Majid Razzak

A prolonged immune response to antigen delivered in poly ( ∈ -caprolactone) microparticles

A single dose vaccine formulation which induces both humoral and cell‐mediated immune responses over a prolonged period would provide a potent weapon against infectious disease. We have used a water‐in‐oil‐in‐oil, solvent evaporation method for generating poly ∊‐caprolactone microparticles and tested their ability to induce an immune response against the model antigen ovalbumin. We hypothesized that the initial release of antigen from the surface of the poly ∊‐caprolactone microparticles would act as the priming dose and that the delayed release over the following months, due to diffusion from or break‐down of the microparticles, would act as a boost to the immune response. Ovalbumin encapsulated in the poly ∊‐caprolactone microparticles was able to induce both antibody and cell‐mediated immune responses. However our results suggest that the spontaneous release had little effect on the immune response. Despite this the response was maintained for at least 8 months following a single immunization. Both humoral and cell‐mediated immune responses were induced in mice. This simple method of vaccine formulation offers a cost‐efficient way to deliver antigen in a single dose to the immune system.

HPLC Assay of Levamisole and Abamectin in Sheep Plasma for Application to Pharmacokinetic Studies

Abstract High performance liquid chromatography (HPLC) techniques were developed to quantify levamisole and abamectin in sheep plasma. UV detection (225 nm) for levamisole and fluorescence detection (excitation at 365 nm and emission at 470 nm) for abamectin were used. Separation was achieved on a C18 Prodigy ODS column with a mobile phase of phosphate (NaH2PO4 and Na2HPO4)‐acetonitrile (60∶40, v/v) (pH 7.5; 0.01M) for levamisole and methanol‐acetonitrile‐water (95∶3∶2, v/v/v) for abamectin. The retention times were 5.7 minutes for levamisole phosphate, 4.9 minutes for abamectin, 7.4 minutes internal standard (ivermectin). Calibration curves for levamisole phosphate and abamectin were linear over the range 0.05–10 µg/mL for levamisole phosphate and 0.25–20 ng/mL for abamectin, with the correlation coefficients for both drugs exceeding >0.999. The LOQ was achieved as the lowest point on the standard curve, 0.05 µg/mL for levamisole phosphate with the RSD 18.2% and 0.25 ng/mL for abamectin with the RSD 19.6%. The maximum intra‐day and inter‐day coefficients of variation were 9.1% and 15.0%, respectively, at 0.1 µg/mL (lowest concentration) for levamisole phosphate, and 11.9% and 19.3%, respectively, at 0.5 ng/mL (lowest concentration) for abamectin. Accuracies were 107.5% and 91.9% for levamisole and abamectin, respectively, at lowest concentrations of 0.1 µg/mL for levamisole phosphate and 1 ng/mL for abamectin. The recoveries from frozen and thawed plasma samples were 86.3% at 0.1 µg/mL for levamisole phosphate, and 105.8% at 2 ng/mL for abamectin. Both methods were successfully applied for analysis of levamisole and abamectin in plasma after subcutaneous injection to sheep of a formulation of medium chain mono‐ and diglyceride‐propylene glycol‐glycerol formal containing both levamisole phosphate and abamectin.

Isotropic Systems of Medium‐Chain Mono‐ and Diglycerides for Solubilization of Lipophilic and Hydrophilic Drugs

The aim of this study was to investigate isotropic mono‐ and diglyceride‐based (MCMDG) systems, which are potential vehicles for injectable products containing both hydrophilic and lipophilic drugs. For two‐component systems, MCMDG was mixed with various masses of water. For three‐component systems, the samples were prepared by mixing propylene glycol or glycerol formal or short‐chain alcohols with MCMDG prior to the addition of water. The isotropic region was examined by visual inspection and confirmed using polarized light microscopy. Viscosities of formulations were measured. Solubilities of levamisole phosphate (hydrophilic) and abamectin (lipophilic) were determined in the isotropic formulations using high‐performance liquid chromatography assay. The isotropic region in the two‐component systems had a water content of up to 18% at 25°C. Solvents such as propylene glycol (PG), glycerol formal (FG), and ethyl alcohol increased the isotropic region. The area of isotropic region in these three‐component systems increased with increasing temperature. The area of the isotropic region became larger with decreasing dielectric constant and solubility parameter of the series of short‐chain alcohols, except n‐butyl alcohol, at 25°C. The systems exhibited Newtonian behavior. The solubility of both hydrophilic and lipophilic drugs was high in formulations at 25°C. It was concluded that more water was solubilized in MCMDG/short‐chain alcohols/water systems, and the isotropic region in the short‐chain alcohol systems enlarged compared with MCMDG/PG/water or MCMDG/GF/water systems, except the n‐butyl alcohol system. Hydrophilic and lipophilic drugs solubilize in the systems. The isotropic formulations containing MCMDG may represent an alternative to more traditional formulations for injectable formulations containing both lipophilic and hydrophilic drugs.

