ANTIVIRAL DRUGS FOR INFLUENZA VIRUSES

Antiviral drugs have significant action against influenza viruses A and B. Virus spread deadly disease in which many people die, and the country economy greatly suffer. Presently, most of the people need to get vaccination, which is depending on the dose limit in humans. It reacts directly or sometimes indirectly in the form of metabolites. However, it is mandatory to know how much drug is absorbed or metabolites concentration after administered. Therefore, pharmacokinetics data is very crucial for all drugs. Our review discusses the mechanism of drugs action and their activity and also describes how antiviral drugs and their metabolites are determined using highly sensitive instruments such as high-performance liquid chromatography (HPLC), ultra-pressure liquid chromatography (UPLC), and mass spectrometry (MS). Therefore, the present review gives brief information about antiviral drugs, their activity, biotransformation and analytical methods for quantification and this information will be helpful for any future studies done by experts in this field and will be beneficial for research scientists and influenza experts of all over the globe.


INTRODUCTION
Respiratory system is commonly infected with influenza viruses [1] and mainly orthomyxoviridae family (A, B, C). Worldwide, 2.5-5 hundred thousand people were reported death and 3-5 million diagnosed with acute illness [2] annually. Viruses classified with respect to activity of glycoproteins neuraminidase (N/NA), hemagglutinin (H/HA) and M2 ion channel. Influenza A and B are seasonal influenza with different subtypes such as H1-H16 (16 hemagglutinin) and N1-N9 (9 neuraminidase). Well-known viruses H1N1 and H3N2 are generally subtyped of seasonal virus (Influenza A). The avian influenza virus H5N1 [3][4][5] have great interest among the people but swine influenza (novel H1N1) is completely different from seasonal H1N1 influenza virus [6]. In the 20 th century, 20-50 million people from different countries were killed with Spanish flu, 1-2 million from Asian flu, and 7 hundred thousand from Hong Kong flu [7,8]. Virus infection was initiated by hemagglutinin after binding with a receptor called Sialic acid, 1 ( fig. 1). Then other cells infected by new viruses with the help of neuraminidase [9,10]. Life threat influenza can only be overcome with annual immunization. However, the limitation related to efficacy, timeline and design of the vaccine production increase the need for antiviral drugs. The approved antiviral drugs M2 ion channel and NA inhibitors are the basic option for therapeutic treatment with prevention. This classification based on resistance behavior, tolerance study, pharmacokinetics and drugs mechanism of action [11][12][13][14][15][16][17].

Neuraminidase inhibitors
Proteins (H and N) are available on the walls of influenza virus surfaces. New viral molecule was released by host cell due to enzymatic function of neuraminidase, will help to catalyzed glycoside hydrolysis. As a result, α-ketosidically connected sialic acid with glycolipid, glycoprotein, and hemagglutinin divided increased infectious molecule. However, the nascent virus is still securing with host cell due to an interaction between HA/HA receptor. The viral molecule exhibit HA/HA receptors on the surface, which enables new molecules stick together. The viral particles emerged from the clump and host cell when neuraminidase receptor removed sialic acid. In the respiratory, glycosylated components are digested by neuraminidase, contribute viral infection. Therefore, design-based modern drug act on the site of neuraminidase inhibitors, depend on its clinical efficacy, mechanism, genetic role are the key feature for success [18][19][20]. The neuraminidase inhibitors prompt action and excellent mortality is the main reason for its demand [21,22]. The drugs and their pharmaceutical formulations worked for the treatment of deadly disease are Oseltamivir 2, Laninamivir 3, Zanamivir 4, Peramivir 5 [23][24][25] (fig. 1), established on sialic acid transformation [26][27][28]. Influenza experts from different countries set up a neuraminidase inhibitors susceptibility network (NISN) for the outcome of clinical result and monitoring development process. This committee is participating to monitor liability studies for encouraging the people to know the exact method of evaluation and investigation process to control influenza virus.

M2 ion channel inhibitors
Amantadine 6 and Rimantadine 7 ( fig. 2) licensed by United States of America (USA) in the years 1966 and 1993 respectively for the treatment of influenza A as M2 ion inhibitor. Allosteric inhibition controls the movement and activity of M2 by the presence of methyl group in amantadine; consequently, it blocks the release of RNA. It was found that M2 ion is successful for influenza A but ineffective towards influenza B. Therefore, these drugs are precise only for influenza A. M2 inhibitor has extraordinary response to antibiotic resistance [29,30], but it is very less compared to neuraminidase inhibitors [31,32]. Through endocytosis process, host cell allows viruses to enter. At low pH, M2 channel permits the virus to viral interior due to an activity of endosome responsible for maturation and growth of molecule. The reproduction is continued in cytoplasm, viral ribonucleic acid (RNA) and genetic material released from protein matrix [33]. Therefore, it is very important to develop inhibitors functioning against the virus but in native condition. Oseltamivir phosphate 8 is significant ester prodrug, applied orally for prevention and curing of influenza disease (A and B). It's brand name Tamiflu is available in quantity of 75 mg. The approved regimen for adults is 75 mg daily two times for 5 d with respect to influenza (A and B), but it is different for the prophylactic regimen. For control and prevention, daily dose is 75 mg once; for a minimum of 10 d up to 42 d. The intake dose for healthy participants follows linear pharmacokinetics in the range of 75-675 mg [34][35][36].
Oseltamivir is absorbed in the gastrointestinal tract then metabolized and converted into Oseltamivir carboxylate 9 ( fig. 3) in the liver and later on, uniformly distributed in the body. After oral administration, therapeutic action continues for 30 min with the metabolite (80%) but the remaining 20% available 3-4 h. in plasma. The drug half-life is 1.8h. It was found that renal disease may affect active metabolite clearance and found that cytochrome P450 has only 3% interaction with plasma protein. Similar action occurs during glucuronosyl transferases [37][38].

