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Zanamivir is a potent and highly selective inhibitor of influenza neuraminidase in which the inhibition of this enzyme prevents the virus from infecting other cells and specifically prevents release of the new virion from the host cell membrane. It is available as an oral powder for inhalation and intravenous formulations. The current population pharmacokinetic model based on data from eight studies of subjects treated with the intravenous formulation (125 healthy adults and 533 hospitalized adult and pediatric subjects with suspected or confirmed influenza) suggested a decreased zanamivir clearance in pediatric and renal impairment adult subjects. It also indicates that b.i.d. dosing is necessary to keep the exposure in influenza infected subjects above the 90% inhibitory concentration values of recently circulating viruses over the dosing interval. In the exposure-response analysis (phases II and III studies), no apparent relationship was found between zanamivir exposure and clinically relevant pharmacodynamic end points., (2019 GSK. Clinical and Translational Science published by Wiley Periodicals, Inc. on behalf of the American Society for Clinical Pharmacology and Therapeutics.)

Introduction : Influenza represents a major public health threat worldwide. Implementation of good personal health and hygiene habits, together with vaccination, is the most effective tools to reduce influenza burden both in community and in healthcare setting. However, achieving adequate vaccination rates is challenging, and vaccination does not always guarantee complete protection. Neuraminidase inhibitors represent an important measure to reduce the risk of influenza-related complications among high-risk patients developing influenza infection. Areas covered : Neuraminidase inhibitors have been proven to be safe and effective in reducing influenza severity, duration of symptoms, hospitalizations, and influenza-related-mortality. Here the authors review the available data on neuraminidase inhibitors, including the mechanism of action, pharmacokinetics, efficacy, safety and current indications for their use in clinical practice. Expert opinion : Although vaccination is the most effective tool to reduce influenza-associated morbidity and mortality, neuraminidase inhibitors represent an important option for the treatment of patients with influenza infection, particularly in high-risk categories. Moreover, antivirals play an important role in influenza prevention and prophylaxis in selected settings.

A single dose of laninamivir octanoate (LO) inhaled using a dry powder inhaler (DPI) is effective for the treatment and prophylaxis of influenza. Nebulizers are an option for pediatric and elderly patients who may have difficulty in using a DPI. A single-center, open-label study was conducted to evaluate the plasma and intrapulmonary pharmacokinetics (PK) of laninamivir after a single nebulized administration of LO in healthy male Japanese subjects for identifying a safe and effective dosage regimen for a nebulizer. A single dose of LO (40 to 320 mg) was administered using a nebulizer, and plasma concentrations of LO and laninamivir were analyzed up to 168 h after inhalation by validated liquid chromatography-tandem mass spectrometry methods. Subgroups of 6 subjects each underwent bronchoalveolar lavage at specified time intervals over 4 to 168 h following a single nebulized administration of LO (160 mg), and the concentrations in epithelial lining fluid (ELF) were calculated by the urea diffusion method. PK parameters were determined by noncompartment analysis. Inhaled nebulized LO was found to be safe and well tolerated up to the highest dose evaluated (320 mg). Plasma laninamivir concentrations increased almost dose proportionally. Laninamivir concentrations in ELF exceeded the 50% inhibitory concentrations for viral neuraminidase up to 168 h after the nebulized inhalation of 160 mg LO. Thus, similarly to the DPI, ELF concentration profiles of laninamivir after a single nebulized administration support its long-lasting effect against influenza virus infection. This study has been registered at JAPIC Clinical Trials Information (http://www.clinicaltrials.jp/) under registration no. JAPIC CTI-152996., (Copyright © 2017 American Society for Microbiology.)

This work describes a new dry powder for inhalation containing zanamivir, which is less hygroscopic than Relenza®. The powders were prepared via a spray-drying technique using mannitol as the carrier. A 5(3) central composite design was used to optimize the formulations. The final optimized powders, characterized with an angle of repose 37.48°, an aerodynamic diameter of 2.346 μm and in vitro deposition of 58.54%, were obtained by using the predicted variable values. Relenza® absorbed a significant amount of water at 66%, 75% and 85% relative humidity (RH; weight changes of approximately 1.38%, 2.18% and 3.72%, respectively). In contrast, the weight change for the zanamivir dry powder inhalation (DPI) was negligible when the RH was increased to 66%. The in vivo potential for the optimized powders was studied further in rats via the endotracheal administration of an 8.4 mg/kg dose. The bioavailability was 116% relative to Relenza®. Fluorescence imaging monitored the zanamivir dry powder inhalers in rats. The results indicated that the zanamivir DPIs were effectively delivered to the lung. These results indicate that the spray-dried zanamivir DPIs were promising for pulmonary delivery.

