Technique validation by liquid chromatography for the determination of acyclovir in plasma

In this research project, a high-performance liquid chromatography (HPLC) method was developed for the determination of acyclovir (ACV) in plasma. The plasma samples, recharged with acyclovir and in presence of 5'-N-methylcarboxyamidoadenosine (MECA) as an internal standard, were purified using a solid-phase extraction technique with Waters Oasis HLB columns. The separation of the components from the extract was carried out in a LiChrospher 100 RP-18 column for further ultraviolet detection at a wavelength range of 250-260 nm. The mobile phase composition was 18% acetonitrile, sodium dodecylsulphate 5 mM and phosphate buffer at pH 2.6 with an analysis time of 13 min per sample. The average retention time for acyclovir was of 5.0 min and for the internal standard 11.2 min. The calibration curve was linear ranging between 0.05 and 1.80 microg/ml. The detection limit was 0.006 microg/ml with a quantification limit of 0.020 microg/ml. The ACV recuperation percentage for 250 microl of plasma was between 94.7 and 109.7% with a coefficient of variation not higher than 5.2%. This method was developed and validated for use in bioavailability and bioequivalence studies.     

Design of xanthone propionate photolabile protecting group releasing acyclovir for the treatment of ocular herpes simplex virus

We have attached the antiviral drug acyclovir (ACV) to a xanthone photolabile protecting group (or photocage) through the O6 position of acyclovir, a procedure designed for the treatment of ocular herpes simplex virus infections. Acyclovir is photoreleased from the photocage, under physiological conditions, with a quantum yield (Φ(ACV?release)) of 0.1-0.3 and an uncaging cross section (Φ·ε) of 450-1350 M cm(-1). We demonstrate that this photorelease method outcompetes alternative reaction pathways, such as protonation. Furthermore, complete release of the drug is theoretically possible given a sufficient dose of light . Surprisingly the acyclovir photocage, also showed some antiviral activity towards HSV-1.     

青蒿鞣质抗病毒活性研究

从青蒿水提物中分离得到一种缩合鞣质(CTA),在体外进行了抗HSV-2和HBV活性研究。结果表明,CTA具有显著抗HSV-2活性,与阳性对照药物阿昔洛韦(ACV)比较,OC50分别为6．84mg／mL和3．69mg／mL,IG50分别为0．162mg／mL和0．138mg／mL,表明CTA的细胞毒性比ACV小,最低抗病毒活性浓度相当。用含CTA的培养基培养HepC2．2．1．5细胞,结果表明在浓度为2．5mg／mL时有轻微细胞毒性,在浓度为2．5～0．156mg／mL对HepG2．2．1．5细胞表达的HBeAg具有显著的抑制作用,0．625mg／mL浓度档培养第12d对HBeAg抑制率达到90．45％,提示CTA具有潜在的抗HBV活性。     

Determination of acyclovir in human serum by high-performance liquid chromatography using liquid-liquid extraction and its application in pharmacokinetic studies

A fast, simple and sensitive high performance liquid chromatographic (HPLC) method has been described for determination of acyclovir in human serum. Since acyclovir is a polar compound and soluble in aqueous medium and practically insoluble in most of organic solvents, its analysis in biological fluids in currently published HPLC methods, involve pre-treatment of acyclovir plasma sample including deproteinization or solid phase extraction. In present method liquid-liquid extraction of acyclovir and internal standard (vanillin) is achieved using dichloromethane-isopropyl alcohol (1:1, v/v) as an extracting solvent. Analysis was carried out on ODS column using methanol-phosphate buffer (0.05 M) containing sodium dodecyl sulfate (200 mg/L) and triethylamine (2 mL/L, v/v) as mobile phase (pH=2.3; 5:95, v/v) at flow rate of 2 ml/min. The method was shown to be selective and linear into the concentration range of 10-2560 ng/mL. Accuracy and precision of the method were also studied. The limit of quantitation was evaluated to be 10 ng/mL. This method was applied in bioequivalence study of two different acyclovir preparations after administration of 400mg in 12 healthy volunteers.     

