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Biomedical subjects

Patrick Augustijns

Publications and source records attributed to Patrick Augustijns.

18 recordsLinked to original sources
PubMed2006

In vitro behavior of a phosphate ester prodrug of amprenavir in human intestinal fluids and in the Caco-2 system: illustration of intraluminal supersaturation.

As a result of their improved aqueous solubility, the development of phosphate ester prodrugs is an interesting approach to increase intestinal absorption of poorly water-soluble drugs. Absorption of a drug from its phosphate ester prodrug is based on intestinal dephosphorylation of the prodrug which may result in intraluminal supersaturation of the parent drug, followed by an increased absorptive flux across the intestinal mucosa. In this study, we evaluated the behavior of fosamprenavir, a phosphate ester prodrug of amprenavir, in the Caco-2 system and in aspirated human intestinal fluids (HIF), both showing phosphatase activity. Starting from a solution of fosamprenavir in HIF, a supersaturated solution of amprenavir was generated and maintained during a time period sufficient for absorption. Moreover, supersaturation of amprenavir resulted in an enhanced flux across Caco-2 monolayers. To our knowledge, this is the first illustration of supersaturation in real intestinal media. Next, we showed an inhibitory effect of inorganic phosphate on the dephosphorylation of fosamprenavir, both in the Caco-2 model and in HIF. As a consequence, phosphate-buffered media, including fasted state simulated intestinal fluid (FaSSIF), are incompatible with the study of phosphate ester prodrugs and should be replaced with media containing a biorelevant phosphate concentration (0.4-1 mM) and another buffering compound such as 2-morpholinoethanesulfonic acid (MES).

Alkaline Phosphatase↗
PubMed2006

Novel generic UPLC/MS/MS method for high throughput analysis applied to permeability assessment in early Drug Discovery.

A novel generic ultra performance liquid chromatography-tandem mass spectrometric (UPLC/MS/MS) method for the high throughput quantification of samples generated during permeability assessment (PAMPA) has been developed and validated. The novel UPLC/MS/MS methodology consists of two stages. Firstly, running a 1.5min isocratic method, compound-specific multiple reaction monitoring (MRM) methods were automatically prepared. In a second stage, samples were analyzed by a 1.5min generic gradient UPLC method on a BEH C18 column (50mmx2.1mm). Compounds were detected with a Waters Micromass Quattro Premier mass spectrometer operating in positive electrospray ionization using the compound-specific MRM methods. The linearity for the validation compounds (caffeine, propranolol, ampicillin, atenolol, griseofulvin and carbamazepine) typically ranges from 3.05nM to 12,500nM and the limits of detection for all generically developed methods are in the range between 0.61nM and 12nM in an aqueous buffer. The novel generic methodology was successfully introduced within early Drug Discovery and resulted in a four-fold increase of throughput as well as a significant increase in sensitivity compared to other in-house generic LC/MS methods.

Ampicillin↗
PubMed2006

Intraluminal drug and formulation behavior and integration in in vitro permeability estimation: a case study with amprenavir.

The purpose of this study was to assess the effect of biorelevant apical conditions on intestinal permeability estimation in the Caco-2 system for amprenavir, a poorly water-soluble substrate of the efflux carrier P-glycoprotein (P-gp). To establish biorelevant conditions, human intestinal fluids (HIF) were aspirated from the duodenum and jejunum in fasted subjects, before and during 4 h after the intake of a standard formulation of amprenavir (Agenerase). The HIF samples were characterized with respect to the concentrations of phospholipids, individual bile salts, amprenavir, and the excipient d-alpha-tocopheryl polyethyleneglycol 1000 succinate (TPGS); subsequently, the use of these samples in the Caco-2 system during permeability estimation for amprenavir was compared to standard conditions (amprenavir 10 microM dissolved in HBSS-based transport medium). The presence of the solubilizing excipient TPGS resulted in high intraluminal amprenavir concentrations (mM-range) and affected the permeability in a concentration-dependent way. At the observed intraluminal TPGS concentrations (mM-range), TPGS appeared to completely inhibit the interaction between amprenavir and P-gp, suggesting that the effect of P-gp on transepithelial transport of amprenavir in a clinical setting is probably negligible. This study illustrates the importance of the evaluation of intraluminal conditions after drug intake and their integration in permeability estimation in vitro.

Adult↗
PubMed2006

Characterization of fasted-state human intestinal fluids collected from duodenum and jejunum.

