Drug-related problems and pharmacy interventions in non-prescription medication, with a focus on high-risk over-the-counter medications.

Background The risks associated with over-the-counter medication are often underestimated by consumers. The incorrect use of certain medications can lead to significant patient harm. Inappropriate use can be prevented by pharmaceutical counselling. Objective To determine the number and nature of drug-related problems in over-the-counter medication with a special emphasis on high-risk over-the-counter medications. Setting Fifty-two community pharmacies in Finland. Method This observational study was conducted as a questionnaire survey. The pharmacists working in participating pharmacies documented the observed drug-related problems and pharmacy interventions in over-the-counter medication during 1 week using an electronic study form based on the Westerlund drug-related problem classification system. Main outcome measure The prevalence of drug-related problems and problem types in different medication categories. Results The 52 community pharmacies documented 339 drug-related problems in 0.6% of over-the-counter customers, the most common problem being "Uncertainty about the indication for the drug" (39.2%). A significant proportion of the documented problems (26.3%) concerned high-risk over-the-counter medications, and the majority of these cases were associated with non-steroidal anti-inflammatory drugs (21.8%). In total, pharmacies made 641 interventions to resolve the drug-related problems. For majority of drug-related problems (87%), pharmacist's intervention involved counselling. In more than half of the problem cases, the pharmacy intervention was precautionary. Conclusion Pharmacists intervene in and prevent problems related to over-the-counter medications, including high-risk medications like analgesics, in which inappropriate use due to consumers' lack of knowledge can lead to severe consequences. As the selection and use of over-the-counter medications is continuously increasing, pharmaceutical counselling should be readily available and actively provided for consumers to achieve safer self-medication.

The Consequence of Drug-Drug Interactions Influencing the Interplay between P-Glycoprotein and Cytochrome P450 3a: An Ex Vivo Study with Rat Precision-Cut Intestinal Slices.

P-glycoprotein (P-gp) and cytochrome P450 3A (CYP3A) are differentially expressed along the intestine and work coordinately to reduce the intracellular concentration of xenobiotics and the absorption of orally taken drugs. Drug-drug interactions (DDIs) based on P-gp/CYP3A interplay are of clinical importance and require preclinical investigation. We investigated the P-gp/Cyp3a interplay and related DDIs with different P-gp inhibitors in the various regions of the rat intestine ex vivo using precision-cut intestinal slices (PCIS) with quinidine (Qi), a dual substrate of P-gp and Cyp3a, as the probe. The results showed that P-gp efflux was the main factor limiting the intracellular Qi content at concentrations below 5µM, whereas both efflux and metabolism were saturated at [Qi] > 50µM. The selective P-gp inhibitors CP100356 [N-(3,4-dimethoxyphenethyl)-4-(6,7-dimethoxy-3,4-dihydroisoquinolin-2[1H]-yl)-6,7-dimethoxyquinazolin-2-amine] and PSC833 [valspodar, 6-[(2S,4R,6E)-4-methyl-2-(methylamino)-3-oxo-6-octenoic acid]-7-l-valine-cyclosporin A] enhanced the Qi accumulation in slices in line with the different P-gp expression in the intestinal regions and, as a result, also enhanced metabolism in the jejunum and ileum. Dual inhibitors of both P-gp and Cyp3a (verapamil and ketoconazole) increased the concentration of Qi in the jejunum and ileum, but less 3-hydroxy-quinidine was produced due to inhibition of Cyp3a. The results indicate that the P-gp/Cyp3a interplay depends on the concentration of the drug and on the intestinal region under study. Furthermore, due to the P-gp/Cyp3a interplay, DDIs can lead to remarkable changes in the intracellular concentration of both the parent drug and the metabolite, which varies among the intestinal regions and depends on the selectivity of the inhibitors, with potentially important implications for disposition and toxicity of drugs and their metabolites.

Nanoparticle formulation of a poorly soluble cannabinoid receptor 1 antagonist improves absorption by rat and human intestine.

The inclusion of nanoparticles dispersed in a hydrophilic matrix is one of the formulation strategies to improve the bioavailability of orally administered Biopharmaceutics Classification System (BCS) class II and IV drugs by increasing their dissolution rate in the intestine. To confirm that the increased dissolution rate results in increased bioavailability, in vitro and in vivo animal experiments are performed, however, translation to the human situation is hazardous. In this study, we used a range of in vitro and ex vivo methods, including methods applying human tissue, to predict the in vivo oral bioavailability of a model BCS class II CB-1 antagonist, formulated as a nanoparticle solid dispersion. The enhanced dissolution rate from the nanoparticle formulation resulted in an increased metabolite formation in both rat and human precision-cut intestinal slices, suggesting increased uptake and intracellular drug concentration in the enterocytes. In Ussing chamber experiments with human tissue, both the metabolite formation and apical efflux of the metabolite were increased for the nanoparticulate solid dispersion compared with a physical mixture, in line with the results in intestinal slices. The pharmacokinetics of the different formulations was studied in rats in vivo. The nanoparticle formulation indeed improved the absorption of the cannabinoid receptor 1 (CB-1) antagonist and the delivery into the brain compared with the physical mixture. In conclusion, the combined approach provides a valuable set of tools to investigate the effects of formulation on the absorption of poorly soluble compounds in human intestine and may provide relevant information on the oral bioavailability in humans early in the development process.

