An Isolated Perfused Rat Liver Model: Simultaneous LC-MS Quantification of Pitavastatin, Coproporphyrin I, and Coproporphyrin III Levels in the Rat Liver and Bile.

The isolated perfused rat liver (IPRL) model provides a mechanistic understanding of the organic-anion-transporting polypeptide (OATP/Oatp)-mediated pharmacokinetics in the preclinical evaluation, which often requires the use of control substrates (i.e., pitavastatin) and monitoring endogenous biomarkers (coproporphyrin I and III). This study aimed to develop and validate an LC-MS method allowing the simultaneous quantification of pitavastatin, coproporphyrin I (CPI), and coproporphyrin III (CPIII) in rat liver perfusion matrices (perfusate, liver homogenate, bile). The analysis was performed on a C18 column at 60 °C with 20 µL of sample injection. The mobile phases consisted of water with 0.1% formic acid and acetonitrile with 0.1% formic acid with a gradient flow of 0.5 mL/min. The assay was validated according to the ICH M10 Bioanalytical Method Validation Guideline (2022) for selectivity, calibration curve and range, matrix effect, carryover, accuracy, precision, and reinjection reproducibility. The method allowing the simultaneous quantification of pitavastatin, CPI, and CPIII was selective without having carryover and matrix effects. The linear calibration curves were obtained within various calibration ranges for three analytes in different matrices. Accuracy and precision values fulfilled the required limits. After 60 min perfusion with pitavastatin (1 µM), the cumulative amounts of pitavastatin in the liver and bile were 5.770 ± 1.504 and 0.852 ± 0.430 nmol/g liver, respectively. CPIII was a more dominant marker than CPI in both liver (0.028 ± 0.017 vs 0.013 ± 0.008 nmol/g liver) and bile (0.016 ± 0.011 vs 0.009 ± 0.007 nmol/g liver). The novel and validated bioanalytical method can be applied in further IPRL preparations investigating Oatp-mediated pharmacokinetics and DDIs.

Development of a RP-HPLC method for simultaneous determination of atenolol, metoprolol tartrate and phenol red for in-situ rat intestinal perfusion studies.

Single-pass intestinal perfusion (SPIP) method is a widely used experimental model to determine the intestinal permeability of drugs. These studies are performed in the presence of a reference standard (metoprolol, MT) and a zero permeability marker (phenol red, PR). Therefore, it is important to develop a validated method for simultaneous determination of the investigated compound along with MT and PR. The aim of this study was to develop a reversed phase high-performance liquid chromatography (RP-HPLC) method with UV-detection for the simultaneous determination of atenolol (ATN), MT, and PR in the perfusion medium used in SPIP experiments. Separation of compounds were performed using an InertSustain C18 (250 × 4.6 mm, 5 µm) HPLC column at 35 °C. The mobile phase was a mixture of acetonitrile and phosphate buffer (pH 7.0, 12.5 mM) in gradient elution, and was delivered at a flow rate of 1 mL/min. The acetonitrile ratio of the mobile phase increased linearly from 10 to 35 % over 15 min. The injection volume was 20 µL, and ATN, MT and PR were detected at 224 nm. The retention times under optimum HPLC conditions were 5.028 min, 12.401 min, and 13.507 min for ATN, MT and PR, respectively. The developed RP-HPLC method was validated for selectivity, specificity, calibration curve and range, accuracy and precision, carry-over effect, stability, reinjection reproducibility, recovery and robustness. The method was linear for ATN (0.76-50 µg/mL), MT (1.14-50 µg/mL), and PR (0.47-20 µg/mL) with determination coefficients of 0.9999, 0.9994 and 0.9998, respectively. The results obtained for all validation parameters of the developed RP-HPLC method met the required limits of the ICH M10 Guideline.

Evaluation of in vitro dissolution profiles of modified-release metoprolol succinate tablets crushed using mortar and pestle technique.

