Rakhi B. Shah

High-performance liquid chromatographic analysis of cyclosporin A in rat blood and liver using a commercially available internal standard.

All the available HPLC assays of cyclosporin A (CyA) use internal standards that are not commercially available. Our purpose was to develop an HPLC assay for measurements of CyA in rat blood and liver using a commercially available internal standard (I.S.). After the addition of tamoxifen (I.S.), blood (0.25 ml) or the liver homogenate (1 ml) samples were extracted into a mixture of ether:methanol (95:5). The residue after evaporation of the organic layer was dissolved in 200 microl of an injection solution and washed with 1 ml of hexane before analysis. The separation was achieved using an LC-1 column (70 degrees C) with a mobile phase of methanol-acetonitrile-0.01 M KH(2)PO(4) (50:25:25, v/v) and a flow-rate of 1 ml/min. Detection was at 205 nm. Cyclosporin A and I.S. eluted at 5 and 7 min, respectively, free from endogenous peaks. Linear relationships (r>0.98) were observed between the CyA:I.S. peak area ratios and the CyA concentrations within the range of 0.2-10 microg/ml for blood and 0.1-4 microg/ml for the liver homogenates. The intra- and inter-run C.V.s and errors for both the blood and liver samples were <15%. The extraction efficiency (n=5) was close to 100% for both CyA and I.S. in both blood and liver homogenates. The lower limit of quantitation of the assay was 0.2 or 0.1 microg/ml based on 250 microl of blood or 1 ml of liver homogenate, respectively. The assay was capable of measuring blood and liver concentrations of CyA in a rat injected intravenously with a single 5-mg/kg dose of the drug.

Regional permeability of salmon calcitonin in isolated rat gastrointestinal tracts: Transport mechanism using Caco-2 cell monolayer

The objective of the study was to determine the region of maximum permeation of salmon calcitonin (sCT) through the gastrointestinal tract and to investigate the mechanism of permeation. For regional permeability determination, male Sprague-Dawley rats (250–300 g) were anesthetized and the gastrointestinal tissues were isolated. Stomach, duodenum, jejunum, ileum, or colon tissues were mounted on Navicyte side-by-side diffusion apparatus. Salmon calcitonin solutions (50 μM in phosphate-buffered saline, pH 7.4, 37°C) were added to the donor side, and the samples were removed from the receiver compartment and analyzed by competitive radioimmunoassay (RIA). For mechanistic studies, Caco-2 cells were grown on Transwell inserts (0.4-μm pore size, 0.33 cm2 area) in a humidified 37°C incubator (with 5% CO2). Transport experiments were conducted for sCT solutions (50 μM in Dulbeccos modified eagles medium [DMEM], pH 7.4) from the apical-to-basolateral (A-to-B) direction and B-to-A direction at 37°C and from the A-to-B direction at 4°C. Cell monolayer integrity was monitored by mannitol permeability and transepithelial electrical resistance (TEER) measurements. The permeability coefficients (× 10−9, cm/sec) for sCT through rat stomach, duodenum, jejunum, ileum, and colon tissues were 0.482±0.086, 0.718±0.025, 0.830±0.053, 1.537±0.32, and 0.934±0.15, respectively. The region of maximum sCT permeability is ileum followed by colon, jejunum, duodenum, and stomach. The permeability coefficients (× 10−6, cm/sec) for sCT through Caco-2 cell monolayer were 8.57±2.34 (A-to-B, 37°C), 8.01±1.22 (A-to-B, 4°C), and 6.15±1.97 (B-to-A, 37°C). The mechanism of its permeation is passive diffusion through the mucosa as determined from the Caco-2 monolayer permeability of sCT.

Microsomal Cytochrome P450 Levels and Activities of Isolated Rat Livers Perfused with Albumin

AbstractPurpose. We recently showed that the perfusion of isolated rat livers with perfusates containing bovine serum albumin (BSA) would significantly stimulate the release of tumor necrosis factor (TNF)-α. Here, we hypothesize that BSA-induced increase in the release of TNF-α, and possibly other cytokines, would affect cytochrome P450 (CYP)-mediated drug metabolism. Methods. Rat livers were perfused ex vivo for 1, 2, or 3 h with a physiologic buffer containing or lacking 1% BSA (n = 4-5/group). At the end of perfusion, liver microsomes were prepared and analyzed for their total CYP, CYP2E1, CYP3A2, and CYP2C11 protein contents and the activities of cytochrome c reductase, CYP2E1, CYP3A2, CYP2C11, CYP2E1, CYP2D1, CYP1A1, and CYP2B1/2. In addition, the concentrations of various cytokines and nitric oxide were quantified in the outlet perfusate. Results. In the absence of BSA, the perfusate levels of all measured cytokines and nitric oxide were low. However, when the perfusate contained BSA, the levels of TNF-α, interleukin-6, and nitric oxide increased significantly (p < 0.005). Perfusion of the livers for 3 h with the BSA-containing perfusate resulted in significant (p < 0.05) decreases in the total CYP (41%), CYP2E1 (59%), CYP3A2 (68%), and CYP2C11 (50%) protein contents and activities of cytochrome c reductase (31%), CYP2E1 (66%), CYP3A2 (54%), and CYP2C11 (51%). In contrast, perfusion of livers for 1 or 2 h with the BSA perfusate did not have any significant effect on CYP-mediated metabolism. The CYP1A2, CYP2D1, and CYP2B1/2 activities were not affected by BSA, regardless of perfusion time. Conclusion. Addition of BSA to perfusates, which is a routine practice in isolated rat liver studies, can reduce CYP-mediated drug metabolism by a mechanism independent of protein-binding effect.

