Leena Laitinen

N-in-One Permeability Studies of Heterogeneous Sets of Compounds across Caco-2 Cell Monolayers

AbstractPurpose. The purpose of the study was to evaluate several n-in-one cocktails of heterogeneous compounds to increase the throughput of permeability studies across Caco-2 monolayers, to investigate the reliability and applicability of the method, and to develop fast and sensitive analysis for the compounds. Compounds with potential interactions in efflux and/or active transport were chosen. Methods Permeability experiments with verapamil, propranolol, midazolam, hydroxyzine, timolol, buspirone, procaine, naproxen, ketoprofen, and antipyrine as single compounds and in cocktails of 5-10 compounds were performed at 50 μM concentration both in the apical-to-basolateral and basolateral-to-apical direction. The compounds were quantified by liquid chromatography-electrospray tandem mass spectrometry (LC-ESI/MS/MS). Toxicity tests were performed to determine cellular damage. Results The analytical method was sensitive, accurate, and rapid. The individual permeabilities of compounds in cocktails correlated well with permeabilities as single compounds. No significant interactions between the compounds within the mixtures were observed, except for acidic compounds. The studied mixtures did not show any toxicity. Conclusions The use of n-in-one cocktails is a suitable method to improve the capacity in routine permeability experiments and higher throughput screening of drug candidates, although potential interactions should always be borne in mind. The use of LC-ESI/MS/MS technology provides an excellent tool in fast and accurate analysis of small amounts of heterogeneous compounds.

Effects of extracts of commonly consumed food supplements and food fractions on the permeability of drugs across Caco-2 cell monolayers.

AbstractPurpose. Extracts made from berries, herbs, and various plant materials, which might possess a range of activities, are used as health promoting products. Because little is known about their effects on the absorption of co-administered drugs, the effects of some food supplements, Finnish berries, and herbs were studied on the permeability of some commonly used drugs. Methods. The permeabilities of verapamil, metoprolol, ketoprofen, paracetamol, and furosemide were studied across Caco-2 cell monolayers with contemporaneously administered extracts from flax seed, purple loosestrife, and Scots pine bark; bilberries, cowberries, and raspberries; oregano, rosemary, and sage. Toxicological tests were conducted to determine cellular damage. Results. The effects of extracts on drug permeabilities were generally minor. Flax seed decreased the permeability of all drugs except verapamil. Purple loosestrife and pine decreased verapamil and metoprolol permeability. Changes caused by berries were mainly pH-related. Rosemary and oregano enhanced furosemide permeability. Conclusions. Ingestion of extracts of herbs and berries studied are not expected to markedly change the permeabilities of highly permeable drugs. Harmful effects at sites of or during absorption are unlikely. However, if high doses of extracts are administered with low permeable drugs in vitro,effects on drug permeabilities could not be excluded. Use of such extracts should therefore be evaluated during continuous medication.

In-vitro mutagenic potential and effect on permeability of co-administered drugs across Caco-2 cell monolayers of Rubus idaeus and its fortified fractions

This study investigated the mutagenic, anti‐mutagenic and cytotoxic effects of acetone extract of raspberry, Rubus idaeus L. (v. Ottawa) Rosaceae, and the isolated and characterized ellagitannin and anthocyanin fractions thereof, suitable for food applications. The studied raspberry extract and fractions did not show any mutagenic effects determined in the miniaturized Ames test and were not cytotoxic to Caco‐2 cells at the used concentrations. However, the anti‐mutagenic properties were changed (i.e. decreased mutagenicity of 2‐nitrofluorene in strain TA98, and slightly increased mutagenicity of 2‐aminoanthracene in strain TA100) with metabolic activation. Further, their influence on the permeability of co‐administered common drugs (ketoprofen, paracetamol, metoprolol and verapamil) across Caco‐2 monolayers was evaluated. The apical‐to‐basolateral permeability of highly permeable verapamil was mostly affected (decreased) during co‐administration of the raspberry extract or the ellagitannin fraction. Ketoprofen permeability was decreased by the ellagitannin fraction. Consumption of food rich in phytochemicals, as demonstrated here with chemically characterized raspberry extract and fractions, with well‐absorbing drugs would seem to affect the permeability of some of these drugs depending on the components. Thus their effects on the absorption of drugs in‐vivo cannot be excluded.