Isolation and characterization of degradation products of moxidectin using LC, LTQ FT-MS, H/D exchange and NMR

AbstractThis study aimed to evaluate the degradation profile and pathways, and identify unknown impurities of moxidectin under stress conditions. During the experiments, moxidectin samples were stressed using acid, alkali, heat and oxidation, and chromatographic profiles were compared with known impurities given in European Pharmacopeia (EP) monograph. Moxidectin has shown good stability under heat, while reaction with alkali produced 2-epi and ∆2,3 isomers (impurities D and E in EP) by characteristic reactions of the oxahydrindene (hexahydrobenzofuran) portion of the macrocyclic lactone. Two new, previously unreported, unknown degradation products, i.e. impurity 1 and impurity 2, detected after acid hydrolysis of moxidectin (impurity 2 was also observed to a lesser extent after oxidation), were isolated from sample matrices and identified using liquid chromatography, NMR, high-resolution FT-ICR MS, and hydrogen/deuterium exchange studies. FTMS analysis showed accurate mass of molecular ion peaks for moxidectin at m/z 640.38412, impurity 1 at m/z 656.37952 and impurity 2 at m/z 611.35684, giving rise to daughter ions traceable up to the seventh levels of MSn experiments and supporting the proposed structures. Both unknown impurities along with moxidectin were fully characterized by 1H, 13C, 1D HMBC and 2D (NOESY, COSY and HSQC) NMR experiments. The interpretation of experimental data positively identified impurity 1 as 3,4-epoxy-moxidectin and impurity 2 as 23-keto-nemadectin. The identification of new impurities and correlation of their chromatographic profiles with the EP method is very useful to establish the stability profile of moxidectin and its preparations, as well as add value to the forthcoming moxidectin finished product European Pharmacopeia monographs. FigureAcid catalyzed degradation of moxidectin into 23-keto-moxidectin and 3,4-epoxy-moxidectin

Tissue compatibility and pharmacokinetics of three potential subcutaneous injectables for low-pH drug solutions

OBJECTIVES The aim of the study was to investigate the tissue tolerance and bioavailability of four formulations containing 5% ricobendazole solubilised at low pH, following subcutaneous injection in sheep. Formulations were: a water-in-oil emulsion, a microemulsion, a hydroxypropyl-beta-cyclodextrin (HP-beta-CD, 20%) drug solution, and a low-pH drug solution (reference). METHODS In-vitro cytotoxicity of the formulations was investigated in L929 fibroblasts using MTS viability and lactate dehydrogenase leakage assays. Each formulation and respective vehicle was injected into either side of the back of a sheep to investigate the tissue tolerance and pharmacokinetics. KEY FINDINGS In-vitro studies suggested that both the emulsion and the microemulsion are unlikely to give a burst release of the low-pH drug solution in aqueous media. The microemulsion showed the greatest in-vitro cytotoxic effect but no significant difference was observed between the other formulations. In sheep, the three new formulations and vehicles caused little or no injection-site reactions compared with a marked response to the reference formulation. Bioavailabilities of HP-beta-CD formulation, emulsion and microemulsion formulations, relative to the reference formulation, were 194, 155 and 115%, respectively. CONCLUSIONS The three new subcutaneous injectables showed promise for reducing irritation of low-pH solubilised ricobendazole. HP-beta-CD significantly enhanced the drug absorption. Controlling the burst release of the low-pH drug solution may improve tissue tolerance and minimise post-injection precipitation, and hence increase drug bioavailability. The in-vitro cytotoxicity studies did not predict the in-vivo irritation effects.Objectives Our aim was to investigate the effect of etoricoxib on the anticonvulsant activity of phenytoin and diazepam against seizure models in mice. In addition the acute adverse effect of etoricoxib was assessed with a chimney test.

Rapid, Simultaneous Determination of Levamisole and Abamectin in Liquid Formulations Using HPLC

Abstract The aim of the study was to develop a high performance liquid chromatography (HPLC) assay for the rapid, simultaneous determination of levamisole phosphate and abamectin in liquid formulations containing mixtures of solvents and medium chain mono‐ and diglycerides and triglycerides. The experimental procedure involved reversed‐phase‐HPLC with a Zorbax ODS column (7 µm particle size, 4.6 ID × 250 mm), acetonitrile‐water‐ and ammonia 1.0N volumetric solution (80:20:0.1, v/v/v) mobile phase, UV detection at 253 nm for both levamisole phosphate and abamectin. The flow rate of the mobile phase was 2 mL/min. The retention times of levamisole phosphate and abamectin varied from 2.45–2.46 min and 5.78–5.82 min, respectively. Formulation components did not give rise to any interfering peaks. Calibration curves were linear over the range 13.5–270 µg/mL for levamisole phosphate and 0.5–10 µg/mL for abamectin. The maximum intraday and interday coefficients of variation were 0.7% and 1% at 13.5 µg/mL for levamisole phosphate, and 2.2% and 5.8% at 0.5 µg/mL for abamectin, respectively. Accuracies were 98.9 ± 5.2% and 96.2 ± 3.1% (Mean ± SD, n = 14) for levamisole phosphate and abamectin, respectively, at concentrations of 27.3 µg/mL of levamisole phosphate and 1 µg/mL of abamectin. The assay was used to evaluate the stability of these drugs in the liquid formulations. A simple and accurate liquid chromatographic method was developed and validated for simultaneous determination of levamisole and abamectin in liquid formulations. Due to its simplicity and accuracy, the assay method is suitable for routine analysis of both drugs in liquid formulations.