The solubility of drugs in the gastrointestinal tract is very challenging to simulate with artificial media due to the high complexity of human intestinal fluid (HIF). In particular, bile salt composition, pH and buffer capacity are very important characteristics of HIF, since they determine the solubility of drugs in-vivo. In this study, we have measured the concentrations of individual bile salts in human intestinal fluids (n=6) collected from two different locations (duodenum and jejunum) in the fasted state. Total bile salt concentrations ranged from 570 to 5,137 microM in the duodenum and from 829 to 5,470 microM in the jejunum. The following rank order of relative bile salt concentration in duodenum was observed: taurocholic acid > glycocholate >or= glycochenodeoxycholate > glycodeoxycholate > taurochenodeoxycholate > taurodeoxycholate. Cholic acid, tauroursodeoxycholate, chenodeoxycholic acid, and deoxycholic acid represented less than 1% of bile salts present in the samples. Ursodeoxycholate could not be detected in HIF. No statistically significant difference between bile salt composition of duodenal and jejunal aspirates was observed. The buffer capacity of HIF was compared with other media commonly used for solubility/dissolution determinations, indicating a relatively low buffer capacity of HIF (4-13 mmol L(-1)/pH). This low buffer capacity was reflected in the change in pH (between 4 and 9.5) that occurred in HIF after addition of model compounds covering a broad pK(a) range. Interindividual variability in pH, buffer capacity and bile salt contents of HIF will contribute to differences in the rate and extent of absorption of compounds for which dissolution/solubility is the rate limiting step. The variability observed warrants further research to explore the impact of intraluminal conditions on drug solubility.

Adult↗
PubMed2005

Specific antibody modulates absorptive transport and metabolic activation of benzo[a]pyrene across Caco-2 monolayers.

It has been shown that oral anticarcinogen antibodies can decrease intestinal absorption of procarcinogens. So far, no attempts have been made to understand the potential modulatory effect of such antibodies on metabolic activation at mucosal surfaces. Moreover, the influence of naturally induced serosal-specific antibodies on absorptive transport of carcinogens remains unknown. In this study, the prototype food carcinogen benzo[a]pyrene (B[a]P) and a specific monoclonal antibody were used to address these questions in a bicompartmental model of polarized intestinal cells (Caco-2). Apical (i.e., luminal) administration of a 30-fold molar excess antibodies increased about 25-fold recovery of unmetabolized B[a]P, concomitantly with a decrease of 80% in both absorptive transport and formation of phenol metabolites. Interestingly, when metabolism was slowed down by antibodies, cross-reactive antibodies also increased at least 5-fold the extracellular levels of the 7,8-diol-B[a]P, interrupting subsequent activation steps. On the other hand, basolateral antibodies changed by 8-fold the rate of carcinogen appearance in the basolateral compartment, albeit without interfering with the apical cellular uptake or sequestration of either B[a]P or 7,8-diol-B[a]P by apical antibodies. Furthermore, basolateral antibodies reduced exposure of Caco-2 monolayers to B[a]P as demonstrated by a 50% decrease in apical efflux of 3-OH-B[a]P. These data show for the first time that both luminal and serosal antibodies may reduce the carcinogenic process by preventing high local concentrations, which would overload DNA repair mechanisms. This study also sheds light on the relevance of both naturally induced and immunoprophylactic antibodies against polycyclic aromatic hydrocarbon carcinogens.

Absorption↗
PubMed2005

In vitro intestinal transport and antihypertensive activity of ACE inhibitory pea and whey digests.

Angiotensin I converting enzyme (ACE) inhibitory peptides cause an antihypertensive effect if they reach the systemic circulation. This was investigated for the high ACE inhibitory activity present in peas and whey in vitro gastrointestinal digests. The samples retained high ACE inhibitory activity when incubated in Caco-2 homogenates or rat intestinal acetone powder, both sources of small intestine peptidases. Only little ACE inhibitory activity was transported through Caco-2 cell monolayers in 1 h. As the Caco-2 model is tighter than intestinal mammalian tissue, sufficient absorption of these peptides might occur in vivo. After intravenous administration of 50 mg protein kg(-1) BW in spontaneously hypertensive rats (SHR), pea digest exerted a transient, but strong antihypertensive effect of 44.4 mmHg. Whey digest exerted no effect at this dose. These results suggest that pea digest could be a promising source of ACE inhibitory peptides for use in the prevention and treatment of hypertension.

Acetone↗
PubMed2005

High-speed digital imaging method for ciliary beat frequency measurement.