PURPOSE: Clinical practice guidelines advise against crushing modified-release dosage forms. Metoprolol succinate modified-release (MS-MR) tablets are commonly crushed in clinical practice to facilitate administration to patients with swallowing difficulties or using feeding tubes. To date, the effect of this practice remains unexplored. The in vitro effects of crushing commercially available MS-MR tablets were explored using a holistic approach. METHODS: Dissolution profiles of crushed versus whole MS-MR tablets were compared. Tablets were crushed to powder state using pragmatic method mimicking hospital practices. For standardization purposes, the same operator, duration (60 seconds), hand, and mortar-pestle apparatus were used. Dissolution studies were conducted per U.S. Pharmacopeia at pH 1.2, pH 4.5, and pH 6.8 with USP apparatus 2 (paddle) at rotation speed of 50 rpm at 37±0.5 °C in 500 mL dissolution media. Samples were withdrawn at predetermined time points. Percent drug dissolved was measured by validated UV-vis Spectrophotometry. Comprehensive analysis of the dissolution data was conducted using model-independent, model-dependent, and ANOVA-based approaches (SPSS v.23 at α=0.05). Similarity (f2) and difference (f1) factors were calculated to compare the dissolution profiles between crushed (CT) and whole tablets (WT). Goodness of fit (GOF) analysis examined the compliance between in vitro dissolution behaviors and several drug release models. Model selection was based on GOF plots, Akaike criteria and adjusted coefficient of determination (R2adj). Imaging and particle size distribution analysis were conducted to examine associated surface and morphologic changes. RESULTS: The dissolution profiles were not similar at pH 4.5 (f2=45.43, f1=18.97) and pH 6.8 (f2=31.47, f1=32.94). CT best fitted with Higuchi (pH 1.2: R2adj=0.9990), Weibull (pH 4.5: R2adj=0.9884), and Korsmeyer-Peppas (pH 6.8: R2adj=0.9719). Contrastingly, WT best fitted with Hopfenberg (pH 1.2: R2adj=0.9986), logistic (pH 4.5: R2adj=0.9839) and first-order (pH 6.8: R2adj=0.9979) models. A significant difference in the dissolution profiles was found between CT and WT using multivariate analysis of variance per time points and between the tablet forms (p=0.004). This was confirmed by unparalleled dissolution profiles. Crushing resulted in variations in particle size and surface morphological changes to the micropellets. CONCLUSION: Crushing practices change the dissolution profile of MS-MR tablets by deforming the surface morphology of embedded micropellets. Amounts of drug dissolved between CT and WT were not the same at the compared time points across gastrointestinal pH ranges. This suggests potential clinical impact on plasma-concentration profiles of critically ill patients using feeding tube.

Influence of permeability enhancers on the paracellular permeability of metformin hydrochloride and furosemide across Caco-2 cells.

Permeability enhancers can affect absorption of paracellularly transported drugs. This study aims to evaluate effects of permeability enhancers (chitosan, methyl-ß -cyclodextrin, sodium caprate, sodium lauryl sulfate, etc.) on the permeability of paracellularly absorbed furosemide and metformin hydrochloride. Methyl thiazole tetrazolium bromide test was carried out to determine the drug concentrations in permeability study. Trans-epithelial electrical resistance (TEER) values determined to assess the integrity of tight junctions. Permeability enhancers were applied at different concentrations alone, in dual/triple combinations. Permeability was determined using human colorectal adenocarcinoma (Caco-2) cells (TEER > 400 Ω·cm2). Permeability enhancers have no significant effect (<2-fold; p > 0.05) on the permeability of furosemide (1.80 × 10-5 ± 4.55 × 10-7 cm/s); however, metformin permeability (1.36 × 10-5 ± 1.25 × 10-6 cm/s) increased significantly (p < 0.05) with 0.3% and 0.5% (w/v) chitosan (2.0- and 2.7-fold, respectively), 1% methyl-ß -cyclodextrin (w/v) (3.5-fold), 10 and 20 µmol/L sodium caprate (2.2- and 2.8-fold, respectively), and 0.012% sodium lauryl sulfate (w/v) (1.9-fold). Furosemide permeability increased significantly (p < 0.05) with chitosan-sodium lauryl sulfate combination (1.7-fold), and all triple combinations (1.4- to 1.9-fold). Chitosan containing dual/triple combinations resulted in significant increase (p < 0.05) in metformin permeability (1.7 to 2.8-fold). All results indicated that absorption of furosemide and metformin can be improved by the combination of permeability enhancers. Therefore, it can be evaluated for the formulation of development strategies containing furosemide and metformin by the pharmaceutical industry.