Based upon molecular docking, this study aimed to find notable in silico neuraminidase 9 (NA9) point mutations of the avian influenza A H7N9 virus that possess a Zanamivir resistant property and to determine the lead compound capable of inhibiting these NA9 mutations. Seven amino acids (key residues) at the binding site of neuraminidase 9 responsible for Zanamivir-NA9 direct interactions were identified and 72 commonly occurring mutant NA9 versions were created using the Sybyl-X 2.0 software. The docking scores obtained after Zanamivir was bound to all mutant molecules of NA9 revealed 3 notable mutations R292W, R118P, and R292K that could greatly reduce the binding affinity of the medicine. These 3 mutant NA9 versions were then bound to each of 154 different molecules chosen from 5 groups of compounds to determine which molecule(s) might be capable of inhibiting mutant neuraminidase 9, leading to the discovery of the lead compound of potent mutant NA9 inhibitors. This compound, together with other mutations occurring to NA9 identified in the study, would be used as data for further research regarding neuraminidase inhibitors and synthesizing new viable medications used in the fight against the virus., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The neuraminidase (NA) inhibitor zanamivir (1) is potently active against a broad panel of influenza A and B strains, including mutant viruses, but suffers from pharmacokinetic (PK) shortcomings. Here, distinct prodrug approaches are described that aimed at overcoming zanamivir's lack of oral bioavailability. Lowering the high basicity of the 4-guanidino group in zanamivir and of a bioisosteric 4-acetamidine analog (5) by N-hydroxylation was deemed to be a plausible tactic. The carboxylic acid and glycerol side chain were also masked with different ester groups. The bioisosteric amidine 5 turned out to be potently active against a panel of H1N1 (IC50 = 2-10 nM) and H3N2 (IC50 = 5-10 nM) influenza A viruses (NA inhibition assay). In vitro PK studies showed that all prodrugs were highly soluble, exhibited low protein binding, and were bioactivated by N-reduction to the respective guanidines and amidines. The most promising prodrug candidates, amidoxime ester 7 and N-hydroxyguanidine ester 8, were subjected to in vivo bioavailability studies. Unfortunately, both prodrugs were not orally bioavailable to a convincing degree (F ≤ 3.7%, rats). This finding questions the general feasibility of improving the oral bioavailability of 1 by lipophilicity-increasing prodrug strategies, and suggests that intrinsic structural features represent key hurdles., (© 2015 Wiley Periodicals, Inc. and the American Pharmacists Association.)

Influenza is an important cause of annual epidemics of respiratory viral infection associated with significant morbidity and mortality. Three classes of drugs, the M2 ion channel, neuraminidase and RNA-dependent RNA polymerase inhibitors, are approved for the prevention and treatment of influenza. Due to widespread resistance to the class, the M2 ion channel inhibitors are not recommended currently for therapy. The only polymerase inhibitor, favipiravir, is approved only in Japan and its use is highly restricted. Despite significant data to support the early use of the neuraminidase inhibitors, their use in all patient populations is suboptimal. The data to support the early use of neuraminidase inhibitors will be reviewed, as will current data on the utilization rates in ambulatory and hospitalized populations.

Background: Intravenous zanamivir is a neuraminidase inhibitor suitable for treatment of hospitalized patients with severe influenza., Methods: Patients were treated with intravenous zanamivir 600 mg twice daily, adjusted for renal impairment, for up to 10 days. Primary outcomes included adverse events (AEs), and clinical/laboratory parameters. Pharmacokinetics, viral load, and disease course were also assessed., Results: One hundred thirty patients received intravenous zanamivir (median, 5 days; range, 1-11) a median of 4.5 days (range, 1-7) after onset of influenza; 83% required intensive care. The most common influenza type/subtype was A/H1N1pdm09 (71%). AEs and serious AEs were reported in 85% and 34% of patients, respectively; serious AEs included bacterial pulmonary infections (8%), respiratory failure (7%), sepsis or septic shock (5%), and cardiogenic shock (5%). No drug-related trends in safety parameters were identified. Protocol-defined liver events were observed in 13% of patients. The 14- and 28-day all-cause mortality rates were 13% and 17%. No fatalities were considered zanamivir related. Pharmacokinetic data showed dose adjustments for renal impairment yielded similar zanamivir exposures. Ninety-three patients, positive at baseline for influenza by quantitative polymerase chain reaction, showed a median decrease in viral load of 1.42 log10 copies/mL after 2 days of treatment., Conclusions: Safety, pharmacokinetic and clinical outcome data support further investigation of intravenous zanamivir., Clinical Trials Registration: NCT01014988.

Intravenous zanamivir is in clinical development for the treatment of influenza in hospitalized patients, many of whom have renal impairment. This open-label study evaluated zanamivir pharmacokinetics and clinical safety following a single 100-mg intravenous infusion dose in subjects with impaired renal function compared with normal renal function. Male and female subjects between 18 and 79 years of age were recruited, four subjects to each renal function group (normal function and mild, moderate, and severe impairment). Serial blood samples were collected up to 24 h after dose administration (48 h for the severe renal impairment group) to estimate zanamivir serum pharmacokinetic parameters. Urine was collected over the same 24-h (or 48-h) period for estimation of renal clearance (CLR). Zanamivir pharmacokinetics were assessed by regression analysis of systemic clearance (CL) and CLR as a function of creatinine clearance (CLCR). Safety evaluations included adverse-event monitoring, vital signs, electrocardiogram, and clinical laboratory assessments. Zanamivir clearance (total and renal) significantly decreased with decreasing renal function, with corresponding increases in area under the concentration-time curve and elimination half-life. Renal impairment had no apparent effects on peak concentration or volume of distribution. Regression analysis indicated that zanamivir clearance was highly correlated (r(2) = 0.89) with creatinine clearance: CL ≅ 7.08 + 0.826 · CLCR. There were no patterns or trends in adverse events, and no new safety concerns were identified following administration of intravenous zanamivir. Results from this study support the inclusion of subjects with renal impairment, with appropriate dose adjustment, in studies to evaluate intravenous zanamivir in the treatment of influenza.

