Mahasen A. Radwan

Determination of Metoclopramide in Serum by HPLC Assay and Its Application to Pharmacokinetic Study in Rat

Abstract A simple, sensitive, and reproducible HPLC method has been developed for the determination of metoclopramide employing reversed phase high performance liquid chromatography with UV detection at 270 nm. The separation was performed on a Novapak C18, 4 μm (3.9 × 150 mm) column. Acetonitrile (18%) in 0.02 M ammonium acetate containing 0.1% triethylamine was used as the mobile phase and the run time was 7 min. Tramadol was used as the internal standard. The mean retention times of metoclopramide and tramadol were 3.4 and 4.6 min, respectively. Linear response (r > 0.997) was observed over the range of 0.025–5 μg/ml of metoclopramide. There was no significant difference (p < 0.05) between inter- and intra-day studies for metoclopramide. The mean relative standard deviations (RSD%) of the results of within-day precision and accuracy of the drug were < 10%. The applicability of the assay was demonstrated in measuring metoclopramide pharmacokinetics in rats. The elimination half-life was 2.09 ± 0.39 h wi...

High performance liquid chromatographic determination of tramadol in pharmaceutical dosage forms

Abstract A simple, specific and accurate high performance liquid chromatographic (HPLC) method for determination of tramadol in pharmaceutical dosage forms has been developed. Reversed phase chromatography was conducted using (.μ-Bondapak C18 column (3.9 × 150 nm) with an isocratic mobile phase consisting of 0.005 M triethylamine in 0.01 M sodium phosphate buffer (pH 5.5) containing 17% acetonitrile. The effluent was monitored on a UV detector at 230 nm. Each analysis required no longer than 8 minutes. Quantification was achieved by the measurement of the peak-area ratio of the drug to the internal standard (metoclopramide) and the detection limit was 75 ng/mL. Linear response (r > 0.999) was observed over the range of 0.1 – 10 μg/mL and was run on 6 different occasions. There was no significant difference (p < 0.05) between inter- and intra- day studies for tramadol determined for two different concentrations (0.5 and 5.0 mg/mL). The mean relative standard deviations (RSD%) of the results of within-day p...

Enhancement of Absorption of Insulin-Loaded Polyisobutylcyanoacrylate Nanospheres by Sodium Cholate After Oral and Subcutaneous Administration in Diabetic Rats

Polyisobutylcyanoacrylate (PIBCA) nanospheres were employed as biodegradable polymeric carriers for oral (p.o.) and subcutaneous (s.c.) delivery of insulin. The polymerization technique used was able to hold 65%–95% of insulin added 30 min after initiation of polymerization. The percentage drug loading was monomer concentration dependent. Insulin adsorption to the nanospheres was measured by radioimmumoassay. Although Pluronic F68 (0.5%) did not significantly alter the in vitro insulin degradation half-life T50%, sodium cholate (0.5%) increased the degradation T50% of insulin by 56% (from 13.6 ± 1.6 to 22.1 ± 2 min). This study also investigated the in vivo performance of insulin-loaded PIBCA in aqueous suspension with or without sodium cholate (0.5%) and Pluronic F68 (0.5%) surfactants after oral and subcutaneous administration to alloxan-induced diabetic rats. Insulin absorption was evaluated by its hypoglycemic effect. Insulin associated with PIBCA nanospheres retains its biological activity up to 15 h and 24 h after oral and subcutaneous administrations, respectively. Administered orally, insulin-loaded (75 U/kg) nanospheres, in the presence of surfactants, significantly reduced the mean blood glucose level from 392 ± 32 to 80 ± 13 mg/dl within 2 h and maintained it at 100 mg/dl or less for more than 8 h. On the other hand, the subcutaneous administration of insulin-loaded (25 U/kg) nanospheres significantly decreased the blood glucose level from 406 ± 33 to 88.5 ± 12.8 mg/dl within 1 h, and the lowered glucose level was maintained at 100 mg/dl or less for more than 12 h; it returned to its initial value 24 h after administration. Insulin-loaded nanospheres with surfactants showed significant P < 05) pharmacological availability (PA%) of 37.6% ± 3.7% and 65.2% ± 2.7% after oral and subcutaneous dosages, respectively. The existence of surfactants with PIBCA nanospheres improved the oral PA% by 49.2%. These findings suggest that the developed PIBCA, in the presence of surfactants, would be useful not only in improving insulin gastrointestinal absorption, but also in sustaining its systemic action by lowering the blood glucose to an acceptable level.

