Assessing the impact of antiviral drugs commonly utilized during the COVID-19 pandemic on the embryonic development of Xenopus laevis.

The antiviral drugs favipiravir and oseltamivir are widely used to treat viral infections, including coronavirus 2019 (COVID-19), and their levels are expected to increase in the aquatic environment. In this study, the potential toxic and teratogenic effects of these drugs were evaluated using the frog embryo teratogenesis assay Xenopus (FETAX). In addition, glutathione S-transferase (GST), glutathione reductase (GR), catalase, carboxylesterase (CaE), and acetylcholinesterase (AChE) enzyme activities and malondialdehyde levels were measured as biochemical markers in embryos and tadpoles for comparative assessment of the sublethal effects of the test compounds. Prior to embryo exposure, drug concentrations in the exposure medium were measured with high-performance liquid chromatography. The 96-h median lethal concentration (LC50) was 137.9 and 32.3 mg/L for favipiravir and oseltamivir, respectively. The teratogenic index for favipiravir was 4.67. Both favipiravir and oseltamivir inhibited GR, CaE, and AChE activities in embryos, while favipiravir increased the GST and CaE activities in tadpoles. In conclusion, favipiravir, for which teratogenicity data are available in mammalian test organisms and human teratogenicity is controversial, inhibited Xenopus laevis embryo development and was teratogenic. In addition, sublethal concentrations of both drugs altered the biochemical responses in embryos and tadpoles, with differences between the developmental stages.

Adverse Effects of Oseltamivir Phosphate Therapy on the Liver of LDLR-/- Mice Without Any Benefit on Atherosclerosis and Thrombosis.

ABSTRACT: Desialylation, governed by sialidases or neuraminidases, is strongly implicated in a wide range of human disorders, and accumulative data show that inhibition of neuraminidases, such as neuraminidases 1 sialidase, may be useful for managing atherosclerosis. Several studies have reported promising effects of oseltamivir phosphate, a widely used anti-influenza sialidase inhibitor, on human cancer cells, inflammation, and insulin resistance. In this study, we evaluated the effects of oseltamivir phosphate on atherosclerosis and thrombosis and potential liver toxicity in LDLR-/- mice fed with high-fat diet. Our results showed that oseltamivir phosphate significantly decreased plasma levels of LDL cholesterol and elastin fragmentation in aorta. However, no effect was observed on both atherosclerotic plaque size in aortic roots and chemically induced thrombosis in carotid arteries. Importantly, oseltamivir phosphate administration had adverse effects on the liver of mice and significantly increased messenger RNA expression levels of F4/80, interleukin-1ß, transforming growth factor-ß1, matrix metalloproteinase-12, and collagen. Taken together, our findings suggest that oseltamivir phosphate has limited benefits on atherosclerosis and carotid thrombosis and may lead to adverse side effects on the liver with increased inflammation and fibrosis.

Development of Sustained Release Oseltamivir Phosphate Dry Powder Inhaler: In-Vitro Characterization and In-Vivo Toxicological Studies.

BACKGROUND: Oseltamivir Phosphate (OP) is an ethyl ester prodrug prescribed for the treatment of influenza virus infection. Current marketed formulations of OP have been observed to be supplemented with an adverse effect during post-marketing surveillance. These prerequisites are sufficed by developing a sustained release Dry Powder for Inhalation (DPI). OBJECTIVE: The objective of the present study was to develop OP-DPI by an innovative formulation approach comprising of Immediate (IR) and Sustained (SR) Release portions. METHODS: DPI formulation comprising IR and SR portions were prepared by spray drying technique using Hydroxy Propyl Methyl Cellulose (HPMC) as the rate-controlling polymer for SR portion. The spray-dried product was further characterized for various pharmaco-technical, in-vitro and in-vivo parameters. RESULTS: OP-DPI showed a burst release of 49% within 15 min further sustaining the drug release up to 9 hrs. The in-vitro aerodynamic performance of OP-DPI showed maximum deposition at stage 3 and Fine Particle Dose (FPD) of 1.08 mg indicating deposition in the upper respiratory tract. Solid-state characterization by DSC and XRD indicated the partial amorphization of OP due to spray drying. In-vivo toxicological examination revealed no sign of inflammation, indicating the safety of the developed formulation. Accelerated stability study as per ICH guidelines displayed no significant change in the solid-state characterization and drug-related performance of OP-DPI. CONCLUSION: Prepared novel and scalable OP-DPI may have the potential to overcome the problems associated with existing marketed dosage forms of OP. Further, localized drug delivery of the antiviral drug through the pulmonary route might be clinically beneficial in controlling the viral proliferation.