In vitro methods to study the interplay of drug metabolism and efflux in the intestine.

This review provides an overview of the in vitro methods currently used in studies of intestinal drug metabolism and active efflux with a special emphasis on the efflux- metabolism interplay. These methods include e.g. expressed enzymes or efflux transporters, fractionated intestinal cells, cell lines, primary cells, intestinal segments and other tissue preparations. Pharmacokinetics of effluxmetabolism interplay is often very complicated, possibly involving saturation, stimulation and/or inhibition of one or both of these mechanisms. Parent drug and/or metabolite(s) can be substrates for several enzymes and/or efflux proteins. These detoxifying proteins may alter the exposure of drugs to each other and, consequently, their contributions to the overall drug elimination. Depending on the complexity of the in vitro system used, different kinds of information can be extracted from the results. Simple methods concentrating on single mechanisms provide easily interpretable information, but neglect the interplay between various mechanisms influencing the kinetics in a whole organism. More complex experimental systems mimic the mechanistic complexity of in vivo setting better, but at the same time the interpretation and utilization of the results becomes more challenging. Advantages and limitations of various in vitro systems are addressed and consideration is given to the physiological relevance of the results obtained and there is discussion of approaches for in vitro - in vivo translation of the data.

Defining new criteria for selection of cell-based intestinal models using publicly available databases.

BACKGROUND: The criteria for choosing relevant cell lines among a vast panel of available intestinal-derived lines exhibiting a wide range of functional properties are still ill-defined. The objective of this study was, therefore, to establish objective criteria for choosing relevant cell lines to assess their appropriateness as tumor models as well as for drug absorption studies. RESULTS: We made use of publicly available expression signatures and cell based functional assays to delineate differences between various intestinal colon carcinoma cell lines and normal intestinal epithelium. We have compared a panel of intestinal cell lines with patient-derived normal and tumor epithelium and classified them according to traits relating to oncogenic pathway activity, epithelial-mesenchymal transition (EMT) and stemness, migratory properties, proliferative activity, transporter expression profiles and chemosensitivity. For example, SW480 represent an EMT-high, migratory phenotype and scored highest in terms of signatures associated to worse overall survival and higher risk of recurrence based on patient derived databases. On the other hand, differentiated HT29 and T84 cells showed gene expression patterns closest to tumor bulk derived cells. Regarding drug absorption, we confirmed that differentiated Caco-2 cells are the model of choice for active uptake studies in the small intestine. Regarding chemosensitivity we were unable to confirm a recently proposed association of chemo-resistance with EMT traits. However, a novel signature was identified through mining of NCI60 GI50 values that allowed to rank the panel of intestinal cell lines according to their drug responsiveness to commonly used chemotherapeutics. CONCLUSIONS: This study presents a straightforward strategy to exploit publicly available gene expression data to guide the choice of cell-based models. While this approach does not overcome the major limitations of such models, introducing a rank order of selected features may allow selecting model cell lines that are more adapted and pertinent to the addressed biological question.

Transport of the coumarin metabolite 7-hydroxycoumarin glucuronide is mediated via multidrug resistance-associated proteins 3 and 4.

Coumarin (1,2-benzopyrone) is a natural compound that has been used as a fragrance in the food and perfume industry and could have therapeutic usefulness in the treatment of lymphedema and different types of cancer. Several previous pharmacokinetic studies of coumarin have been performed in humans, which revealed extensive first-pass metabolism of the compound. 7-Hydroxycoumarin (7-HC) and its glucuronide (7-HC-G) are the main metabolites formed in humans, and via this route, 80 to 90% of the absorbed coumarin is excreted into urine, mainly as 7-HC-G. Active transport processes play a role in the urinary excretion of 7-HC-G; however, until now, the transporters involved remained to be elucidated. In this study, we investigated whether the efflux transporters multidrug resistance-associated proteins (MRP)1-4, breast cancer resistance protein, or P-glycoprotein play a role in 7-HC and 7-HC-G transport. For this purpose, we measured uptake of the metabolites into membrane vesicles overexpressing these transporters. Our results showed that 7-HC is not transported by any of the efflux transporters tested, whereas 7-HC-G was a substrate of MRP3 and MRP4. These results are in line with the pharmacokinetic profile of coumarin and suggest that MRP3 and MRP4 are the main transporters involved in the excretion of the coumarin metabolite 7-HC-G from liver and kidney.

Caco-2 cell monolayers as a tool to study simultaneous phase II metabolism and metabolite efflux of indomethacin, paracetamol and 1-naphthol.