Determination of free and total warfarin concentrations in plasma using UPLC MS/MS and its application to a patient samples

Warfarin is routinely monitored by assessing its pharmacologic effects on the international normalized ratio. However, having a patient with INR not responding to increasing warfarin dose mandates a direct measurement of warfarin concentrations (total and free) for better patient clinical management of warfarin therapy. Therefore, a new fully validated specific, precise and accurate ultra-performance liquid chromatography tandem mass spectrometry was developed for the determination of free and total warfarin in human plasma. Free warfarin was measured in plasma filtrate, prepared by ultrafiltration, and sample pretreatment involved protein precipitation with acetonitrile. Linear response (r(2) ≥0.99) was observed over the studied range of free and total warfarin, with the lower limit of detection of 0.25 ng/mL. The intra- and inter-day precision (relative standard deviation) values were <10% and the accuracy (relative error) was ≤6.6 for free and total warfarin. There was no significant difference (p>0.05) between inter- and intra-day studies for the free and total warfarin, which confirmed the reproducibility of the assay method. The mean extraction efficiency was 88.6-107.2% of free and total warfarin. The assay was sensitive to follow warfarin pharmacokinetics (free and total) in a patient with resistance to warfarin up to 24 h after a daily dose of warfarin.

Stealth Amphotericin B nanoparticles for oral drug delivery: In vitro optimization.

Purpose Amphotericin B (AmB) is an effective anti-fungal and anti-leishmanial agent. However, AmB has low oral bioavailability (0.3%) and adverse effects (e.g., nephrotoxicity). The objectives of this study were to improve the oral bioavailability by entrapping AmB in pegylated (PEG) poly lactide co glycolide copolymer (PLGA–PEG) nanoparticles (NPs). The feasibility of different surfactants and stabilizers on the mean particle size (MPS) and entrapment efficiency were also investigated. Materials and methods NPs of AmB were prepared by a modified emulsification diffusion method employing a vitamin E derivative as a stabilizer. Physicochemical properties and particle size characterization were evaluated using Fourier Transform Infra-Red spectroscopy (FTIR), differential scanning calorimetry, scanning electron microscopy and transmission electron microscopy. Moreover, in vitro dissolution profiles were performed for all formulated AmB NPs. Results MPS of the prepared spherical particles of AmB ranged from 26.4 ± 2.9 to 1068 ± 489.8 nm. An increased stirring rate favored AmB NPs with a smaller MPS. There was a significant reduction in MPS, drug content and drug release, when AmB NPs were prepared using the diblock polymer PLGA–PEG with 15% PEG. Addition of three emulsifying agents poly vinyl pyrrolidone (PVP), Vitamin E (TPGS) and pluronic F-68 to AmB formulations led to a significant reduction in particle size and increase in drug entrapment efficiency (DEE) compared to addition of PVP alone. FTIR spectroscopy demonstrated a successful loading of AmB to pegylated PLGA–PEG copolymers. PLGA–PEG copolymer entrapment efficiency of AmB was increased up to 56.7%, with 92.7% drug yield. After a slow initial release, between 20% and 54% of AmB was released in vitro within 24 h phosphate buffer containing 2% sodium deoxycholate and were best fit Korsmeyer–Peppas model. In conclusion, PLGA–PEG diblock copolymer with 15% PEG produced a significant reduction (>70%) in MPS with highest drug content. The percentage of PEG in the copolymer and the surfactant/stabilizer used had a direct effect on AmB release in vitro, entrapment efficiency and MPS. These developed formulations are feasible, effective and improved alternatives to other carriers for oral delivery of AmB.