Separation and identification of degradation products in eprinomectin formulation using LC, LTQ FT-MS, H/D exchange, and NMR

The aim of this study was to evaluate the suitability of the compendial active pharmaceutical ingredient (API) method for the analysis of finished products and characterization of degradation products in eprinomectin (EPM) samples. Heat stressed sample tests revealed a limitation of the API method in distinguishing an impurity merging with the principal analyte peak. A new selective, specific and sensitive method was therefore developed for the determination of EPM in formulations that separates its degradation products currently undetectable with the official method. The determination was carried out by reversed-phase HPLC using an isocratic solvent elution. The method was validated and found to be precise, accurate and specific; the detector response was linear over 50-150 μg/ml (EPM) and 0.1-3 μg/ml (degradation product) range of concentrations. Two major degradation products detected with the new method were isolated from sample matrices and characterized using LC-PDA, high resolution FT-ICR MS, NMR and hydrogen/deuterium exchange (HX-MS) studies. FTMS analysis showed accurate mass of molecular ion peaks for EPM and its two degradation products at m/z 914.52505 (mass error ≤ 1 ppm) with almost identical fragmentation patterns. Given the isomeric nature of the compounds, all three were further evaluated by ¹H, ¹³C, 1D NOESY and 2D (COSY) NMR experiments. The interpretation of experimental data positively identified Unknown 1 as the 2-epimer of EPM and Unknown 2 as the structural isomer Δ2,3-EPM containing a conjugated enoate. The new HPLC method and identification exercise is useful for analysis of EPM and its degradation products.

Absorption and tissue tolerance of ricobendazole in the presence of hydroxypropyl-β-cyclodextrin following subcutaneous injection in sheep

Post-injection precipitation may cause poor and erratic drug absorption and tissue irritation at the injection site. Tissue tolerance and pharmacokinetics of a low pH ricobendazole (RBZ) injectable containing 20% hydroxypropyl-beta-cyclodextrin (HP-beta-CD) were simultaneously investigated after subcutaneous injection in sheep compared to a reference formulation without HP-beta-CD. Each animal received a RBZ containing formulation on one side of the back and the respective vehicle on the contralateral side. The HP-beta-CD vehicle showed good tissue tolerance and the acidic solution caused minimal injection site reactions. Both RBZ containing formulations caused pain on injection and tissue histological changes in some animals. Lack of elevation of plasma creatine kinase indicated that none of the formulations caused significant damage to the underlying muscle tissue. Compared to the reference formulation, AUC and C(max) of the HP-beta-CD formulation were 1.6 and 2.2 times higher, respectively, whereas t(max), MRT and t(1/2) were significantly shorter suggesting faster and greater absorption of RBZ in the presence of HP-beta-CD. This was attributed to the effect of inhibition of post-injection drug precipitation and drug absorption enhancement of HP-beta-CD. In conclusion, HP-beta-CD was shown to be a tissue-compatible excipient with potential to inhibit post-injection precipitation and increase absorption of poorly water soluble drugs. Additionally, the HP-beta-CD formulation showed promise as an injectable that potentially minimizes irritation by reducing the dose required.

Stability of ricobendazole in aqueous solutions

The chemical stability of ricobendazole (RBZ) was investigated using a stability-indicating high performance liquid chromatographic (HPLC) assay with ultraviolet detection. The degradation kinetics of RBZ in aqueous solution was evaluated as a function of pH, buffer strength and temperature. The oxidation reaction in hydrogen peroxide solution was also studied. Degradation products were analyzed by mass spectroscopy and degradation pathways are proposed. Degradation of RBZ followed pseudo first-order kinetics and Arrhenius behavior over the temperature range 24-55 degrees C. A V-shaped pH-rate profile over the pH range 2-12 was observed with maximum stability at pH 4.8. The shape of the pH-rate profile was rationalized by catalytic effects of various components in the solution on each RBZ species. At pH 11 the activation energy for hydrolysis was 79.5 kJ/mol, and phosphate catalysis was not observed. Oxidation occurred in hydrogen peroxide solutions and was catalyzed by the presence of copper (Cu(2+)) ions. Ricobendazole amine and albendazole sulfone were identified by MS assay to be the degradation products of hydrolysis and oxidation respectively.

Tissue compatibility and pharmacokinetics of three potential subcutaneous injectables for low-pH drug solutions: Subcutaneous injectables for low-pH drug solutions

Objectives  The aim of the study was to investigate the tissue tolerance and bioavailability of four formulations containing 5% ricobendazole solubilised at low pH, following subcutaneous injection in sheep. Formulations were: a water‐in‐oil emulsion, a microemulsion, a hydroxypropyl‐β‐cyclodextrin (HP‐β‐CD, 20%) drug solution, and a low‐pH drug solution (reference).