The human intestinal cell line, Caco-2, was used to study compounds - indomethacin, paracetamol and 1-naphthol - that undergo intestinal phase II metabolism followed by apical and/or basolateral efflux of the metabolites and/or parent compounds. The interplay was studied during permeability experiments across fully differentiated Caco-2 cell monolayers. The parent compounds and their glucuronide and/or sulfate metabolites were detected by LC-MS/MS. Conjugation of the model compounds and effluxes of their metabolites were observed. The efflux of indomethacin glucuronide was apical, but complementary basolateral efflux was observed at the highest indomethacin concentration (500 microM), probably due to apical saturation. Paracetamol glucuronide was not formed in these experiments, but apical and basolateral effluxes of paracetamol sulfate were observed. A typical bell-shaped inhibition curve was observed for the formation of 1-naphthol glucuronides, indicating substrate or product inhibition of the UGT enzyme(s) at higher 1-naphthol concentrations (200 microM and 500 microM). Based on these results, the fully differentiated Caco-2 cell monolayers can be applied as a platform for qualitative in vitro studies, where phase II metabolism and efflux activities are ongoing simultaneously.

A Caco-2 cell based screening method for compounds interacting with MRP2 efflux protein.

The aim of this work was to develop a screening method for MRP2 efflux substrates using the well-characterized, human-based intestinal Caco-2 cell model as a platform. MRP2 has a significant role in drug absorption and disposition and is known to co-operate with phase II metabolic enzymes. Caco-2 cells grown in a 96-well plate were loaded with non-fluorescent CDCFDA (diacetate ester of 5(6)-carboxy-2',7'-dichlorofluorescein), which is hydrolyzed to fluorescent CDCF by intracellular esterases. De-esterification in Caco-2 was comparable to that in porcine liver esterases. CDCFDA enters the cells passively, while CDCF is effluxed out of the cells by the apically localized MRP2 and/or basolateral MRPs. The method was optimized with regard to several factors. In the concluding protocol, Caco-2 cells are grown on clear 96-well plates for 8 days. The loading conditions were optimized to 10 min incubation with 5 microM CDCFDA. The highest responses were obtained for samples taken at t=30 min. The samples were analyzed in black 96-well plates with a fluorescence plate reader. The Caco-2 based method utilizing the probe pair CDCFDA/CDCF provides a fast screening tool for MRP2 substrates and/or inhibitors, along with compounds having metabolites formed in Caco-2 that interact with MRP2.

The expression of most UDP-glucuronosyltransferases (UGTs) is increased significantly during Caco-2 cell differentiation, whereas UGT1A6 is highly expressed also in undifferentiated cells.

The human colon carcinoma cell line Caco-2 is often used as a model for intestinal drug absorption. To better understand xenobiotic glucuronidation in Caco-2 cells, we have examined the expression levels of different UDP-glucuronosyltransferases (UGTs) in them. The effects of two main factors were investigated, namely, passage number and cell differentiation. Hence, the mRNA levels of 15 human UGTs of subfamilies 1A and 2B were assessed in both undifferentiated and fully differentiated cells at four passage levels: P31, P37, P43, and P49. Quantitative reverse transcriptase-polymerase chain reaction was used to determine the mRNA levels of individual UGTs, and the values were normalized using beta-actin as a reference gene. The results indicate that although passage number in the tested range exerts a mild effect on the expression level of several UGTs, the contribution of cell differentiation is much larger. The expression of nearly all the UGTs that were examined in this study was significantly, sometimes greatly, increased during cell differentiation. UGT1A6 was a distinct exception to this rule, however, because it was already highly expressed in the undifferentiated cells. The mRNA findings were confirmed at the enzyme activity level by measuring the glucuronidation of 1-naphthol, a very good substrate for UGT1A6, as well as estradiol that is not glucuronidated by this enzyme. The results revealed that 1-naphthol glucuronidation activity was high in both the differentiated and undifferentiated cells, whereas estradiol glucuronidation was only detected in the differentiated cells. Thus, Caco-2 cell differentiation plays a major role in UGT expression and ensuing metabolic reactions.

Effect of cell differentiation and passage number on the expression of efflux proteins in wild type and vinblastine-induced Caco-2 cell lines.

The mRNA level expression of MDR1, MRP1-6, BCRP and CYP3A4 was determined by quantitative PCR in wild type (Caco-2WT) and vinblastine-treated (Caco-2VBL) Caco-2 cells at different passage levels (32-53). Differentiation increased the mRNA levels of MDR1, BCRP and all the MRPs except MRP4. Corresponding mRNA levels were observed in Caco-2WT and Caco-2VBL, except that the expression of MRD1 was higher in Caco-2VBL than in Caco-2WT cells. CYP3A4 was barely detected in either cell line. MDR1 functionality was studied using rhodamine123 and verapamil as a substrate-inhibitor pair. Corresponding to the observed differences in mRNA levels, MDR1 activity was higher in the Caco-2VBL cells. In Caco-2WT, MDR1 functionality was elevated at low passage numbers (32-35) compared to higher ones (49-53). Verapamil inhibited MDR1 efflux except at higher passage Caco-2WT cells, where no MDR1 activity could be observed. The results support the use of Caco-2VBL cells in MDR1 screening. The functional expression is higher than in Caco-2WT and remains consistent across the studied passages without major differences in mRNA levels of other efflux proteins. As both the passage number and the level of cell differentiation affect the expression profile of efflux proteins, short-term cell growth protocols should be evaluated accordingly.