Zanamivir
Relenza is the trade name of Zanamivir, an anti-influenza inhibitor, which obtained the regulatory approval in 1999. It targets respiratory site and available as inhaled powder. The pharmaceutical formulation has high potential against people diagnosed with deadly disease influenza A (H5N1). The maximum amount of 20 mg (10 mg twice) is allowed daily as inhalation, continued for 5 consecutive days. However, for prophylaxis, time and quantity limit is 10 mg daily for 28 d for children age five or more, and same for adults [39][40]. The nasal spray set down into posterior nasal and nostril approximately 88% but 13% in lung, 78% in oropharynx deposited active site for replication of influenza virus [41]. Settle down in stomach (17%) and lung (2%) dose intake orally after fifty minutes [42]. Sputum and nasal wash sample resulted concentration about 50% inhibitory after 24 and 12 h. respectively [43] for healthy volunteers with 10 mg daily dose but 20 mg amount with infected sample quantified after 4 d [44]. The unchanged drug (90%) was found with excreted urine sample recommended without biotransformation [41]. Therefore, the drug not metabolized in the liver.

Peramivir
Peramivir is a predominant cyclopentane based neuraminidase inhibitor. The binding capacity of the inhibitor will be half (IC50) with a concentration in the range 0.09-1.39 nM and 0.  [45]. The drug is productive without any complication for seasonal influenza viruses [46]. The Peramivir IV was first launched with trade name Rapiacta in Japan (2010). The drug prescribed for acute treatment in adults is 300 mg daily but for severe cases, hospitalized patients, 600 mg daily dose is recommended. The drug eliminated through renal and not metabolized in the human liver.

Laninamivir
The function of Laninamivir 3 is similar to Zanamivir, potent neuraminidase inhibitor for influenza viruses (A and B) [47][48].
Laninamivir octanoate is an inactive prodrug and the commercially approved product (inavir) is available in Japan since 2010 [49]. Single dose of Laninamivir octanoate hydrate is limited to children with age above 10 y is 40 mg and below 10 y is 20 mg. Laninamivir, active metabolite of Laninamivir octanoate (LO) formed within 24 h due to hydrolysis in the octanoyl ester site. When LO is mixed with water in the human body, equilibrated to form 3-acyl form 10, 2-acyl form 11 ( fig. 4) known as major and minor metabolites [50].

Fig. 4: Chemical structures of Laninamivir octanoate 3-acyl form 10 and 2-acyl form 11
Amantadine Amantadine 6 is a very important drug as M2 proton channel inhibitor for influenza A. Symmetrel is the brand name normally used for amantadine. Its rate of absorption is high for young people compared to elder independents. Renal insufficiency does not appear to affect absorption, nor does the formulation used. First relative bioavailability studies of the oral administration reported that 86% recovery of the original dose (2-4 mg/kg) within 96 h from urine samples of five healthy men [51]. In case of ingestion after 72 h, 105 mg (52.5%) of the original dose was found in old man (84 y) urine [52]. Eight metabolites originated during metabolism.
Besides the major metabolite produced by N-acetylation which is Nacetylamantadine 12; other metabolites were observed. These are N

High-performance liquid chromatography
High-performance liquid chromatography (HPLC) is an accurate instrument for pharmaceutical analysis. The inhibitor demand is increasing with time in the world because influenza viruses causes significant health alert. All determined drugs either applying ultraviolet detector or fluorescence detector. As well as used C18 and C8 column with a different manufacturer. The single mobile phase is usually a combination of two or more solvents. The ratio of the mobile phase is changed with time; gradient method investigation resulted in drugs with a short time. During the analysis buffer also added for the preparation of the mobile phase. Therefore, for the quantification of neuraminidase and M2 ion channel inhibitor, numerous HPLC methods are developed based on the mobile phase composition and the stationary phase for pure bulk drug, tablet dosage form and biological fluids (table 1) [ .

High-performance thin layer chromatography
Currently, the high-performance thin layer chromatography (HPTLC) has high demand because it involves fewer samples cleanup, high throughput value, low-cost methodology. The fundamental advantage of HPTLC leads to compute several samples at one time.
Oseltamivir study performed on silica gel 60F-254 coated aluminum plate consists of mobile phase (toluene: methanol: ammonia=3.5:1.5:0.2, v/v/v), detected at Rf value (0.45±0.02) for bulk and its dosage form [103]. This technique applied to evaluate Zanamivir in a pharmaceutical formulation having a correlation coefficient close to one [90,104].

