Metabolism and disposition of oseltamivir (OS) in rats, determined by immunohistochemistry with monospecific antibody for OS or its active metabolite oseltamivir carboxylate (OC): A possibility of transporters dividing the drugs' excretion into the bile and kidney.

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.

Preliminary results on the uptake and biochemical response to water-exposure of Tamiflu® (oseltamivir phosphate) in two marine bivalves.

Tamiflu® (oseltamivir phosphate, OST) is an antiviral drug used for the pandemic treatment of avian influenza but few data are available regarding its toxicity. It should be noted that acute adverse responses are not likely to occur due to low environmental presence of this drug. Nonetheless, water concentration levels of this compound may reach the µg/L range under influenza episodes. Bivalves are reliable sentinels of chemical exposure due to their low metabolism; however, biotransformation of drugs does occur in these aquatic invertebrates. Two species of bivalves, namely mussels Mytilus galloprovincialis and clams Ruditapes philippinarum, were exposed for 48 h to 100 µg/L OST. Hemolymph from control and treated bivalves was withdrawn and the presence of OST and its metabolite oseltamivir carboxylate (OST-C) determined by LC-MS/MS. Gills and digestive gland were excised from control and exposed bivalves and carboxylesterase (CE) activities measured using different substrates. In addition, antioxidant defences and lipid peroxidation levels were determined. Higher metabolism of OST seemed to occur in mussels, since both OST and OST-C were found in hemolymph, whereas in clams only the parent compound was detected. In contrast, biomarker responses were more evident in exposed clams which indicate that this species may be considered as more sensitive to OST exposure. CE-related activities successfully reflected OST exposure, with substrates 1-naphthyl acetate (1NA) and 1-naphthyl butyrate (1NB) displaying the highest sensitivity in the two bivalve species. Data thus indicate the usefulness of CE-related activities as biomarkers for OST exposure in bivalves.

H3N2 influenza viruses in humans: Viral mechanisms, evolution, and evaluation.

Annual seasonal influenza vaccines are composed of two influenza A strains representing the H1N1 and H3N2 subtypes, and two influenza B strains representing the Victoria and Yamagata lineages. Strains from these Influenza A and Influenza B viruses currently co-circulate in humans. Of these, strains associated with the H3N2 subtype are affiliated with severe influenza seasons. H3N2 influenza viruses pre-dominated during 3 of the last 5 quite severe influenza seasons. During the 2016/2017 flu season, the H3N2 component of the influenza vaccine exhibited a poor protective efficacy (∼28-42%) against preventing infection of co-circulating strains. Since their introduction to the human population in 1968, H3N2 Influenza viruses have rapidly evolved both genetically and antigenically in an attempt to escape host immune pressures. As a result, these viruses have added numerous N-linked glycans to the viral hemagglutinin (HA), increased the overall net charge of the HA molecule, changed their preferences in receptor binding, and altered the ability of neuraminidase (NA) to agglutinate red blood cells prior to host entry. Over time, these adaptations have made characterizing these viruses increasingly difficult. This review investigates these recent changes in modern H3N2 influenza viruses and explores the methods that researchers are currently developing in order to study these viruses.

Reductive amination assistance for quantification of oseltamivir phosphate and oseltamivir carboxylate by HPLC-MS/MS.

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 (R2) 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.

Different Inhibitory Potencies of Oseltamivir Carboxylate, Zanamivir, and Several Tannins on Bacterial and Viral Neuraminidases as Assessed in a Cell-Free Fluorescence-Based Enzyme Inhibition Assay.

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.

Discovery of acylguanidine oseltamivir carboxylate derivatives as potent neuraminidase inhibitors.

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 (IC50 <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.

In vitro and in vivo stability of oseltamivir within a bioequivalence trial.