In vivo performance of parenteral theophylline-loaded polyisobutylcyanoacrylate nanoparticles in rats

Theophylline-loaded polyisobutylcyanoacrylate (PICA) nanoparticles were prepared by emulsifier-free polymerization in aqueous media at ambient conditions. PICA nanoparticles were shown (in vitro) to be a promising controlled delivery system for theophylline. Therefore, this study was conducted to investigate the feasibility of PICA nanoparticles as a parenteral controlled drug delivery system in rats. Wistar rats were given intraperitoneal (i.p.) injections of theophylline solution (4 mg/kg) and theophylline nanospheres suspension (8 mg/kg) on two different occasions. Theophylline serum concentrations were measured by an HPLC assay. The drug solution was rapidly absorbed, distributed, and eliminated. The peak concentration (Cmax), 5.34+/-1.9 mg/l, was achieved 20 min following administration. The mean residence time was 2.94 h, and the apparent clearance was 0.31 (l/h)/kg. After nanospheres administration the mean Cmax, 2.53+/-1.1 mg/l, was attained at 3 h. The drug was successfully maintained around this elevated serum drug concentration up to 11 h in rats. The drug concentration was only reduced to 1.43+/-0.98 mg/l (i.e. reduced by 43.5%) after 20 h of administration. This present study provides evidence that the sorption of theophylline to PICA nanoparticles could control the drug release in rats.

In Vitro Performance of Carbamazepine Loaded to Various Molecular Weights of Poly (D, L-Lactide-Co-Glycolide)

The purpose of this study was to develop and assess the in vitro characteristics of carbamazepine-loaded microspheres. A solvent evaporation method was used to incorporate carbamazepine (CBZ) into poly (D,L-lactide-co-glycolide) (PLGA) with different molecular weights. The optimum conditions for CBZ-PLGA microspheres preparation were considered and the in vitro release of CBZ of PLGA microspheres were followed up to 24 hr in USP dissolution medium. The effect of using different ratios of PLGA microspheres, prepared with different molecular weights, for optimizing CBZ release also was investigated. CBZ encapsulation efficiency was 68 to 82% for all prepared formulations. Thermograms of CBZ-PLGA microspheres suggest that CBZ was totally entrapped with the PLGA polymer. The presence of Pluronic F-68 has improved the encapsulation of CBZ, resulted in better and smoother microspheres surfaces and enhanced its release pattern. CBZ release profiles were biphasic patterns; after an initial burst, a constant CBZ release rate was observed up to 24 hr. The release from these PLGA-based spherical matrices was consistent with the diffusion mechanism. CBZ dissolution T50% was significantly affected (> 3-fold) by increasing the lactide percent from 33.3 to 66.6% from different microspheres mixtures. The present study provides evidence that the encapsulation of CBZ to PLGA microspheres, either as a single polymer or mixture of two, was a successful attempt to control the release of CBZ.

The effect of absorption enhancers on the initial degradation kinetics of insulin by α-chymotrypsin

The goal of this investigation was to establish a fast method to screen various insulin absorption enhancers by following their effect on the initial kinetics of insulin incubated with alpha-chymotrypsin at 37 degrees C. A simple, sensitive and reproducible reversed phase high performance liquid chromatography (HPLC) method has been developed to carry out this goal. Linear responses (r > 0.999) were observed over the range of 0.4-4 U/ml for insulin. There was no significant difference (P < 0.05) between inter- and intra-day studies for insulin. The mean relative standard deviations (RSD%) of the results of within-day precision and accuracy of insulin were 12%. The assay was sensitive to detect the existence of any metabolite due to the addition of any absorption enhancers, even if it was not seen with insulin alone. Three metabolites (A-C) were detected only when insulin was incubated with alpha-chymotrypsin at 37 degrees C. Metabolite D was observed when either glycocholic acid (0.5, 1%) or taurochenodeoxycholate (0.5, 1%) was incubated with insulin in the absence of alpha-chymotrypsin at 37 degrees C. The compounds that significantly increased insulin T50% were glycyrrhizic acid (0.5%) > deoxycholic acid (1%) > deoxycholic acid (0.5%) > glycyrrhizic acid (1%) > cholic acid (0.5, 1%). Capric acid (0.5%), hydroxypropyl-alpha-cyclodextrin (0.5, 1%) and dimethyl-alpha-cyclodextrin (0.5, 1, 5%) did not significantly affect insulin T50%. The bile salts increased insulin T50% in this order: deoxycholate > cholate > glycocholate > taurocholate > taurodeoxycholate > taurochenodeoxycholate > glycodeoxycholate. The results obtained would support the feasibility of utilizing such method for screening any compound incorporated in insulin formulation. These compounds should be used in the minimum possible concentration to avoid or minimize insulin degradation.