Study Objective: To assess the effect of a therapeutic and supratherapeutic intravenous dose of the neuraminidase inhibitor zanamivir on QT and rate-corrected QT intervals., Design: Randomized, placebo-controlled, single-dose, four-period, balanced crossover study., Setting: Clinical research unit., Subjects: Forty healthy adults were randomized to receive intravenous zanamivir at two dose levels, oral moxifloxacin, and placebo; 38 subjects completed all four study treatments., Intervention: Subjects were randomized to receive a single intravenous dose of zanamivir 600 mg (therapeutic dose) with oral moxifloxacin placebo, a single intravenous dose of zanamivir 1200 mg (supratherapeutic dose) with oral moxifloxacin placebo, oral moxifloxacin 400 mg (positive control) with intravenous zanamivir placebo, or intravenous zanamivir placebo with oral moxifloxacin placebo. Subjects crossed over to all other treatments, with each treatment separated by a 7-day washout period., Measurements and Main Results: Zanamivir pharmacokinetics were dose proportional; the pharmacokinetic exposure from zanamivir 1200 mg was 2 times higher than that from 600 mg, the maximum dose under clinical evaluation. For both 600-mg and 1200-mg doses of intravenous zanamivir, the upper limit of the 90% confidence interval (CI) for the placebo-adjusted mean change from baseline of the QT interval corrected for heart rate using Fridericia's formula (ΔΔQTcF) was less than 10 msec at all time points. The sensitivity of the study to detect modest increases in QT interval was established with the positive control, moxifloxacin. The maximum ΔΔQTcF value for zanamivir 1200 mg was 1.73 msec (90% CI -0.40 to 3.87 msec), which was observed within 30 minutes after dosing, and 11.21 msec (90% CI 8.81-13.60) for moxifloxacin, observed at 4 hours after dosing. No relationship was observed between zanamivir serum concentration and ΔΔQTcF. Zanamivir was generally well tolerated, with very few adverse events; none were serious or severe., Conclusion: Intravenous zanamivir does not affect cardiac repolarization. Accordingly, treatment with intravenous zanamivir does not require additional cardiac monitoring beyond the standard of care., (© 2013 Pharmacotherapy Publications, Inc.)

What Is Known and Objective: Neuraminidase inhibitors are important options for the treatment of infection by the influenza virus. For the treatment of severe influenza, parenteral administration of a neuraminidase inhibitor may be desirable. This study was conducted to evaluate the pharmacokinetic and safety profiles of intravenous zanamivir, an influenza viral neuraminidase inhibitor, in Japanese subjects to further characterize these profiles particularly following relatively high-doses when compared with inhalation doses and to provide reassurance that there are no marked differences with profiles reported for other ethnically different populations., Methods: Single doses of 100, 300, 600 mg zanamivir were administered to healthy Japanese men in a randomized, double-blind, ascending dose, placebo-controlled, incomplete three-period cross-over study. In period 3, subjects were given 600 mg of zanamivir on day 1, followed by a 60 h washout period and then a 5-day course of 600 mg zanamivir twice daily. Each subjects received two of three active dosages of zanamivir from 100, 300 and 600 mg, and placebo., Results: Adverse events reported in the study were all mild in intensity and resolved without any treatment. The mean AUC0-∞ values after single intravenous administration of 100, 300 and 600 mg were 16768, 53462 and 100400 ng·h/mL, respectively, demonstrating dose proportionality. No accumulation or time variance was observed after 5 days of twice-daily administration of 600 mg zanamivir. Urinary concentrations of zanamivir after single doses ranging from 100 to 600 mg indicated that over 94% of the zanamivir administered was excreted in urine within 24 h., What Is New and Conclusion: Single and 5-day BID repeat dosing of 600 mg were safely administered in Japanese healthy subjects. The pharmacokinetic profile of zanamivir after intravenous administration was consistent with previously reported findings in non-Japanese subjects., (© 2013 Blackwell Publishing Ltd.)

We have demonstrated that simple formulations composed of the parent drug in combination with generally regarded as safe (GRAS) permeability enhancers are capable of dramatically increasing the absolute bioavailability of zanamivir. This has the advantage of not requiring modification of the drug structure to promote absorption, thus reducing the regulatory challenges involved in conversion of an inhaled to oral route of administration of an approved drug. Absolute bioavailability increases of up to 24-fold were observed when Capmul MCM L8 (composed of mono- and diglycerides of caprylic/capric acids in glycerol) was mixed with 1.5 mg of zanamivir and administered intraduodenally to rats. Rapid uptake (t(max) of 5 min) and a C(max) of over 7200 ng/mL was achieved. Variation of the drug load or amount of enhancer demonstrated a generally linear variation in absorption, indicating an ability to optimize a formulation for a desired outcome such as a targeted C(max) for enzyme saturation. No absorption enhancement was observed when the enhancer was given 2 hr prior to drug administration, indicating, in combination with the observed tmax, that absorption enhancement is temporary. This property is significant and aligns well with therapeutic applications to limit undesirable drug-drug interactions, potentially due to the presence of other poorly absorbed polar drugs. These results suggest that optimal human oral dosage forms of zanamivir should be enteric-coated gelcaps or softgels for intraduodenal release. There continues to be a strong need and market for multiple neuraminidase inhibitors for influenza treatment. Creation of orally available formulations of inhibitor drugs that are currently administered intravenously or by inhalation would provide a significant improvement in treatment of influenza. The very simple GRAS formulation components and anticipated dosage forms would require low manufacturing costs and yield enhanced convenience. These results are being utilized to design prototype dosage forms for initial human pharmacokinetic studies.

