resistance mutations., Results: No evidence for transfer of OE resistance mutations from avian to human N genes was obtained, and events suggesting recombination between human and avian influenza virus variants could not be traced in the sequence material studied., Conclusions: The results indicate that resistance in influenza viruses infecting humans is due to the selection pressure posed by the global OE administration in humans rather than transfer from avian influenza A virus strains carrying mutations induced by environmental exposure to OC.