Design, synthesis and biological evaluation of "Multi-Site"-binding influenza virus neuraminidase inhibitors.

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-NH2 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 EC50 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.

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.

Assessing exposure risks for aquatic organisms posed by Tamiflu use under seasonal influenza and pandemic conditions.

Environmental pollution by anti-influenza drugs is increasingly recognized as a threat to aquatic environments. However, little is known about empirical data on risk effects posed by environmentally relevant concentrations of anti-influenza drug based on recently published ecotoxicological researches in Taiwan. Here we linked ecotoxicology models with an epidemiological scheme to assess exposure risks of aquatic organisms and environmental hazards posed by antiviral oseltamivir (Tamiflu) use in Taiwan. Built on published bioassays, we used probabilistic risk assessment model to estimate potential threats of environmentally relevant hazards on algae, daphnid, and zerbrafish. We found that Tamiflu use was unlikely to pose a significant chronic environmental risk to daphnia and zebrafish during seasonal influenza. However, the chronic environmental risk posed by Tamiflu use during pandemic was alarming. We conclude that no significant risk to algal growth was found during seasonal influenza and high pandemic Tamiflu use.

Novel neuraminidase inhibitors: identification, biological evaluation and investigations of the binding mode.

BACKGROUND: The pathogenicity of influenza A and B viruses depends on the function of influenza neuraminidase (NA). Emerging resistant influenza A viruses of subtype H1N1 increasingly challenge the effectiveness of established NA inhibitors. Recent computational studies have indicated several weak points of NA that can be exploited for rational inhibitor design to conquer this imminent threat, such as the opening of the binding pocket due to the flexibility of the 150-, 245- and 430-loops. METHODS: We employed shape-focused virtual screening based on a recently discovered lead compound, katsumadain A, to identify novel promising compounds with significant inhibitory efficacy on NA and resistance-breaking capacity on oseltamivir-resistant strains. A potential binding mode of these compounds was derived employing ligand-based techniques and protein-ligand docking using representative protein conformations selected from molecular dynamics simulations. RESULTS: Five novel compounds were identified by virtual screening. Their IC(50) values, determined in chemiluminescence-based NA inhibition assays, are in the range of 0.18-17 µM. In particular, artocarpin exhibits high affinity toward three H1N1 oseltamivir-sensitive influenza A viruses. It also inhibits the NA of an oseltamivir-resistant H1N1 isolate.

Effect of oseltamivir on catecholamines and select oxidative stress markers in the presence of oligoelements in the rat brain.

The effect that osteltamivir has on the metabolism of catecholamines and oxidative damage in the brains of young patients remains unclear. The purpose of this study was to measure the effects of oseltamivir, in the presence of oligoelements, on biogenic amines and select oxidative biomarkers in the brains of uninfected, young rats under normal conditions. The study was conducted using male Wistar rats intraperitoneally treated for three days with either a control dose of 0.9 % NaCl, oseltamivir (50 mg/kg), oligoelements (50 µL/rat), or oseltamivir (50 mg/kg) and oligoelements (50 µL/rat). The brain tissue extracted from the treated rats was used to determine the concentrations of adrenaline, noradrenaline, and dopamine, as well as the levels of GSH, lipid peroxidation, and ATPase activity. An increase in the concentration of adrenaline and noradrenaline and in the level of GSH in the group treated with oligoelements (p < 0.001) was observed, while the group treated with oseltamivir and oligoelements, the levels of dopamine increased (p < 0.001), and in the groups treated with oligoelements alone or combination with oseltamivir a decrease in lipid peroxidation was observed (p < 0.001). The results of this study suggest that the consumption of oseltamivir and oligoelements induce biphasic changes in the metabolism of catecholamines; thereby, inducing a protective mechanism against oxidative damage in the brains of young rats.

Effect of the antiviral drug oseltamivir (Tamiflu) on the bacterial community structure of a surface water ecosystem analyzed using fluorescence in situ hybridization.