Development and characterization of pullulan-based orally disintegrating films containing amlodipine besylate.

The aim of this study was to prepare pullulan-based orally disintegrating films (ODFs) containing amlodipine besylate, an anti-hypertensive drug, by the solvent casting method. For this purpose, nine different ODF formulations (F1-F9) were prepared by using different plasticizers (glycerol, sorbitol, propylene glycol) and different superdisintegrants (croscarmellose sodium, sodium starch glycolate, crospovidone). FD&C Green and aspartame were used as coloring agent and sweetener, respectively. According to the results of preformulation studies, the optimum ODF (F9) was determined and various characterization studies such as uniformity of mass, film thickness, surface pH of films, and mechanical properties (such as elongation at break, tensile strength, Young's modulus, and folding endurance), moisture content, disintegration time, uniformity of content and dissolution test, X-ray, DSC, SEM and short term stability analysis were performed on this formulation. Cytotoxicity and permeability studies for the F9 formulation were performed on the human epithelial colorectal adenocarcinoma (Caco-2) cell line. The formulation F9 had appropriate morphological and mechanical properties and disintegrated within 51.3 s according to the petri dish method, and 28.8 s according to the drop method. Dissolution studies revealed that 78.1 % of amlodipine besylate was dissolved in 20 min from F9 formulation. Cell culture studies showed that the formulation had no significant toxic effect on the Caco-2 cells. Also, there was no significant difference between the Caco-2 permeabilities of amlodipine besylate powder and amlodipine besylate ODFs. As a result of all these studies, we suggest to use the pullulan based amlodipine besylate ODFs to enhance ease of administration and patient compliance.

Development and characterization of metformin hydrochloride- and glyburide-containing orally disintegrating tablets.

Diabetes is characterized by chronic hyperglycemia. Although metformin hydrochloride (MHCl)- and glyburide (GLB)-containing conventional tablets are available in the market and used to treat diabetes, orally disintegrating tablets (ODTs) containing the combination of these drugs are not commercially available. Therefore, the aim of this study was to prepare ODTs containing MHCl and GLB by direct-compression (DC-ODTs) and freeze-drying (FD-ODTs) methods. Physical properties of the powder mixture of DC-ODT formulation were determined (Angle of repose: 37.18 ± 1.27°; compressibility index: 20.31 ± 1.06%; Hausner ratio: 1.25 ± 0.03). Its moisture content was 0.3 ± 0.09%. The hardness values and the disintegration times for DC-ODTs and FD-ODTs were 221.60 ± 40.82 and 66.54 ± 2.68 N, and 80 and 30 s, respectively. Friability values were 0.24% for DC-ODTs and 0.38% for FD-ODTs. In uniformity-of-mass for single-dose-preparations test, the average weight was 684.38 ± 1.97 mg for DC-ODTs and 342.93 ± 2.4 mg for FD-ODTs, with less than 5% deviation for all 20 tablets. Water-absorption ratio for DC-ODTs was 1.30 ± 0.05. More than 90% of MHCl and GLB were dissolved within 5 min in both DC-ODTs and FD-ODTs. Although Caco-2 permeability of MHCl was influenced by the ODTs, GLB permeability was not. These results indicated that MHCl- and GLB-containing ODTs may be used as promising formulations for the treatment of diabetes.

Effects of curcumin-loaded PLGA nanoparticles on the RG2 rat glioma model.