In order to exploit the 430-cavity in the active sites of neuraminidases, 22 zanamivir analogs with C-1 and C-4 modification were synthesized, and their inhibitory activities against both group-1 (H5N1, H1N1) and group-2 neuraminidases (H3N2) were determined. Compound 9f exerts the most potency, with IC(50) value of 0.013, 0.001, and 0.09 μM against H3N2, H5N1, and H1N1, which is similar to that of zanamivir (H3N2 IC(50) = 0.0014 μM, H5N1 IC(50) = 0.012 μM, H1N1 IC(50) = 0.001 μM). Pharmacokinetic studies of compound 9f in rats showed a much longer plasma half-life (t(1/2)) than that of zanamivir following administration (po dose). Molecular modeling provided information about the binding model between the new inhibitors and neuraminidase, with the elongated groups at the C-1-position being projected toward the 430-loop region. This study may represent a novel starting point for the future development of improved antiflu agents.

Laninamivir octanoate (LO) is a new neuraminidase inhibitor for inhalation. The objectives of this study were to model the population pharmacokinetics of LO and its active metabolite laninamivir after inhaled administration of LO using a pooled population of healthy subjects, and adult and pediatric patients with influenza virus infection from 8 clinical studies, and to evaluate covariate effects on pharmacokinetics. The pharmacokinetics of LO and laninamivir in plasma and urine are well-described by structural models that consist of a 2-compartment model for LO with instantaneous bolus input and first-order elimination; and a 1-compartment model for laninamivir with formation of laninamivir via the metabolic pathway from LO in systemic circulation, entry of laninamivir from the respiratory tract compartment, and linear elimination. Creatinine clearance was identified as a covariate of apparent total clearance for LO and renal clearances for LO and laninamivir, with the largest effect on laninamivir exposure. Body weight was identified to affect distribution volumes of LO and laninamivir and the metabolic clearance of LO; however there was no notable effect on exposures across the wide body weight range evaluated. The population pharmacokinetic model also provides insight into the likely kinetics of drug disposition in the respiratory tract following inhaled administration.

Background: During a pandemic, the need for available anti-influenza medications increases. There has been extensive use of the approved zanamivir Rotadisk/Diskhaler but no clinical data are available for administration by an alternative Rotacap/Rotahaler presentation., Methods: In this randomized three-way crossover study, each healthy adult received zanamivir 10 mg every 12 h for 5 days via Rotadisk/Diskhaler, via Rotacap/Rotahaler and placebo via Rotacap/Rotahaler, with a washout period between treatments. Safety assessments were conducted throughout the study and at follow-up. Serial blood samples for pharmacokinetic analysis were collected over a 12-h dose interval on day 5 of each treatment period. Pharmacokinetic parameters were compared using a mixed-effects model., Results: A total of 18 healthy adults were recruited and 17 subjects completed the study. A total of 20 adverse events (AEs) were reported (all grade 1) by nine subjects, with no AE reported ≥1× in any treatment group. Nasal congestion, reported by one subject in the zanamivir Rotadisk/Diskhaler group, was the only drug-related AE. No serious AEs or withdrawals due to AEs occurred during the study. There were no significant changes in clinical laboratory values, vital signs or spirometry. Serum zanamivir exposures were similar after administration via Rotacap/Rotahaler and Rotadisk/Diskhaler. Both oral inhalation presentations are likely to deliver similar zanamivir concentrations to sites of influenza infection in the respiratory tract., Conclusions: The safety and pharmacokinetic results from this study support the use of the Rotacap/Rotahaler presentation, potentially allowing an increased number of zanamivir treatment courses to be supplied in the event of an influenza pandemic.

Laninamivir octanoate (LO) (Inavir; Daiichi Sankyo, Japan) is an ester prodrug of the neuraminidase inhibitor laninamivir. We previously reported that a prolonged high retention of laninamivir in mouse respiratory tissues was achieved by intranasal administration of LO. In this study, we evaluated intrapulmonary pharmacokinetics both in vivo and in vitro to investigate the potential mechanism involved in such a preferable retention. After intranasal administration of LO to mice (0.5 μmol/kg), the drug was distributed from the airway space into the lungs, and laninamivir remained in the lung at 24 hours postdose (2680 pmol/g), with a higher concentration than that in the epithelial lining fluid. The laninamivir was localized mainly on the epithelial cells of airway tracts, determined by microautoradiography using (14)C-labeled LO. In mouse airway epithelial cells, the cellular uptake and hydrolysis of LO were observed over incubation time without any apparent saturation at the highest concentration tested (1000 μM). Furthermore, after additional incubation in drug-free medium, the intracellular laninamivir was released very slowly into the medium with an estimate rate constant of 0.0707 h(-1), which was regarded as a rate-limiting step in the cellular retention. These results demonstrated that the prolonged high retention of laninamivir in the respiratory tissues was attributed to a consecutive series of three steps: uptake of LO into the airway epithelial cells, hydrolysis of LO into laninamivir by intracellular esterase(s), and limited efflux of the generated laninamivir due to its poor membrane permeability. This prodrug approach could be useful for lung-targeting drug delivery.