The antiviral drug Tamiflu has received particular attention because of its recommended use against the influenza A H5N1 and H1N1 viruses. Given its resistance to degradation and its hydrophilicity the active metabolite, Oseltamivir Carboxylate (OC), is expected to enter the aquatic ecosystem from sewage treatment plants. In the present paper the bacterial community of surface water samples, treated with OC (1.5 mg L⁻¹), was characterized by Fluorescence In Situ Hybridization (FISH) in microcosm experiments. The α-, ß- and γ-Proteobacteria increased in OC-treated versus non-treated water samples during the incubation period, suggesting these bacterial groups had an active role in OC degradation.

Mixture toxicity of the antiviral drug Tamiflu((R)) (oseltamivir ethylester) and its active metabolite oseltamivir acid.

Tamiflu (oseltamivir ethylester) is an antiviral agent for the treatment of influenza A and B. The pro-drug Tamiflu is converted in the human body to the pharmacologically active metabolite, oseltamivir acid, with a yield of 75%. Oseltamivir acid is indirectly photodegradable and slowly biodegradable in sewage works and sediment/water systems. A previous environmental risk assessment has concluded that there is no bioaccumulation potential of either of the compounds. However, little was known about the ecotoxicity of the metabolite. Ester hydrolysis typically reduces the hydrophobicity and thus the toxicity of a compound. In this case, a zwitterionic, but overall neutral species is formed from the charged parent compound. If the speciation and predicted partitioning into biological membranes is considered, the metabolite may have a relevant contribution to the overall toxicity. These theoretical considerations triggered a study to investigate the toxicity of oseltamivir acid (OA), alone and in binary mixtures with its parent compound oseltamivir ethylester (OE). OE and OA were found to be baseline toxicants in the bioluminescence inhibition test with Vibrio fischeri. Their mixture effect lay between predictions for concentration addition and independent action for the mixture ratio excreted in urine and nine additional mixture ratios of OE and OA. In contrast, OE was an order of magnitude more toxic than OA towards algae, with a more pronounced effect when the direct inhibition of photosystem II was used as toxicity endpoint opposed to the 24h growth rate endpoint. The binary mixtures in this assay yielded experimental mixture effects that agreed with predictions for independent action. This is consistent with the finding that OE exhibits slightly enhanced toxicity, while OA acts as baseline toxicant. Therefore, with respect to mixture classification, the two compounds can be considered as acting according to different modes of toxic action, although there are indications that the difference is a toxicokinetic effect, not a true difference of mechanism of toxicity. The general mixture results illustrate the need to consider the role of metabolites in the risk assessment of pharmaceuticals. However, in the concentration ratio of parent to metabolite excreted by humans, the experimental results confirm that the active metabolite does not significantly contribute to the risk quotient of the mixture.

An environmental risk assessment for oseltamivir (Tamiflu) for sewage works and surface waters under seasonal-influenza- and pandemic-use conditions.

In the event of an influenza pandemic, anti-viral medications such as oseltamivir (Tamiflu) are expected to be used in high amounts over a duration of several weeks. Oseltamivir has been predicted to reach high concentrations in surface waters and sewage works. New oseltamivir environmental fate and toxicity studies permit an environmental risk assessment (ERA) under seasonal- and pandemic-use scenarios. The environmental fate data for sewage works (no removal), surface waters (no significant degradation), and water/sediment systems (>50% primary degradation in 100 days) were used for the derivation of new predicted environmental concentrations (PECs) for western Europe and the River Lee catchment in the UK. Existing worst-case PECs for western Europe, the River Lee catchment in the UK and the Lower Colorado basin in the USA under pandemic conditions (< or =98.1 microg/L for surface waters, < or =348 microg/L for sewage works) were also considered for the ERA. PECs were compared with predicted no-effect concentrations (PNECs) based on new chronic ecotoxicity data (no observed effect concentration for algae, daphnia, and fish > or =1 mg/L). Based on all PEC/PNEC risk ratios, no significant risk is evident to surface waters or sewage works during both regular seasonal-use and high pandemic-use of oseltamivir.

Extending the environmental risk assessment for oseltamivir (Tamiflu) under pandemic use conditions to the coastal marine compartment.