BACKGROUND: Curcumin, the active ingredient of turmeric, has a remarkable antitumor activity against various cancers, including glioblastoma. However, it has poor absorption and low bioavailability; thus, to cross the blood-brain barrier and reach tumor tissue, it needs to be transferred to tumor site by special drug delivery systems, such as nanoparticles. OBJECTIVE: We aimed to evaluate the antitumor activity of curcumin on glioblastoma tissue in the rat glioma-2 (RG2) tumor model when it is loaded on poly(lactic-co-glycolic acid)-1,2-distearoyl-glycerol-3-phospho-ethanolamine-N-[methoxy (polyethylene glycol)-2000] ammonium salt (PLGA-DSPE-PEG) hybrid nanoparticles. METHODS: Glioblastoma was induced in 42 adult female Wistar rats (250-300g) by RG2 tumor model. The curcumin-loaded nanoparticles were injected by intravenous (n=6) or intratumoral route (n=6). There were five control groups, each containing six rats. First control group was not applied any treatment. The remaining four control groups were given empty nanoparticles or curcumin alone by intravenous or intratumoral route, respectively. The change in tumor volume was assessed by magnetic resonance imaging and histopathology before and 5days after drug injections. RESULTS: Tumor size decreased significantly after 5days of intratumoral injection of curcumin-loaded nanoparticle (from 66.6±44.6 to 34.9±21.7mm3, p=0.028), whereas it significantly increased in nontreated control group (from 33.9±21.3 to 123.7±41.1mm3, p=0.036) and did not significantly change in other groups (p>0.05 for all). CONCLUSION: In this in vivo experimental model, intratumoral administration of curcumin-loaded PLGA-DSPE-PEG hybrid nanoparticles was effective against glioblastoma. Curcumine-loaded nanoparticles may have potential application in chemotherapy of glioblastoma.

Dry powders for the inhalation of ciprofloxacin or levofloxacin combined with a mucolytic agent for cystic fibrosis patients.

OBJECTIVE: This study aimed to design and characterize an inhalable dry powder of ciprofloxacin or levofloxacin combined with the mucolytics acetylcysteine and dornase alfa for the management of pulmonary infections in patients with cystic fibrosis. METHODS: Ball milling, homogenization in isopropyl alcohol and spray drying processes were used to prepare dry powders for inhalation. Physico-chemical characteristics of the dry powders were assessed via thermogravimetric analysis, differential scanning calorimetry (DSC), Fourier transform infrared spectroscopy (FT-IR), X-ray diffractometry and scanning electron microscopy. The particle size distribution, dissolution rate and permeability across Calu-3 cell monolayers were analyzed. The aerodynamic parameters of dry powders were determined using the Andersen cascade impactor (ACI). RESULTS: After the micronization process, the particle sizes of the raw materials significantly decreased. X-ray and DSC results indicated that although ciprofloxacin showed no changes in its crystal structure, the structure of levofloxacin became amorphous after the micronization process. FT-IR spectra exhibited the characteristic peaks for ciprofloxacin and levofloxacin in all formulations. The dissolution rates of micro-homogenized and spray-dried ciprofloxacin were higher than that of untreated ciprofloxacin. ACI results showed that all formulations had a mass median aerodynamic diameter less than 5 µm; however, levofloxacin microparticles showed higher respirability than ciprofloxacin powders did. The permeability of levofloxacin was higher than those of the ciprofloxacin formulations. CONCLUSION: Together, our study showed that these methods could suitably characterize antibiotic and mucolytic-containing dry powder inhalers.

Microparticulate and nanoparticulate drug delivery systems for metformin hydrochloride.