Hybrid MC/QC simulations of water-assisted proton transfer in nucleosides. Guanosine and its analog acyclovir

To provide an in-depth insight into the molecular basis of spontaneous tautomerism in DNA and RNA base pairs, a hybrid Monte Carlo (MC)–quantum chemical (QC) methodology is implemented to map two-dimensional potential energy surfaces along the reaction coordinates of solvent-assisted proton transfer processes in guanosine and its analog acyclovir in aqueous solution. The solvent effects were simulated by explicit inclusion of water molecules that model the relevant part of the first hydration shell around the solute. The position of these water molecules was estimated by carrying out a classical Metropolis Monte Carlo simulation of dilute water solutions of the guanosine (Gs) and acyclovir (ACV) and subsequently analyzing solute–solvent intermolecular interactions in the statistically-independent MC-generated configurations. The solvent-assisted proton transfer processes were further investigated using two different ab initio MP2 quantum chemical approaches. In the first one, potential energy surfaces of the ‘bare’ finite solute–solvent clusters containing Gs/ACV and four water molecules (MP2/6-31+G(d,p) level) were explored, while within the second approach, these clusters were embedded in ‘bulk’ solvent treated as polarizable continuum (C-PCM/MP2/6-31+G(d,p) level of theory). It was found that in the gas phase and in water solution, the most stable tautomer for guanosine and acyclovir is the 1H-2-amino-6-oxo form followed by the 2-amino-6-(sZ)-hydroxy form. The energy barriers of the water-assisted proton transfer reaction in guanosine and in acyclovir are found to be very similar – 11.74 kcal mol^?1 for guanosine and 11.16 kcal mol^?1 for acyclovir, and the respective rate constants (k = 1.5?×?10¹ s^?1, guanosine and k = 4.09?×?10¹ s^?1, acyclovir), are sufficiently large to generate the 2-amino-6-(sZ)-hydroxy tautomer. The analysis of the reaction profiles in both compounds shows that the proton transfer processes occur through the asynchronous concerted mechanism.     

Determination of ampicillin and amoxycillin by flow injection chemiluminescence method based on their enhancing effects on the luminol–periodate reaction

In this work, a new flow injection chemiluminescence method is described for the determination of ampicillin and amoxycillin. The method is based on the strong enhancing effects of these antibiotics on the luminol–periodate reaction. The present method allows the measurements of ampicillin in the range 0.02–1.0 mg/L range and amoxycillin in the range 0.1–10.0 mg/L range with the relative standard deviations within 0.8–2.0%. The sampling frequency was calculated about 90/h. The method was successfully applied to the determination of ampicillin and amoxycillin in pharmaceutical preparations. A brief discussion on the possible chemiluminescence reaction mechanism is presented. Copyright © 2003 John Wiley & Sons, Ltd.     

Synthesis,Biological Activity and Decomposition Studies of Amino Acid Phosphomonoester Amidates of Acyclovir

Abstract

Highly stable and water soluble amino acid phosphomonoester amidates of acyclovir (ACV) were synthesized and shown to function predominantly as prodrugs of AC V and not acyclovir monophosphate (AC V-MP) with activities within two fold of the amino acid prodrug of ACV, valaciclovir (VACV). Metabolism studies revealed that incubation of cell-free extracts of Vero cells with the L-leucine phosphomonoester amidate of ACV (3c), resulted in a burst of ACV-MP production followed by the rapid generation of ACV.     

Determination of parathion residues in rice samples using a flow injection chemiluminescence method

A sensitive ?ow injection chemiluminescence method is described for the determination of parathion pesticide, based upon its direct chemiluminescence reaction with luminol and hydrogen peroxide in the presence of non‐ionic surfactant polyethylene glycol 400. Under the selected experimental conditions, the concentration of parathion is proportional to the CL intensity in the range 0.02–1.0 mg/L. The detection limit was 0.008 mg/L and the relative standard deviation was 2.8% for 0.2 mg/L parathion solution (n = 11). This method was successfully applied to the determination of parathion residue in rice samples. Copyright © 2003 John Wiley & Sons, Ltd.     

Efficacy of topical acyclovir monophosphate, acyclovir, or penciclovir in orofacial HSV-1 infections of mice and genital HSV-2 infections of guinea pigs

The purpose of these studies was to compare the efficacy of acyclovir monophosphate (ACVMP), acyclovir (ACV), or penciclovir (PCV) against HSV-1 in an orofacial infection of mice and against ACV sensitive and resistant genital HSV-2 infections of guinea pigs. Treatment was initiated 24, 48, or 72 hours post inoculation with 5% ACVMP, 5% ACV (Zovirax) or 1% PCV (Denavir). In all experiments, similar efficacy was obtained for ACVMP and ACV, whereas PCV was considerably less effective.     

Inhibition of the system luminol-H2O2-Fe(CN)63− chemiluminescence by the Mn(II) indirect determination of isoniazid in a pharmaceutical formulation

A flow injection procedure for the indirect chemiluminescent determination of isoniazid is proposed. The method is performed in a flow-injection manifold provided with a solid-phase reactor. The reactor was made from manganese dioxide physically entrapped by polymerization; the redox reaction isoniazid–manganese dioxide released Mn(II) which was monitored through its inhibitory effect on the reaction between luminol and hydrogen peroxide in presence of potassium hexacyanoferrate(III). The procedure resulted in a linear calibration graph over the range 5–15 mg/L of isoniazid with a sample throughput of 43 samples/h. The influence of foreign compounds was studied and the method was applied to determination of the drug in a pharmaceutical formulation. © 1998 John Wiley & Sons, Ltd.     