Programming an anti-flu strategy: A new and potent neuraminidase inhibitor that maintains long-term systemic exposure of an antibody and the therapeutic activity of the neuraminadase inhibitor zanamivir has been created. This strategy could provide a promising new class of influenza A drugs for therapy and prophylaxis, and validates enzyme inhibitors as programming agents in synthetic immunology., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

With the introduction of bioisosteres of the guanidinium group together with scaffold hopping, 35 zanamivir analogs with C-4-modification were synthesized, and their inhibitory activities against both group-1 and group-2 neuraminidase (H5N1 and H3N2) were determined. Compound D26 exerts the most potency, with IC(50) values of 0.58 and 2.72 μM against N2 and N1, respectively. Further preliminary anti-avian influenza virus (AIV, H5N1) activities against infected MDCK cells were evaluated, and D5 exerts ∼58% protective against AIV infection, which was comparable to zanamivir (∼67%). In a rat pharmacokinetic study, compound D5 showed an increased plasma half-life (t(1/2)) compared to zanamivir following either intravenous or oral administration. This study may represent a new start point for the future development of improved anti-AIV agents., (Copyright © 2012 Elsevier Masson SAS. All rights reserved.)

A single inhaled dose of laninamivir octanoate (LO), a long-acting neuraminidase inhibitor, exhibits efficacy in treating both adult and pediatric patients with influenza virus infection. The intrapulmonary pharmacokinetics (PK) of LO and laninamivir, a pharmacologically active metabolite, were investigated by a single-center, open-label study of healthy adult volunteers. Subgroups of five subjects each underwent bronchoalveolar lavage (BAL) 4, 8, 24, 48, 72, 168, and 240 h following a single inhaled administration of LO (40 mg). Plasma, BAL fluid, and alveolar macrophages (AM) were analyzed to determine LO and laninamivir concentrations, using validated liquid chromatography-tandem mass spectrometry methods. The concentrations in epithelial lining fluid (ELF) and AM from the first and subsequent BAL fluid samples were determined separately to explore the drug distribution in airways. Mean laninamivir concentrations in ELF, calculated using the first BAL fluids and BAL fluids collected 4 h after inhaled administration, were 8.57 and 2.40 μg/ml, respectively. The laninamivir concentration in ELF decreased with a longer half-life than that in plasma, and it exceeded the 50% inhibitory concentrations for viral neuraminidases at all time points examined for 240 h after the inhalation. Laninamivir exposure in ELF from the first BAL samples was 3.2 times higher than that in ELF from the subsequent BAL fluid samples. ELF concentration profiles of laninamivir support its long-lasting effect for treatment of patients with influenza virus infection by a single inhaled administration.

Laninamivir octanoate (Inavir(®); Daiichi Sankyo, Tokyo, Japan) is an anti-influenza drug that provides complete treatment by a single inhalation. It works as a long-acting neuraminidase (NA) inhibitor by means of high and continuous exposure of laninamivir, its active metabolite, in the lungs of mice after intranasal administration. Even after 6 days after intranasal administration of 236 μg/kg laninamivir octanoate, the concentration of laninamivir in the lungs was maintained more than 2-3 orders higher than 50% inhibitory concentrations of laninamivir to N1 NAs, about 2 orders higher than N2 NA of seasonal influenza A viruses, and more than about 50 times higher than influenza B virus NA. From A/H1N1 influenza virus-infected and laninamivir octanoate-treated mice, no low-susceptibility mutants to laninamivir were obtained. In contrast, four different mutants to oseltamivir were obtained from mice administered oseltamivir phosphate, which required repeated administration for treatment under the experimental condition, showing similar virus load reduction between both compounds. This finding suggested the unique characteristics of laninamivir octanoate in mice may work suppressively to generate low-susceptibility mutants.