A simple, precise, and rapid method to simultaneously determine the levels of oseltamivir (OS) and oseltamivir carboxylate (OSC) in human plasma was developed. Additionally, the stability of both substances in plasma was investigated under different conditions. The method involved protein precipitation (0.01 % HCl in acetonitrile), and then the supernatant was injected into the high-performance liquid chromatography (HPLC)-MS/MS. The chromatographic separation was achieved on a YMC-Triart C18 (100 × 2.0 mm, 5 µm) column using acetonitrile/water (30:70, v/v) containing 0.1 % formic acid as the mobile phase. Sample volume was 5 µl. The linearity of the method was established in the concentration range of 0.5-100 ng/mL for OS and 1.0-1000 ng/mL for OSC. The intra-day precision and accuracy for oseltamivir were 1.5-8.9 and 94.4-101.0 %, respectively. For oseltamivir carboxylate, the intra-day precision and accuracy were 3.2-12.7 and 92.8-108.8 %, respectively, whereas the inter-day precision and accuracy were 5.5-11.5 and 94.6-104.0 % for oseltamivir and 4.7-11.5 and 99.9-103.9 % for oseltamivir carboxylate, respectively. The application of this method was demonstrated by a bioequivalence study in 28 healthy humans with 75 mg oseltamivir phosphate capsules (Tamiflu®). Sodium fluoride (2.4 mg/mL) with potassium oxalate (3 mg/mL) was used as anticoagulant within sampling of trial. The assay reproducibility was established by reanalysis of 80 incurred samples.

1,2,3-Triazolyl-4-oxoquinolines: A feasible beginning for promising chemical structures to inhibit oseltamivir-resistant influenza A and B viruses.

We described the synthesis of a new congener series of 1,2,3-triazolyl-4-oxoquinolines and evaluated their ability to inhibit oseltamivir (OST)-resistant influenza strains. Oxoquinoline derivative 1i was the most potent compound within this series, inhibiting 94% of wild-type (WT) influenza neuraminidase (NA) activity. Compound 1i inhibited influenza virus replication with an EC50 of 0.2µM with less cytotoxicity than OST, and also inhibited different OST-resistant NAs. These results suggest that 1,2,3-triazolyl-4-oxoquinolines represent promising lead molecules for further anti-influenza drug design.

Adsorption removal of antiviral drug oseltamivir and its metabolite oseltamivir carboxylate by carbon nanotubes: Effects of carbon nanotube properties and media.

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.

Development and evaluation of a dry powder formulation of liposome-encapsulated oseltamivir phosphate for inhalation.

This study aims to develop oseltamivir phosphate (OP) liposomes as inhalation powders by spray-drying based on the single factor investigation, which was mainly composed of lactose, L-leucine and mannitol. It was found that the ratio of OP and liposomes (1:10), inlet temperature (110 °C) and airflow rate (2.3 mL/min) showed optimized physical properties of OP liposomes. Deposition was evaluated after the aerosolization of powders at 600 L/h via the Aerolizer® into a twin-stage impinger. The concentrations of OP and oseltamivir carboxylate (OSCA) in rats plasma using LC-MS have been determined and performed via pharmacokinetic software DAS 2.0 package. The liposomal OP dry powders displayed an average particle size around 3.5 µm with fine particle fraction (FPF = 35.40%). In vitro evaluation demonstrated a sustained release pattern accounting for 20% drug release compared to that of OP solution up to 90% drug release in 2 h. And the cumulative release percentage was up to 50% in 20 h. Atrioventricular fitting results indicated that all preparations were best fitted with a two-compartment model. There was a significant difference in MRT, Cmax and Tmax (p < 0.01) between the two groups of liposomal OP dry powders and OP solution with t-test, which indicated that the drug released slowly from liposomal OP dry powders in the lung. To sum up, dry powders formulation of liposome-encapsulated OP for inhalation was suitable for pulmonary administration, which offering the opportunity to reduce dosing frequency.

Highlights from the United States Food and Drug Administration's public workshop on the development of animal models of pregnancy to address medical countermeasures in an "at-risk" population of pregnant women: Influenza as a case study.

The U.S. Food and Drug Administration (FDA) and other federal agencies partner to ensure that medical countermeasures (e.g., drug therapies and vaccines) are available for public health emergencies (FDA, 2014). Despite continuing progress, providing medical countermeasures and treatment guidelines for certain populations (e.g., pregnant women) is challenging due to the lack of clinical and/or animal data. Thus, a workshop was convened to discuss animal models of pregnancy for the evaluation of disease progression and medical countermeasures.

In vitro anti-influenza A activity of interferon (IFN)-λ1 combined with IFN-ß or oseltamivir carboxylate.