In case of an avian-influenza-derived human flu pandemic, an inordinately high use of medicines over several weeks is predicted, in particular for the recommended influenza antiviral oseltamivir (Tamiflu). While the risk of oseltamivir to sewage works and freshwater bodies has already been assessed, the fact that a large percentage of the human population worldwide lives relatively close to the sea raises concern for its environmental compatibility in coastal marine waters. The potential risk of high oseltamivir use to the marine compartment is assessed in this publication, based on the 2003 European Community Technical Guidance Document (TGD) for risk assessment. Subchronic embryo-larval ecotoxicity tests with three marine invertebrates (Pomatoceros triqueter, Annelida; Mytilus edulis, Mollusca; Paracentrotus lividus, Echinodermata) and chronic growth inhibition tests with two different groups of marine microalgae (Isochrysis galbana, Haptophyta; Skeletonema costatum, Heterokontophyta) were performed with the active substance oseltamivir carboxylic acid to derive a dependable marine predicted no-effect concentration (PNEC). This was compared to a predicted environmental concentration (PEC) for oseltamivir in coastal waters, based on the worst-case freshwater PEC. The PEC/PNEC risk characterisation ratio for the marine compartment is well below 1, which in the terminology of the TGD signifies no immediate concern. Further, while oseltamivir may be persistent (P), it is not bioaccumulative (B) nor highly ecotoxic (T) and therefore not a PBT substance. In conclusion, even a high pandemic use of oseltamivir would not lead to a significant risk for the marine compartment, in confirmation of the risk assessment for sewage works and freshwaters.

Synaptic and behavioral interactions of oseltamivir (Tamiflu) with neurostimulants.

Oseltamivir (Tamiflu), a neuraminidase inhibitor, is widely used for treatment of influenza. Because abnormal behaviors have been observed in some Japanese teenagers following oseltamivir use, its safety has been questioned. Oseltamivir is known to alter neuronal function and behavior in animals, particularly when administered in combination with ethanol. Based on this, it has been hypothesized that interactions of oseltamivir with other drugs may result in altered CNS excitability in this study. It has been found that injection of ephedrine and caffeine overcame inactivity induced by oseltamivir and ethanol but did not alter changes in novelty seeking behavior in a Y-maze test. In ex-vivo hippocampal slices, oseltamivir carboxylate (OTC), an active form of oseltamivir, alters excitability in the absence of ethanol. In slices pretreated with OTC, long-term depression (LTD), a form of synaptic plasticity that is correlated with Y-maze performance was not altered if caffeine or ephedrine was administered individually. However, LTD could not be induced in slices pretreated with OTC if caffeine and ephedrine were administered simultaneously. These observations suggest that combination of oseltamivir with other neurostimulants may alter synaptic plasticity and this may contribute to behavioral changes associated with the drug.

Prevention and treatment of influenza in high-risk groups: children, pregnant women, immunocompromised hosts, and nursing home residents.

The pediatric population experiences preventable hospitalizations and serves as a reservoir for influenza and its transmission to other children as well as adults. As a consequence, the Advisory Committee on Immunization Practices has recommended initiating influenza immunization of children as young as 6 months of age through 23 months of age and, recently, up to 5 years of age. However, immunization of older children has not yet become a priority of the US Public Health Service. As a consequence, the importance of antiviral agents, particularly neuraminidase (NA) inhibitors, cannot be overemphasized. From an epidemiological perspective, influenza resulted in higher childhood mortality than did Bordetella pertussis infection in 2003-2004. During that season, 153 children died of influenza, and two-thirds were <5 years of age. Importantly, nearly 50% of these children were previously healthy, with no underlying illness. Currently, 2 NA inhibitors are approved for the treatment of influenza in children. Zanamivir is approved for children >7 years of age, and oseltamivir is approved for children >1 year of age. Arguably, the younger children are at particular risk for influenza complications and hospitalization. In placebo-controlled studies in children >1 year of age, oseltamivir therapy accelerated resolution of clinical illness and defervescence and decreased both the incidence of otitis media and the concomitant use of antibiotics. However, oseltamivir is not currently approved for children <1 year of age. Three clinical toxicology studies identified neurotoxicity in newborn rats administered this medication. In these preclinical toxicology studies, the dose of oseltamivir exceeded that which would be used in humans. In addition, the metabolism of oseltamivir is different in rats than in humans. A key component of influenza therapy is the possibility for development of resistance. Although in studies performed in North America, resistance was not a frequent event, it has been documented in Japanese children treated with this medication; the adequacy of the dose used has been questioned. Children represent only one unique study population among others. Individuals who are at increased risk for influenza infection include the elderly, the immunocompromised, and pregnant women. Collectively, antiviral medications must be evaluated in populations in which they have not yet been assessed. The development of additional antiviral drugs is an important recommendation for the future, so that antiviral resistance can be circumvented. Similarly, availability of drugs for children <1 year of age is mandatory.