The purpose of this study was to improve the membrane permeability and oral absorption of the poorly permeable anti-influenza agent, zanamivir. The poor oral bioavailability is attributed to the high polarity (cLogP ∼ -5) resulting from the polar and zwitterionic nature of zanamivir. In order to improve the permeability of zanamivir, prodrugs with amino acids were developed to target the intestinal membrane transporter, hPepT1. Several acyloxy ester prodrugs of zanamivir conjugated with amino acids were synthesized and characterized. The prodrugs were evaluated for their chemical stability in buffers at various pHs and for their transport and tissue activation by enzymes. The acyloxy ester prodrugs of zanamivir were shown to competitively inhibit [(3)H]Gly-Sar uptake in Caco-2 cells (IC(50): 1.19 ± 0.33 mM for L-valyl prodrug of zanamivir). The L-valyl prodrug of zanamivir exhibited ∼3-fold higher uptake in transfected HeLa/hPepT1 cells compared to wild type HeLa cells, suggesting, at least in part, carrier mediated transport by the hPepT1 transporter. Further, enhanced transcellular permeability of prodrugs across Caco-2 monolayer compared to the parent drug (P(app) = 2.24 × 10(-6) ± 1.33 × 10(-7) cm/s for L-valyl prodrug of zanamivir), with only parent zanamivir appearing in the receiver compartment, indicates that the prodrugs exhibited both enhanced transport and activation in intestinal mucosal cells. Most significantly, several of these prodrugs exhibited high intestinal jejunal membrane permeability, similar to metoprolol, in the in situ rat intestinal perfusion system, a system highly correlated with human jejunal permeability. In summary, this mechanistic targeted prodrug strategy, to enhance oral absorption via intestinal membrane carriers such as hPepT1, followed by activation to parent drug (active pharmaceutical ingredient or API) in the mucosal cell, significantly improves the intestinal epithelial cell permeability of zanamivir and has the potential to provide the high oral bioavailability necessary for oral zanamivir therapy.

Zanamivir serum and pulmonary pharmacokinetics were characterized following intravenous (i.v.) or oral inhaled administration. I.v. zanamivir was given as intermittent doses of 100 mg, 200 mg, and 600 mg every 12 h (q12h) for two doses or as a continuous infusion (6-mg loading dose followed by 3 mg/h for 12 h). Oral inhaled zanamivir (two 5-mg inhalations q12h for two doses) was evaluated as well. Each zanamivir regimen was administered to six healthy subjects with serial pharmacokinetic sampling. In addition, a single bronchoalveolar lavage (BAL) fluid sample was collected at various time points and used to calculate epithelial lining fluid (ELF) drug concentrations for each subject. For intermittent i.v. administration of 100 mg, 200 mg, and 600 mg zanamivir, the median zanamivir concentrations in ELF collected 12 h after dosing were 74, 146, and 419 ng/ml, respectively, each higher than the historic mean 50% inhibitory concentrations for the neuraminidases of wild-type strains of influenza A and B viruses. Median ELF/serum zanamivir concentration ratios ranged from 55 to 79% for intermittent i.v. administration (when sampled 12 h after the last dose) and 43 to 45% for continuous infusion (when sampled 6 to 12 h after the start of the infusion). For oral inhaled zanamivir, the median zanamivir concentrations in ELF were 891 ng/ml for the first BAL fluid collection and 326 ng/ml for subsequent BAL fluid collections (when sampled 12 h after the last dose); corresponding serum drug concentrations were undetectable. This study demonstrates zanamivir's penetration into the human pulmonary compartment and supports the doses selected for the continuing development of i.v. zanamivir in clinical studies of influenza.

Oseltamivir and zanamivir are well-established and well-researched drugs for the treatment of influenza in Japan and the rest of the world. A new neuraminidase inhibitor, laninamivir octanoate, has been approved for use in Japanese clinics. Laninamivir octanoate is an inhaled drug with unique characteristics. The inhaled laninamivir octanoate is converted into its active form, laninamivir, in the lungs where a high concentration persists for a long period of time. The concentration of laninamivir exceeds the level necessary for influenza virus replication inhibition for at least 5 days, thus influenza can be treated with a single administration. The drug is delivered using one device requiring four inhalations for children and two devices requiring eight inhalations for adults. Clinical trials have shown comparable efficacy for laninamivir octanoate and oseltamivir. Laninamivir octanoate also displayed a sufficient antiviral effect to treat infection with H275Y-mutated oseltamivir-resistant virus. Laninamivir octanoate has displayed clinical efficacy comparable to that of oseltamivir and zanamivir against the H1N1 pandemic influenza strain from 2009, seasonal H3N2 influenza and influenza B viruses. The prophylactic efficacy of laninamivir octanoate has been shown in animal models. The effectiveness of laninamivir against the highly pathogenic avian influenza virus H5N1 has also been shown in vitro and in animal models. A major clinical benefit of this drug is that the single administration is very convenient for both the patient and doctor, which leads to improved compliance. Furthermore, this drug shows promise for the treatment of influenza in future pandemics.

There is no parenteral formulation of the neuraminidase inhibitor oseltamivir, the most widely used anti-influenza virus drug. Oseltamivir resistance is an increasing problem. Zanamivir is effective against the most prevalent oseltamivir-resistant influenza viruses. A parenteral formulation of zanamivir is in development for the treatment of severe influenza. It is not known if there is any pharmacokinetic interaction between the two drugs. Sixteen healthy Thai adult volunteers were studied in an open-label, four-period, randomized two-sequence crossover pharmacokinetic study in which zanamivir was given by constant-rate infusion or slow intravenous injection either alone or together with oral oseltamivir. Plasma concentration profiles of oseltamivir, the active metabolite oseltamivir carboxylate, and zanamivir were measured by liquid chromatography-mass spectrometry-mass spectrometry. Both drugs were well tolerated alone and in combination. The maximum plasma concentrations and the areas under the plasma concentration-time curves (AUC) of oseltamivir and oseltamivir carboxylate were not significantly different when oseltamivir was given separately or together with zanamivir. Maximum plasma concentrations of zanamivir were 10% (95% confidence interval, 7 to 12%) higher when zanamivir was infused concurrently with oral oseltamivir than with infusions before or after oral oseltamivir. The plasma zanamivir total AUC was positively correlated with the total oseltamivir carboxylate AUC (Pearson's correlation coefficient [r(P)] = 0.720, P = 0.002, n = 16) but not with the oseltamivir AUC (r(p) = 0.121, n = 16). There is no clinically significant pharmacokinetic interaction between oseltamivir and zanamivir.