Capillary zone electrophoresis
Capillary zone electrophoresis (CZE) is strong enough to large molecules additionally for small (organic/inorganic), separation technique controlled by electric field inside narrow bore capillaries. The method has several benefits with respect to fewer amounts of solvent, analyte and high resolution, applicable to forensic, environmental, clinical and pharmaceutical industry. It is a specific technique for the separation of the molecule along with enantiomers. The generic version of Oseltamivir was separated on fused capillary under potential (−15 kV) at room temperature. The analysis time is 1.5 min and detected at 226 nm included phosphate buffer [105]. Unmodified silica capillary, 4methylbenzylamine in ethanol (20%) as background electrolyte, maintained+20 V for separation at 210 nm within 3 min in Rimantadine tablets [106]. Pharmaceutical products can be estimated with derivatization agent 1, 2-naphthoquinone-4sulfonic acid by CZE in alkaline medium [107]. Rat plasma [100] Ultra pressure liquid chromatography Ultra pressure liquid chromatography (UPLC) is obligatory in respect to the analysis time; resolution enhanced its reliability in the pharmaceutical industry. The achieved data from developed methods are robust and accurate. The common available columns for UPLC are acquity BEH C8, acquity BEH C18, acquity BEH RP18 and acquity BEH phenyl. UV-Visible detector is commonly used detector for UPLC analysis. The method for Oseltamivir and its degraded product is established within 5 minute using mobile phase composition phosphate buffer, pH 3.5 and methanol (80: 20, v/v), detected at 207 nm [108].

Liquid chromatography-mass spectrophotometry
Liquid chromatography-mass spectrophotometry technique is involved in the physical separation of compounds by liquid chromatography (LC) combined mass spectrometry (MS) for mass analysis [109][110][111][112][113]. Liquid chromatography can be high pressure or ultra-pressure. The structure of molecule elucidate after confirming the chemical composition of charged particles with respect to massto-charge ratio. The molecule ionized by MS to produce fragments and its mass-to-charge ratio measured. This technique maintains high sensitivity compared to traditional techniques and selective to many applications. It has an excellent impact in the bioanalysis field mainly confronted to pharmacokinetics investigation of pharmaceuticals. Generally, LC installed octadecyl (C18) silica column but nowadays hydrophilic interaction based liquid chromatography (HILIC) has received more attention and recommended due to its potential to retain highly polar molecule with hydrophilic nature, but it needs to apply buffer as the mobile phase to minimize analyte and stationary phase ionic interaction. The LCMS method that was reported for inhibitors is presented in table 2 . The LCMS/MS investigation revealed that low concentration of amantadine present in plasma sample, does not require any derivatization during solid phase extraction, the developed method reported successfully bioequivalence study of healthy volunteers [114]. Amantadine-d15 internal standard can be used based on protein precipitation [115]. QuEChERS extraction method applied for chicken muscle, egg, pet treat sample [116,117]. Zanamivir found in effluent sample and water from river in Japan [118]. The antiviral drugs and metabolite quantified applied flow rate (0.2-1 ml/min) having run time between 1-20 minute with biological fluids and water sample [119][120][121][122][123][124][125][126][127][128][129]. Ultra pressure liquid chromatography combined with the mass spectrophotometer (triple quadrupole ion trap) reported in feed sample illegal addition of amantadine and rimantadine [130]. Fourteen antiviral drugs detected within short time of 11 minute, multiple reaction monitoring ensured method specificity [131]. After winter season the inhibitors found in river water [132][133][134]. Amantadine and rimantadine are quantified in chicken muscle using different sorbent [135][136]. The UPLC method was first reported with whole blood sample, assay calculated using dried blood spot [137]. The minimum amount of sample required, elute from the column within 6 minute. The correlation coefficient of Oseltamivir and its active metabolites Oseltamivir carboxylate was greater than 0.99. Sample collection is very important parameter for this analysis. Therefore, the developed method validated successfully for human plasma of healthy volunteers.

CONCLUSION
Antiviral drugs are very useful versus influenza viruses and molecules that are actively participated for deadly diseases. The developed novel drugs are showing great potential for the prevention of influenza. It was evident that for controlling influenza, administered single dose drug to human and all animals including farm animals is required. All inhibitors can be applied during epidemic or seasonal conditions. Their main function, through vaccination, is to control the activity of viruses, reduce and manage pandemic viruses. All these mentioned drugs are also important for patients hospitalized due to influenza. Although the existing drugs are doing well, influenza experts are looking forward to identify other new drugs that exert antiviral activity like the approved ones and can be applicable as future antiviral agents for influenza viruses. Therefore, the present review gives brief information about antiviral drugs, their activity, biotransformation and analytical methods for quantification and this information will be helpful for any future studies done by experts in this field.