Stability of ranitidine syrup repackaged in unit-dose containers

PURPOSE The stability of ranitidine syrup re-packaged in unit-dose containers was studied. METHODS Oral ranitidine hydrochloride syrup containing 16.8 mg/mL of ranitidine hydrochloride (equivalent to 15 mg of ranitidine) in original bulk containers and re-packaged in unit-dose amber-colored glass bottles sealed with aluminum caps were obtained from commercial sources. For extended-stability determinations, samples were stored for 52 weeks at 25 degrees C and 40% relative humidity and analyzed at 0, 4, 13, 26, 39, and 52 weeks. For accelerated stability determinations, samples were stored for 13 weeks at 40 degrees C and 25% relative humidity and analyzed at 0, 4, 9, and 13 weeks. Stability was assessed using high-performance liquid chromatography and by measuring changes in pH and sample weight. The principal impurity and total impurities were also measured. RESULTS No significant changes in pH were demonstrated, and all values remained well within acceptable limits. The weight change in samples was greater for re-packaged samples stored in accelerated conditions compared with that of samples in the original packaging; however, the differences were not significant. Ranitidine hydrochloride samples in both types of packaging remained stable when stored at 25 degrees C and 40% relative humidity for 52 weeks and at 40 degrees C and 25% relative humidity for 13 weeks. The impurity profiles remained within acceptable limits for all samples. CONCLUSION Re-packaged ranitidine syrup was stable for up to 52 weeks when stored at 25 degrees C and 40% relative humidity and for up to 13 weeks when stored at 40 degrees C and 25% relative humidity.

A Dual Controlled Gastrointestinal Therapeutic System of Salmon Calcitonin. II. Screening of Process and Formulation Variables

An important aspect of the “Desired State” of manufacturablity as defined by the International Committee of Harmonization is the mechanistic understanding and predictability of dosage forms at the laboratory scale. The accomplishment of that aspect is often preceded by a formulation knowledge and previous history of the project or by screening of the variables to identify the critical ones. Osmotically controlled drug delivery systems provide a means of eliminating the effect of pH, food as well as transit time on drug release. Salmon calcitonin, a hypocalcemic peptide, was formulated as an osmotically controlled bilayered enteric‐coated dosage form with turkey ovomucoid (enzyme inhibitor) and glycyrrhetinic acid (permeation enhancer) along with other excipients. Drug release from the dosage form is generally affected by formulation and process variables. However, the literature information is very limited for the effects of these variables on the release kinetics of peptide drugs from osmotically controlled systems. The objective of this study was to evaluate the factors that influence the release of the drug from bilayered, osmotically controlled tablets coated with a semipermeable membrane of cellulose acetate. A seven‐factor‐12‐run Plackett‐Burman screening technique was employed to evaluate the effects of orifice size, coating level, amounts of sodium chloride, Polyox® N10 and N80 and Carbopol® 934P and 974P on drug release. Response variables was cumulative percent released in 24 hr with constraints on time for 25% and 50% drug release. Factors showing maximum influence on drug release were amounts of Carbopol® 934P and Polyox® N10 in the drug layer, orifice size and coating level showing negative effects with the main effect magnitudes of − 30.85, − 10.97, − 9.61, and − 9.95, respectively.

An Enteric Dual‐Controlled Gastrointestinal Therapeutic System of Salmon Calcitonin‐I: Preparation, Characterization, and Preclinical Bioavailability in Rats

Conventional dosage forms are disadvantageous for peptide drugs having short half‐lives because of cyclic under‐ or overdosing and problems of patient compliance. The matrix or reservoir type of controlled‐release dosage forms provide an alternative. However, bioavailability fluctuations due to gastric pH variations continue to be a problem for many drug candidates. Osmotically controlled drug delivery systems provide a means of eliminating the effect of pH on drug release. The objective of this study was to formulate salmon calcitonin (sCT), a hypocalcemic hormone, and turkey ovomucoid (serine protease inhibitor) along with glycyrrhetinic acid (absorption enhancer) as a bilayered, osmotically controlled tablet coated with a semipermeable membrane of cellulose acetate. Coating was achieved using a Strea‐1 fluidized bed coater. Orifices were drilled for drug release and enteric coating was applied in order to bypass the duodenum region. Comparative studies for the drug–additive interactions were performed using X‐ray diffraction, differential scanning calorimetry (DSC), Fourier transform infrared (FTIR) spectroscopy, and gel electrophoresis. Dissolution was carried out in HCl (0.1 N, pH 1.2) for 2 hr followed by phosphate buffer (pH 7.4) for 4 hrs. Results of X‐ray diffraction, DSC, FTIR, and gel electrophoresis did not indicate a strong interaction or defragmentation. Dissolution studies confirmed the enteric and dual‐controlled release of sCT and ovomucoid.