Zanamivir is currently used for the treatment of H1N1 and H5N1 influenza viruses. Due to its highly hydrophilic property, zanamivir has poor oral bioavailability. Liposomal formulations are known to improve oral absorption of hydrophilic drugs. The present study investigates the effect of liposomes encapsulating zanamivir on the permeation of zanamivir across Caco-2 monolayers. Among the formulations studied, neutral liposomes composed of Phospholipon(®) 90 G and cholesterol at molar ratio of 7:3 gave the highest entrapment efficiency of zanamivir. The extrusion of liposomes loading zanamivir (LZV) resulted in the reduced-size liposomal zanamivir (RLZV), which had mean diameter at 283±42 nm and gave higher encapsulation efficiency of zanamivir at 34.69±6.37% compared to 28.32±5.25%. Transport studies across Caco-2 cell monolayers showed that the apparent permeation coefficients (P(app)) of LZV and RLZV were respectively 2.2- and 3.0-fold greater than that of zanamivir solution. The P(app) of RLZV was 1.4-fold higher than that of LZV. On the basis of these results, liposomes are able to improve permeability of zanamivir across the Caco-2 monolayers, thereby possibly enhancing oral bioavailability of zanamivir.

This open-label, single-dose study assessed the safety and pharmacokinetics of laninamivir, a new long-acting neuraminidase inhibitor, after an inhaled 20-mg dose of its prodrug, CS-8958, to a total of 20 subjects with normal, mild, moderate, or severe renal impairment. CS-8958 and laninamivir concentrations were measured in plasma and urine by validated liquid chromatography tandem mass spectrometry methods. The area under the concentration-time curve extrapolated to infinity (AUC(0-inf)), maximum concentration (C(max)), and time to C(max) of CS-8958 did not change with the degree of renal impairment, whereas the half-life (t(1/2)) of CS-8958 increased with increasing renal insufficiency. The AUC(0-inf) and C(max) of laninamivir tended to increase along with the decrease of creatinine clearance. The AUC(0-inf) of laninamivir compared with normal subjects increased 1.10-, 2.03-, and 4.92-fold in subjects with mild, moderate, and severe renal impairment, respectively, without changing t(1/2) among the subjects. Renal clearance of both CS-8958 and laninamivir was well correlated with creatinine clearance. These data indicate that the rate-limiting step for the elimination of laninamivir would not be the renal excretion rate but rather the drug release rate to plasma from the retained tissues. CS-8958 was well tolerated by all the subjects, although increasing renal dysfunction leads to increasing systemic exposure to laninamivir, particularly in severe renal insufficiency.

Phase 1 studies of laninamivir, a novel long-acting neuraminidase inhibitor, were carried out to assess its safety, tolerability, and pharmacokinetics after inhaled administration of its prodrug, CS-8958. Healthy male volunteers (total N = 76) participated in double-blind, randomized, placebo-controlled trials and received 5, 10, 20, 40, 80, or 120 mg of a single dose or 20 or 40 mg of a twice-daily dose for 3 days. The clinical and laboratory parameters and plasma and urinary concentrations of CS-8958 and laninamivir for 144 hours post dosing were measured. There were no adverse events related to the test drug. CS-8958 disappeared from plasma with a half-life of about 2 hours, although laninamivir was slowly eliminated from the body, lasting for even up to 144 hours after administration with a half-life of about 3 days. Area under the curve and maximum concentration increased almost linearly with the dose administered. The cumulative urinary excretion amounts of CS-8958 and laninamivir were 2.3% to 3.6% and 10.7% to 14.6% of the dose, respectively. The half-life of the urinary excretion rates of laninamivir at higher single dose is comparable to plasma half-life. CS-8958, when inhaled by healthy volunteers, is well tolerated and exhibits a suitable pharmacokinetic profile, suggesting potential for long-lasting anti-influenza activity.