Permeability profiles of M-alkoxysubstituted pyrrolidinoethylesters of phenylcarbamic acid across caco-2 monolayers and human skin.

AbstractPurpose. The purpose of the present research was to study 10 m-alkoxysubstituted pyrrolidinoethylesters of phenylcarbamic acid—potential local anesthetics. The relationships between the structure of the molecule, its physicochemical parameters (log Doct, log k, RM, solubility) were correlated to the permeability data obtained from permeation experiments in Caco-2 monolayers and excised human skin in vitro. Methods. The extent and mechanism(s) of permeability of the series were studied through a Caco-2 monolayer in the apical-to-basolateral (a-b) and basolateral-to-apical (b-a) directions. The MTT test was performed to determine cellular damage. In vitro transdermal permeability data were obtained from permeation experiments on excised human skin by using side-by-side chambers. Passive diffusion and iontophoretically enhanced permeability were measured. Results. In Caco-2 monolayers, similar results in the shape of the permeability curves were obtained for the two directions. In the b-a direction, the values of Papp were ∼2-6 times greater than in the a-b direction. A plot of drug permeability vs. the number of carbons in the alkoxychain plateaued first, after which the permeability decreased by the increasing lipophilicity of the drug. If the log Doct of the ester was ≥ 3.4 and the MW > 385 Da, no measurable Caco-2 permeability was found. Cell damage was also higher by the more lipophilic compounds. In excised human skin, the relationship between the passive diffusion of the drugs and the number of carbons in the alkoxychain was parabolic (r2 = 0.95). Introducing low-level electrical current (iontophoresis), transdermal permeability of the more hydrophilic phenylcarbamic acid esters increased clearly. Conclusions. Lipophilicity and solubility of a compound have crucial roles in the permeation process. A very high lipophilicity has, however, a negative influence on the permeability, both intestinally and transdermally. Iontophoresis significantly increases the diffusion of small and less lipophilic compounds.

Prolonged low-molecular-weight heparin use during pregnancy and subsequent bone mineral density ☆

INTRODUCTION In contrast to unfractionated heparin (UFH), use of low-molecular weight heparin (LMWH) during pregnancy has not been reported to be associated with a significant decrease in bone mineral density (BMD). The aim of this study was to investigate whether long-term use of LMWH during pregnancy is associated with subsequent decrease in BMD or with increased number of osteoporotic fractures. MATERIALS AND METHODS In this observational cohort study BMD was measured by dual energy X-ray absorptiometry (DEXA) 4-7years after the last delivery in 152 women. Ninety-two women had prolonged LMWH-exposure during pregnancy - 75 as prophylaxis and 17 as treatment for venous thromboembolic event (VTE). Dalteparin and enoxaparin were the LMWH-preparations used. Sixty women without LMWH-exposure served as controls. A questionnaire about lifestyle factors and medical history was filled out by the subjects. RESULTS Lumbar spine BMD in the LMWH users was lower than that in the controls both in the prophylactic group (1.22g/cm(2) vs. 1.27g/cm(2); p=0.03), and in the treatment group (1.20g/cm(2) vs. 1.27g/cm(2); p=0.07). BMD in femoral neck did not differ between the LMWH-users and controls. However, after adjusting for potential confounding factors, LMWH-exposure did not remain associated with decreased BMD in lumbar spine. Use of contraceptive pills was positively associated with BMD in lumbar spine. Incidence of osteopenia was 13% in the LMWH-group and 8% in the control-group, (p=0.4). No osteoporosis or osteoporotic fractures were found. CONCLUSIONS Prolonged use of LMWH during pregnancy was not associated with subsequent decrease in BMD, osteopenia, osteoporosis, or osteoporotic fractures.