Pharmacokinetics of ketorolac loaded to polyethylcyanoacrylate nanoparticles using UPLC MS/MS for its determination in rats.

Polyethylcyanoacrylate (PECA) nanoparticles (NPs) have been employed as biodegradable polymeric carriers for oral (PO) delivery of ketorolac. The nanoparticles were prepared by polymerization technique at room temperature in a continuous aqueous phase at pH 2.5. This polymerization technique was able to hold 76-96% of ketorolac and the drug loading was a monomer concentration dependent. The feasibility of PECA NPs as PO controlled drug delivery systems of ketorolac was investigated in two groups of rats which were given orally either ketorolac tromethamine solution (1.5 mg/kg) or the selected ketorolac NPs aqueous dispersion (1.6 mg/kg). Ketorolac plasma concentrations were measured by a new fully validated specific, precise and accurate ultra-performance liquid chromatography tandem mass spectrometry (UPLC MS/MS) assay. The detection was performed on Waters TQ detector via negative electrospray ionization in a multiple reaction monitoring mode. Linear response (r(2)> or =0.995) was observed over the range of 10-10,000 ng/ml of ketorolac, with the lower limit of quantification of 5 ng/ml with 1 microl injection volume. The intra- and inter-day precision (relative standard deviation, R.S.D.) values were <10% and the accuracy (relative error) was < or =8 for ketorolac concentrations. The drug solution is rapidly absorbed, distributed, and eliminated and shows a monophasic elimination phase. The assay was sensitive to follow pharmacokinetics of ketorolac in rats up to 24 h after a PO dose of its aqueous solution or NPs suspension. After NPs administration the mean Cmax, 5.0+/-1.3 mg/l, was attained at 1 h. The drug was successfully maintained around this elevated plasma drug concentration up to 6h (>2t(1/2)), in rats. The AUC was significantly higher after the NPs suspension than the solution of ketorolac. This present study provides evidence that the sorption of ketorolac to PECA NPs could control the drug release/elimination in rats.

Oral administration of amphotericin B nanoparticles: antifungal activity, bioavailability and toxicity in rats

Abstract Amphotericin B (AMB) is used most commonly in severe systemic life-threatening fungal infections. There is currently an unmet need for an efficacious (AMB) formulation amenable to oral administration with better bioavailability and lower nephrotoxicity. Novel PEGylated polylactic-polyglycolic acid copolymer (PLGA-PEG) nanoparticles (NPs) formulations of AMB were therefore studied for their ability to kill Candida albicans (C. albicans). The antifungal activity of AMB formulations was assessed in C. albicans. Its bioavalability was investigated in nine groups of rats (n = 6). Toxicity was examined by an in vitro blood hemolysis assay, and in vivo nephrotoxicity after single and multiple dosing for a week by blood urea nitrogen (BUN) and plasma creatinine (PCr) measurements. The MIC of AMB loaded to PLGA-PEG NPs against C. albicans was reduced two to threefold compared with free AMB. Novel oral AMB delivery loaded to PLGA-PEG NPs was markedly systemically available compared to Fungizone® in rats. The addition of 2% of GA to the AMB formulation significantly (p < 0.05) improved the bioavailability from 1.5 to 10.5% and the relative bioavailability was > 790% that of Fungizone®. The novel AMB formulations showed minimal toxicity and better efficacy compared to Fungizone®. No nephrotoxicity in rats was detected after a week of multiple dosing of AMB NPs based on BUN and PCr, which remained at normal levels. An oral delivery system of AMB-loaded to PLGA-PEG NPs with better efficacy and minimal toxicity was formulated. The addition of glycyrrhizic acid (GA) to AMB NPs formulation resulted in a significant oral absorption and improved bioavailability in rats.