Influenza viruses, which can cross species barriers and adapt to new hosts, pose a constant potential threat to human health. The influenza pandemic of 2009 highlighted the rapidity with which an influenza virus can spread worldwide. Currently available antivirals have a number of limitations against influenza, and novel antiviral strategies, including novel drugs and drug combinations, are urgently needed. Here, we evaluated the in vitro effects of interferon (IFN)-ß, IFN-λ1, oseltamivir carboxylate (a neuraminidase (NA) inhibitor), and combinations of these agents against two seasonal (i.e., H1N1 and H3N2) influenza A viruses. We observed that A/California/04/09 (H1N1) and A/Panama/2007/99 (H3N2) isolates were equally sensitive to the antiviral activity of IFN-ß and oseltamivir carboxylate in A549 and Calu-3 cells. In contrast, IFN-λ1 exhibited substantially lower protective potential against the H1N1 strain (64-1030-fold ↓, P<0.05), and was ineffective against H3N2 virus in both cell lines. Three dimensional analysis of drug-drug interactions revealed that IFN-λ1 interacted with IFN-ß and oseltamivir carboxylate in an additive or synergistic manner, respectively, to inhibit influenza A virus replication in human airway epithelial cells. Overall, the present study demonstrated that anti-influenza agents with different mechanisms of action (e.g., a NA inhibitor combined with IFN-λ1) exerted a significantly greater (P<0.05) synergistic effect compared to co-treatment with drugs that target the same signaling pathway (i.e., IFN-ß plus IFN-λ1) in vitro. Our findings provide support for the combined use of interferon plus oseltamivir as a potential means for treating influenza infections.

Mass balance of anti-influenza drugs discharged into the Yodo River system, Japan, under an influenza outbreak.

In February 2011, at the peak of an influenza outbreak, we performed a comprehensive analysis of the mass balances of four anti-influenza drugs-oseltamivir (OS), oseltamivir carboxylate (OC), amantadine (AMN), and zanamivir (ZAN)-in the urban area of the Yodo River system. This area includes three main river catchments (the Katsura, Uji, and Kidzu Rivers) and is home to 12 million people, about 10% of Japan's population. Water was sampled at six main rivers and 13 tributary sites and eight sewage treatment plants (STPs). We concluded that the STP effluents were the major sources of the anti-influenza drug load in the Yodo River system (68-94% of total mass fluxes). Extended measurement throughout the Yodo River system further showed only small fluctuations of the ratio of OS to OC from 0.2 to 0.3, suggesting that OS and its metabolite are environmentally stable. The results also clearly showed the importance of reducing the levels of anti-influenza drugs in the water environment by reducing their emission at STPs.

Simultaneous quantification of prodrug oseltamivir and its metabolite oseltamivir carboxylate in human plasma by LC-MS/MS to support a bioequivalence study.

A simple, precise and rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the simultaneous determination of oseltamivir and oseltamivir carboxylate, a neuraminidase inhibitor, using their deuterated analogs as internal standards (ISs). The method involved solid phase extraction of the analytes and ISs from 200 µL human plasma with no reconstitution and drying steps. The chromatographic separation was achieved on a Symmetry C18 (100 mm×4.6 mm, 5 µm) column using 10 mM ammonium formate and acetonitrile (30:70, v/v) as the mobile phase in a run time of 2.0 min. Quantitation of analytes and ISs were done by multiple reaction monitoring on a triple quadrupole mass spectrometer in the positive ionization mode. The linearity of the method was established in the concentration range of 0.5-200 ng/mL and 2.0-800 ng/mL for oseltamivir and oseltamivir carboxylate respectively. The mean extraction recovery for oseltamivir (94.4%) and oseltamivir carboxylate (92.7%) from spiked plasma samples was consistent and reproducible. The application of this method was demonstrated by a bioequivalence study in 42 healthy Indian subjects with 75 mg oseltamivir phosphate capsules. The assay reproducibility was established by reanalysis of 151 incurred subject samples.

Oseltamivir (Tamiflu(®)) in the environment, resistance development in influenza A viruses of dabbling ducks and the risk of transmission of an oseltamivir-resistant virus to humans - a review.

The antiviral drug oseltamivir (Tamiflu(®)) is a cornerstone in influenza pandemic preparedness plans worldwide. However, resistance to the drug is a growing concern. The active metabolite oseltamivir carboxylate (OC) is not degraded in surface water or sewage treatment plants and has been detected in river water during seasonal influenza outbreaks. The natural influenza reservoir, dabbling ducks, can thus be exposed to OC in aquatic environments. Environmental-like levels of OC induce resistance development in influenza A/H1N1 virus in mallards. There is a risk of resistance accumulation in influenza viruses circulating among wild birds when oseltamivir is used extensively. By reassortment or direct transmission, oseltamivir resistance can be transmitted to humans potentially causing a resistant pandemic or human-adapted highly-pathogenic avian influenza virus. There is a need for more research on resistance development in the natural influenza reservoir and for a prudent use of antivirals.