Rapid determination of valaciclovir and acyclovir in human biological fluids by high-performance liquid chromatography using isocratic elution

A rapid high-performance liquid chromatographic assay with isocratic elution is developed for the simultaneous quantification of valaciclovir (VACV) prodrug and its active converted compound, acyclovir (ACV), in biological fluids of treated patients. For serum, the samples are deproteinized with perchloric acid in presence of 1-methylguanosine as the internal standard (IS). For urine and dialysis liquid, the samples are diluted with a mobile phase containing the IS, then filtered. VACV, ACV and the IS are separated on a SymmetryShield™ RP-8 column with acetonitrile–ammonium phosphate buffer as the mobile phase and detected at 254 nm. The chromatographic time is about 12 min. The relative standard deviations (RSD) of VACV and ACV standards are between 0.5 and 3.5%. Most endogenous nucleosides and their metabolites, psychotropic drugs and drugs of abuse are shown not to interfere with this technique. The method has been applied to study the pharmacokinetics of VACV and ACV in serum, dialysis liquid and urine of renal failure patients on continuous ambulatory peritoneal dialysis (CAPD) under oral treatment of VACV.     

Stability evaluation and sensitive determination of antiviral drug,valacyclovir and its metabolite acyclovir in human plasma by a rapid liquid chromatography–tandem mass spectrometry method

A simple, sensitive and high throughput liquid chromatography/positive-ion electrospray ionization mass spectrometry (LC–ESI-MS/MS) method has been developed for the simultaneous determination of valacyclovir and acyclovir in human plasma using fluconazole as internal standard (IS). The method involved solid phase extraction of the analytes and IS from 0.5 mL human plasma with no reconstitution and drying steps (direct injection of eluate). The chromatographic separation was achieved on a Gemini C18 analytical column using isocratic mobile phase, consisting of 0.1% formic acid and methanol (30:70 v/v), at a flow-rate of 0.8 mL/min. The precursor → product ion transition for valacyclovir (m/z 325.2 → 152.2), acyclovir (m/z 226.2 → 152.2) and IS (m/z 307.1 → 220.3) were monitored on a triple quadrupole mass spectrometer, operating in the multiple reaction monitoring (MRM) mode. The method was validated over the concentration range 5.0–1075 ng/mL and 47.6–10225 ng/mL for valacyclovir and acyclovir respectively. The mean recovery of valacyclovir (92.2%), acyclovir (84.2%) and IS (103.7%) from spiked plasma samples was consistent and reproducible. The bench top stability of valacyclovir and acyclovir was extensively evaluated in buffered and unbuffered plasma. It was successfully applied to a bioequivalence study in 41 healthy human subjects after oral administration of 1000 mg valacyclovir tablet formulation under fasting condition.     

Investigation on the interaction between dihydroxybenzene and Fe3+- H2O2-Rh6G system based on enhancing chemiluminescence.

A highly sensitive flow-injection chemiluminescence (FI-CL) method has been developed for the determination of dihydroxybenzene, based on the hydroxyl radical reaction. Hydroxyl radical (.OH) produced by the reaction of Fe(3+) and H(2)O(2) oxidize rhodamine 6G to produce weak CL. It was observed that catechol and hydroquinone greatly enhanced the weak CL reaction. However, the proposed CL system is not suitable for determination of resorcinol because the enhancement reaction is very slow. The proposed procedure has a linear range of 0.01-2 mg/L for catechol, with a detection limit of 0.006 mg/L, and 0.008-1 mg/L for hydroquinone, with a detection limit of 0.004 mg/L. The possible mechanism of the CL system is discussed.     

Acyclovir transport into human erythrocytes

The mechanism of transport of the antiviral agent acyclovir (ACV) into human erythrocytes has been investigated. Initial velocities of ACV influx were determined with an "inhibitor-stop" assay that used papaverine to inhibit ACV influx rapidly and completely. ACV influx was nonconcentrative and appeared to be rate-saturable with a Km of 260 +/- 20 microM (n = 8). However, two lines of evidence indicate that ACV permeates the erythrocyte membrane by means other than the nucleoside transport system: 1) potent inhibitors (1.0 microM) of nucleoside transport (dipyridamole, 6-[(4-nitrobenzyl)thio]-9-beta-D-ribofuranosylpurine, and dilazep) had little (less than 8% inhibition) or no effect upon the influx of 5.0 microM ACV; and 2) a 100-fold molar excess of several purine and pyrimidine nucleosides had no inhibitory effect upon the influx of 1.0 microM ACV. However, ACV transport was inhibited competitively by adenine (Ki = 9.5 microM), guanine (Ki = 25 microM), and hypoxanthine (Ki = 180 microM). Conversely, ACV was a competitive inhibitor (Ki = 240-280 microM) of the transport of adenine (Km = 13 microM), guanine (Km = 37 microM), and hypoxanthine (Km = 180 microM). Desciclovir and ganciclovir, two compounds related structurally to ACV, were also found to be competitive inhibitors of acyclovir influx (Ki = 1.7 and 1.5 mM, respectively). These results indicate that ACV enters human erythrocytes chiefly via the same nucleobase carrier that transports adenine, guanine, and hypoxanthine.     