CONTEXT: Metformin hydrochloride is a biguanide derivative widely used for the treatment of type 2 diabetes, prescribed nearly to 120 million people worldwide. Metformin has a relatively low oral bioavailability (about 50-60%). Although the major effect of metformin is to decrease hepatic glucose output as an antihyperglycemic agent, its inhibitory effects on the proliferation of some cancer cells (e.g. prostate, breast, glioma cells) have been demonstrated in the cell culture studies. Development of novel formulation (e.g. microparticles, nanoparticles) strategies for metformin might be useful to improve its bioavailability, to reduce the dosing frequency, to decrease gastrointestinal side effects and toxicity and to be helpful for effective use of metformin in cancer treatment. OBJECTIVE: The main aim of this review is to summarize metformin HCl-loaded micro- and nanoparticulate drug delivery systems. METHOD: The literature was rewieved with regard to the physicochemical, pharmacological properties of metformin, and also its mechanism of action in type 2 diabetes and cancer. In addition, micro- and nanoparticulate drug delivery systems developed for metformin were gathered from the literature and the results were discussed. CONCLUSION: Metformin is an oral antihyperglycemic agent and also has potential antitumorigenic effects. The repeated applications of high doses of metformin (as immediate release formulations) are needed for an effective treatment due to its low oral bioavailability and short biological half-life. Drug delivery systems are very useful systems to overcome the difficulties associated with conventional dosage forms of metformin and also for its effective use in cancer treatment.

HPLC method development for the simultaneous analysis of amlodipine and valsartan in combined dosage forms and in vitro dissolution studies

A simple, rapid and reproducible HPLC method was developed for the simultaneous determination of amlodipine and valsartan in their combined dosage forms, and for drug dissolution studies. A C18 column (ODS 2, 10 μm, 200 x 4.6 mm) and a mobile phase of phosphate buffer (pH 3.6 , 0.01 mol L-1):acetonitrile: methanol (46:44:10 v/v/v) mixture were used for separation and quantification. Analyses were run at a flow-rate of 1 mL min-1 and at ambient temperature. The injection volume was 20 μL and the ultraviolet detector was set at 240 nm. Under these conditions, amlodipine and valsartan were eluted at 7.1 min and 3.4 min, respectively. Total run time was shorter than 9 min. The developed method was validated according to the literature and found to be linear within the range 0.1 - 50 μg mL-1 for amlodipine, and 0.05 - 50 μg mL-1 for valsartan. The developed method was applied successfully for quality control assay of amlodipine and valsartan in their combination drug product and in vitro dissolution studies.

Desenvolveu-se método de HPLC rápido e reprodutível para a determinação simultânea de anlodipino e valsartana em suas formas de associação e para os estudos de dissolução dos fármacos. Utilizaram-se coluna C18 (ODS 2, 10 μm, 200 x 4,6 mm) e fase móvel tampão fosfato (pH 3,6, 0,01 mol L-1):acetonitrila: metanol para a separação e a quantificação. As análises foram efetuadas com velocidade de fluxo de 1 mL min-1 e à temparatura ambiente O volume de injeção foi de 20 μL e utilizou-se detector de ultravioleta a 240 nm. Sob essas condições, anlodipino e valsartana foram eluídas a 7,1 min e 3,4 min, respectivamente. O tempo total de corrida foi menor que 9 min. O método desenvolvido foi validado de acordo com a literatura e se mostrou linear na faixa de 0,1-50 μg mL-1 para anlodipino e de 0,05-50 μg mL-1 para valsartana. O método desenvolvido foi aplicado com sucesso para ensaios de controle de qualidade de associações de anlodipino e valsartana e nos estudos de dissolução in vitro.

Formulation and characterization of formaldehyde cross-linked degradable starch microspheres containing terbutaline sulfate.

Preparation of starch microspheres using epichlorohydrin is a time consuming method and requires around 18 hr for cross-linking reaction. To reduce reaction time, terbutaline sulfate (TBS) loaded degradable starch microspheres (DSM) were prepared using formaldehyde as the cross-linking agent. All microspheres were spherical in shape and had a porous, rough surface with a mean particle size of 18-24 microm. Whatever the cross-linking time, it was seen that the release of the TBS was not complete during the release experiments. The influence of enzyme on the degradation of microspheres was moderate. Following intravenous administration, initial uptake of microspheres by the lung was higher than those of other organs.