Sensitive and rapid determination of amantadine without derivatization in human plasma by LC–MS/MS for a bioequivalence study

A highly sensitive, rapid and rugged liquid chromatography-tandem mass spectrometw （LC-ESI-MS/MS） method was developed for reliable estimation of amantadine （AMD）, an antiviral drug in human plasma. The analyte and internal standard （IS）, amantadine-d6 （AMD-d6）, were extracted from 200μL plasma by solid phase extraction on Phenomenex Strata-X-C 33 ~t cartridges. Chromatography was performed on Synergi^TM Hydro-RP C18 （150 mm×4.6 mm, 4 μm） analytical column using a mixture of acetonitrile and 10 mM ammonium formate, pH 3.0 （80：20, v/v） as the mobile phase. Detection and quantitation was done by multiple reaction monitoring in the positive ionization mode for AMD （m/z 152.1→ 135.1 ） and IS （m/z 158.0 → 141.1） on a triple quadrupole mass spectrometer. The assay was linear in the concentration range of 0.50-500 ng/mL with correlation coefficient （r^2） 〉 0.9969. The limit of detection of the method was 0.18 ng/mL The intra-batch and inter-batch precisions were 〈 5.42% and the accuracy varied from 98.47% to 105.72%. The extraction recovery of amantadine was precise and quantitative in the range of 97.89%-100.28%. IS-normalized matrix factors for amantadine varied from 0.981 to 1.012. The stability of AMD in whole blood and plasma was evaluated under different conditions. The developed method was successfully applied for a bioequivalence study with 100 mg of AMD in 32 healthy volunteers. The reproducibility of the assay was determined by reanalysis of 134 subject samples.

Use of Graphene and Cucurbit[7]uril Electrodes for the Determination of Amantadine in Biological Fluids

A differential pulse voltammetry (DPV) method for amantadine (AT) determination is developed. To this end, all the chemical and instrumental variables affecting the determination of amantadine are optimized. These studies have used three types of glassy-carbon electrode, first electrode which has not undergone surface modification or coating, to then modify the working electrode surface with two kinds of suspensions: graphene and graphene-cucurbit[7]uril (CB[7]). From studies of the mechanisms governing the electrochemical response of amantadine, it was concluded that it is an electrochemically system with a diffusive reduction phenomenon. Under optimal conditions and with the appropriate electrode modification, we proceed to study the relation between the peak intensity with the analyte concentration. Thus, we find that when the electrode surface is covered with graphene-CB[7], two linear sections are obtained, the first one in the concentration range of between 0.05 μg·mL﹣1 and 0.75 μg·mL﹣1;and the second one between 1.00 μg·mL﹣1 and 6.00 μg·mL﹣1, with Er (%) = 87 and R.S.D. = 0.94% (n = 10 at 0.5 μg·mL﹣1 level). The minimum detectable amount was 15 ng·mL﹣1 while a concentration of 44 ng·mL﹣1 was calculated as determination limit. The optimized method was applied to the determination of amantadine in biological fluids.

Synthesis and Crystal Structures of Novel Keggin and Dawson Polyoxometalates Containing Amantadine

Two novel polyoxometalates containing pharmaceutical component amantadine, formulated with (C10H18N) 5PMo12O40Cl2 5H2O (I) and (C10H18N) 6As2Mo18O62 6CH3CN 6H2O (II) were first synthesized and characterized by IR, UV-Vis spectra and X-ray diffraction. Structural analyses of I and II suggested that polyanions in these compounds were reserved their Keggin or Dawson structures and were linked to amantadine through electrostatic interaction and hydrogen bonding.

Determination of Amantadine Residue in Honey by Solid-phase Extraction and High-performance Liquid Chromatography with Pre-column Derivatization and Fluorometric Detection

Amantadine （AMA） is an anti-viral drug used in apiculture to protect honeybee against the sacbrood virus （Morator aetatulae）. This study described a reliable high-performance liquid chromatographic （HPLC） method for analyzing AMA in honey using a solid-phase extraction （SPE） cartridge （Plexa PCX） for purification, 4-fluoro-7- nitro-2,1,3-benzoxadiazole （NBD-F） as a pre-column derivatization agent, and fluorometric detection （λex =470 nm, λem=530 nm）. The chromatographic separation was performed on an XDB C18 column （150×4.6 mm i.d.） using 0.1% trifluoroacetic acid/acetonitrile （35 ; 65, V/V） as the mobile phase at a flow rate of 1.0 mLomin 1 with a run time of 20 min. Under these optimal conditions, a linear relationship was observed in the range of 0.025--1.0μg·mL-1 with a good correlation coefficient （0.998） and low limit of detection （0.0080 μg·g-1）, the recoveries were all above 90%, and the intra-day and inter-day precision （RSD） ranged from 3.4%--5.1%.