Determination of free cholesterol based on a novel flow‐injection chemiluminescence method by immobilizing enzyme

A novel flow injection chemiluminescence method is proposed for determination of cholesterol in this paper. The cholesterol oxidase was immobilized onto sol–gel and prepared as an enzymatic reaction column. The determination of cholesterol was performed by quantitative determination of hydrogen peroxide produced from an enzymatic reaction. The luminol–H₂O₂–metal chelate diperiodatocuprate(III) system ensured that the method was highly sensitive and selective. Free cholesterol was determined over the range 5.0 × 10^–8 mol/L–5.0 × 10^–7 mol/L, with a limit of detection (3σ) of 1.9 × 10^–8 mol/L. The relative standard deviation (RSD) for 2.5 × 10^–7 mol/L was 2.7% (n = 7). The proposed method offered the advantages of sensitivity, selectivity, simplicity and rapidity for free cholesterol determination, and was successfully applied to the direct determination of free cholesterol in serum. Copyright © 2008 John Wiley & Sons, Ltd.     

Prophylactic and therapeutic treatment with acyclovir of genital herpes in the guinea pig

The antiviral drug, acyclovir, was tested on experimentally infected guinea pigs with either of two herpes simplex virus type 1 (HSV-1) isolates following intravaginal inoculation. The drug was continuously infused subcutaneously utilizing an osmotic pump. Infusion was begun either prior to virus inoculation (prophylactic) or after virus inoculation at the time of first appearance of lesions (therapeutic). Prophylactic treatment markedly reduced the severity of the genital lesions, the appearance of acute neurologic sequellae, and the virus excretion in the genital tract of guinea pigs infected with either of the two strains tested. Therapeutic acyclovir treatment, however, did not decrease the incidence of acute neurologic sequellae with one of the two HSV-1 strains tested, nor did it reduce the severity of the genital lesions of either strain. These neurologic sequellae may be due to insufficient levels of ACV in the central nervous system as the concentration of ACV in the dorsal root ganglia was found to exceed that of the plasma, but only trace amounts of acyclovir were present in the brain and spinal cord. Continuous perfusion of ACV gave far higher tissue levels than intermittent injections. These findings suggest that prophylactic ACV is far more effective than therapeutic treatment for genital herpes in the guinea pig model.     

First enzymatic galactosylation of acyclic nucleoside drugs by β-galactosidase: Synthesis of water-soluble β-D-galactosidic prodrugs

Acyclic nucleoside analogs constitute an important group of antiviral agents. However, these nucleoside drugs suffer from poor water solubility and low oral bioavailability in the clinic use. In the present work, the enzymatic synthesis of the water-soluble galactosidic prodrugs of acyclic nucleosides by using bovine liver β-galactosidase was described. In the enzymatic transgalactosylation between acyclovir (ACV) and o-nitrophenyl β-galactopyranoside (oNPGal), the optimum enzyme dosage, buffer pH, temperature and molar ratio of ACV to oNPGal were 0.225 U/mL, 7.0, 40°C and 2.5, respectively, under which the initial reaction rate and the yield reached 0.40 mM/h and 29%, respectively. In addition, this enzyme could accept ganciclovir (GCV) and penciclovir (PCV) as substrates, affording the corresponding 4’-β-galactosylated derivatives with the yields of 26% and 71%, respectively.     

Direct chemiluminescence determination of ibuprofen by the enhancement of the KMnO(4)-sulphite reaction.

A simple, rapid, flow-injection chemiluminescence (CL) method is described for the determination of ibuprofen. A strong CL signal was detected when a mixture of the analyte and sulphite was injected into acidic KMnO(4). The CL signal is proportional to the concentration of ibuprofen in the range 0.1-10.0 mg/L. The detection limit is 0.02 mg/L ibuprofen, the relative standard deviation is 1.8% (0.5 mg/L ibuprofen; n = 11) and the sample measurement frequency is 120/h. The proposed method was successfully applied to the determination of ibuprofen in pharmaceutical preparations and in spiked urine samples. The mechanism of the CL reaction is also discussed.