Antiviral drugs often suffer from poor intestinal permeability, preventing their delivery via the oral route. The goal of this work was to enhance the intestinal absorption of the low-permeability antiviral agents zanamivir heptyl ester (ZHE) and guanidino oseltamivir (GO) utilizing an ion-pairing approach, as a critical step toward making them oral drugs. The counterion 1-hydroxy-2-naphthoic acid (HNAP) was utilized to enhance the lipophilicity and permeability of the highly polar drugs. HNAP substantially increased the log P of the drugs by up to 3.7 log units. Binding constants (K(11(aq))) of 388 M(-1) for ZHE-HNAP and 2.91 M(-1) for GO-HNAP were obtained by applying a quasi-equilibrium transport model to double-reciprocal plots of apparent octanol-buffer distribution coefficients versus HNAP concentration. HNAP enhanced the apparent permeability (P(app)) of both compounds across Caco-2 cell monolayers in a concentration-dependent manner, as substantial P(app) (0.8-3.0 x 10(-6) cm/s) was observed in the presence of 6-24 mM HNAP, whereas no detectable transport was observed without counterion. Consistent with a quasi-equilibrium transport model, a linear relationship with slope near 1 was obtained from a log-log plot of Caco-2 P(app) versus HNAP concentration, supporting the ion-pair mechanism behind the permeability enhancement. In the rat jejunal perfusion assay, the addition of HNAP failed to increase the effective permeability (P(eff)) of GO. However, the rat jejunal permeability of ZHE was significantly enhanced by the addition of HNAP in a concentration-dependent manner, from essentially zero without HNAP to 4.0 x 10(-5) cm/s with 10 mM HNAP, matching the P(eff) of the high-permeability standard metoprolol. The success of ZHE-HNAP was explained by its >100-fold stronger K(11(aq)) versus GO-HNAP, making ZHE-HNAP less prone to dissociation and ion-exchange with competing endogenous anions and able to remain intact during membrane permeation. Overall, this work presents a novel approach to enable the oral delivery of highly polar antiviral drugs, and provides new insights into the underlying mechanisms governing the success or failure of the ion-pairing strategy to increase oral absorption.

CS-8958, a prodrug of laninamivir (R-125489), is currently under development as an inhaled anti-influenza drug. In this study, the pharmacokinetics and disposition of CS-8958 were characterized in rats. After intratracheal administration of 14C-CS-8958, radioactivity was retained over long periods in the target tissues (trachea and lung) as its active metabolite R-125489 - 19.12% of the dose was retained in the lung at 24 h. After intratracheal administration of CS-8958, plasma R-125489 concentration was slowly eliminated, and its half-life (14.1 h) was considerably longer than that after intravenous administration of R-125489. The radioactivity of intratracheally administered 14C-CS-8958 was mainly excreted into the urine (67.5% of dose), and this excretion lasted over long periods. R-125489 accounted for most of the urinary radioactivity recovered after 24 h. These results demonstrated that CS-8958 administered intratracheally to rats was converted/hydrolysed to R-125489 in the target tissues, and that the R-125489 was slowly excreted into the urine via an absorption rate-limiting process. Such distinctive pharmacokinetics attributed to the slow release of R-125489 suggests the potential for a long-acting anti-influenza drug.

CS-8958 is a prodrug of the pharmacologically active form R-125489, a selective neuraminidase inhibitor, and has long-acting anti-influenza virus activity in vivo. In this study, the tissue distribution profiles after a single intranasal administration of CS-8958 (0.5 micromol/kg of body weight) to mice were investigated, focusing especially on the retention of CS-8958 in the respiratory tract by comparing it with R-125489 and a marketed drug, zanamivir. After administration of [(14)C]CS-8958, radioactivity was retained in the respiratory tract over long periods. At 24 h postdose, the radioactivity concentrations after administration of [(14)C]CS-8958 were approximately 10-fold higher in both the trachea and the lung than those of [(14)C]R-125489 and [(14)C]zanamivir. The [(14)C]CS-8958-derived radioactivity present in these two tissues consisted both of unchanged CS-8958 and of R-125489 at 1 h postdose, while only R-125489, and no other metabolites, was detected at 24 h postdose. After administration of unlabeled CS-8958, CS-8958 was rapidly eliminated from the lungs, whereas the lung R-125489 concentration reached a maximum at 3 h postdose and gradually declined, with an elimination half-life of 41.4 h. The conversion of CS-8958 to R-125489 was observed in mouse trachea and lung S9 fractions and was inhibited by esterase inhibitors, such as diisopropylfluorophosphate and bis-p-nitrophenylphosphate. These results demonstrated that CS-8958 administered intranasally to mice was efficiently converted to R-125489 by a hydrolase(s) such as carboxylesterase, and then R-125489 was slowly eliminated from the respiratory tract. These data support the finding that CS-8958 has potential as a long-acting neuraminidase inhibitor, leading to significant efficacy as an anti-influenza drug by a single treatment.

Oseltamivir and zanamivir are highly potent inhibitors of influenza A and B neuraminidase and operate by inhibiting viral replication, and more specifically, the release and the movement of the virus through mucus. Neuraminidase inhibitors reduce the severity and duration of symptoms, and prevent clinical influenza as post-exposure and seasonal prophylaxis. Both have similar efficacy; oseltamivir has a more convenient route of administration, and zanamivir a more favourable resistance profile. Pending availability of effective vaccines, neuraminidase inhibitors are the only specific antiviral drugs which might be opposed to a possible pandemic that could emerge from the current highly pathogenic H5N1 virus. Although the effectiveness of oseltamivir and zanamivir for the therapy of clinical H5N1 influenza is questionable, simulation models suggest that a combination of targeted antiviral prophylaxis and quarantine might be able to contain an emerging influenza strain at the source. As a consequence, after an initial lack of commercial success probably related to the mild intensity of seasonal influenza during the last winters, neuraminidase inhibitors are now stockpiled by many countries to prepare for an outbreak.