Triple antiviral therapy in HCV positive patients who failed prior combination therapy

AIM： To assess the efficacy of triple therapy （peginterferon or high dose standard interferon, plus ribavirin and amantadine） in nonresponders to prior combination therapy. METHODS： A total of 196 patients were enrolled in a multicenter, open, randomized study. Patients were given 180 μg/wk of peginterferon-alpha-2a （40 kD） plus ribavirin （800-1000 rag/d） and amantadine （200 rag/d） for 48 wk （group A） or interferon-alpha-2a （6 MU/d for 4 wk, 3 MU/d for 20 wk, and 3 MU tiw for 24 wk） plus ribavirin （800-1000 rag/d） and amantadine （200 rag/d） for 48 wk （group B）. RESULTS： Overall sustained virologic response （SVR） was 26.6% （32.1% and 19.5% in group A and B, P = 0.057）. Baseline ALT 〉120 UI/L （OR 2.4; 95% CI：1.11 to 5.20; P = 0.026） and HCV RNA negativity after 12 wk （OR 8.7; 95% CI： 3.87 to 19.74; P 〈 0.0001） were independently associated with SVR. Therapy discontinuation occurred less frequently in patients treated with peginterferon than standard interferon （P = 0.036）. CONCLUSION： More than 25% of nonresponders to combination therapy can eradicate HCV infection when retreated with triple therapy, especially if they have a high baseline ALT and are treated with pegylated interferon.

Ion Channels as Antivirus Targets

Ion channels are membrane proteins that are found in a number of viruses and which are of crucial physiological importance in the viral life cycle. They have one common feature in that their action mode involves a change of electrochemical or proton gradient across the bilayer lipid membrane which modulates viral or cellular activity. We will discuss a group of viral channel proteins that belong to the viroproin family, and which participate in a number of viral functions including promoting the release of viral particles from cells. Blocking these channel-forming proteins may be ＂lethal＂, which can be a suitable and potential therapeutic strategy. In this review we discuss seven ion channels of viruses which can lead serious infections in human beings： M2 of influenza A, NB and BM2 of influenza B, CM2 of influenza C, Vpu of HIV-1, p7 of HCV and 2B of picomaviruses.

Flu channel drug resistance:a tale of two sites

The M2 proteins of influenza A and B virus,AM2 and BM2,respectively,are transmembrane proteins that oligomerize in the viral membrane to form proton-selective channels.Proton conductance of the M2 proteins is required for viral replication;it is believed to equilibrate pH across the viral membrane during cell entry and across the trans-Golgi membrane of infected cells during viral maturation.In addition to the role of M2 in proton conductance,recent mutagenesis and structural studies suggest that the cytoplasmic domains of the M2 proteins also play a role in recruiting the matrix proteins to the cell surface during virus budding.As viral ion channels of minimalist architecture,the membrane-embedded channel domain of M2 has been a model system for investigating the mechanism of proton conduction.Moreover,as a proven drug target for the treatment of influenza A infection,M2 has been the subject of intense research for developing new anti-flu therapeutics.AM2 is the target of two anti-influenza A drugs,amantadine and rimantadine,both belonging to the adamantane class of compounds.However,resistance of influenza A to adamantane is now widespread due to mutations in the channel domain of AM2.This review summarizes the structure and function of both AM2 and BM2 channels,the mechanism of drug inhibition and drug resistance of AM2,as well as the development of new M2 inhibitors as potential anti-flu drugs.

Direct-acting Antivirals and Host-targeting Agents against the Hepatitis A Virus

Hepatitis A virus (HAV) infection is a major cause of acute hepatitis and occasionally leads to acute liver failure in both developing and developed countries.Although effective vaccines for HAV are available,the development of new antivirals against HAV may be important for the control of HAV infection in developed countries where no universal vaccination program against HAV exists,such as Japan.There are two forms of antiviral agents against HAV:direct-acting antivirals (DAAs) and host-targeting agents (HTAs).Studies using small interfering ribonucleic acid (siRNA) have suggested that the HAV internal ribosomal entry site (IRES) is an attractive target for the control of HAV replication and infection.Among the HTAs,amantadine and interferon-lambda 1 (IL-29) inhibit HAV IRES-mediated translation and HAV replication.Janus kinase (JAK) inhibitors inhibit La protein expression,HAV IRES activity,and HAV replication.Based on this review,both DAAs and HTAs may be needed to control effectively HAV infection,and their use should continue to be explored.