The aim of this study was to develop a high-speed digital imaging system and related software for ciliary beat frequency (CFB) analysis in order to establish an automated and reliable method that is observer independent and faster compared to the conventional computerized microscope photometry method. Using primary human nasal epithelial cell cultures, the CBF was recorded with a computerized microscope photometry system and a high-speed digital imaging system. To obtain a wide range of frequencies, glycocholate (0.5%) and chlorocresol (0.005%) were used as ciliostimulatory and cilio-inhibitory reference compounds, respectively. The mean values in hertz (+/- s.d.) obtained with the photometry and high-speed digital imaging systems were: controls 8.2 +/- 0.9 and 7.9 +/- 1.1; chlorocresol 5.0 +/- 0.9 and 5.1 +/- 1.1; glycocholate 9.8 +/- 1.0 and 9.7 +/- 0.8. A similar increase (by 20 and 24%) and decrease (by 38 and 35%) in CBF was determined by the two methods after glycocholate and chlorocresol treatment, respectively. The mean difference between the photometry and high-speed digital imaging methods was 0.2 +/- 0.6 Hz, and the Bland-Altman limits of agreement were from -1.0 to +1.4 Hz, suggesting that the results obtained by these two methods could be used interchangeably. These results show the reliability of the high-speed digital imaging system and the software developed for in-vitro CBF measurements. The advantages of the system include: (i) fast data acquisition and calculation, (ii) whole field automated CBF analysis and (iii) reduction in selection bias.

Cells, Cultured↗
PubMed2005

Determination of intraluminal theophylline concentrations after oral intake of an immediate- and a slow-release dosage form.

The purpose of this study was to evaluate a protocol which enables determining luminal drug concentrations after oral drug administration in man. Human intestinal fluids were aspirated from two sampling sites (duodenum and jejunum) at different time points after oral intake of theophylline; an immediate- and a slow-release dosage form were used to demonstrate the feasibility of discriminating between different formulations. Osmolarity and pH of the aspirates were measured and theophylline concentrations were determined by HPLC-UV. After intake of the immediaterelease formulation of theophylline, duodenal maximum concentrations up to 3 mM were reached within 30 min. Theophylline appeared to be almost completely absorbed before it reached the second sampling site in the jejunum, as observed jejunal concentrations were lower than 10% of the maximal duodenal concentrations. These results are in agreement with fast dissolution and fast absorption through the intestinal mucosa, which could be expected as theophylline belongs to class I of the Biopharmaceutical Classification System. In contrast to the immediate-release formulation, administering the slow-release dosage form resulted in a gradual appearance of theophylline, reaching maximal intestinal concentrations below 300 muM. The proposed methodology can be used to assess luminal drug concentrations and to monitor the time- and site-dependent composition of intestinal fluids after intake of an oral dosage form. This approach may contribute to a better understanding of the behaviour of oral drug formulations in the gastrointestinal tract and may be exploited to further unravel the complexity of the gastrointestinal absorption process. In addition, knowledge of luminal drug concentrations may assist in the selection of drug concentrations applied in in-vitro permeability assays.

Administration, Oral↗
PubMed2005

Sulfasalazine transport in in-vitro, ex-vivo and in-vivo absorption models: contribution of efflux carriers and their modulation by co-administration of synthetic nature-identical fruit extracts.

Sulfasalazine is characterised by low oral bioavailability. In this study, its intestinal transport characteristics were studied in an in-vitro, ex-vivo and in-situ system. The absorptive transport of sulfasalazine across Caco-2 monolayers appeared to be lower than the secretory transport (P(app-abs) = 0.21 +/- 0.02 x 10(-6) cm s(-1) and P(app-secr) = 2.97 +/- 0.30 x 10(-6) cm s(-1), respectively). This polarity in transport of sulfasalazine was not mediated by P-glycoprotein (P-gp), as inclusion of verapamil (100 microM) did not have any effect on the transport polarity of sulfasalazine. However, inclusion of the multidrug resistance-associated protein (MRP) inhibitors benzbromarone (50 microM) and sulfinpyrazone (1 mM), and the glutathione-depleting agent chlorodinitrobenzene (100 microM), resulted in an increased absorptive transport of sulfasalazine in the Caco-2 system (P(app-abs) = 0.64 +/- 0.02, 0.51 +/- 0.04 and 0.60 +/- 0.03 x 10(-6) cm s(-1), respectively). The interference of carriers implies that, during absorption, interactions with food components may occur at the level of this carrier. Therefore, the effect of food extracts was studied in a parallel set of experiments. For two standardized nature-identical fruit extracts (pineapple and apricot extract) a concentration-dependent absorption-enhancing effect could be observed in the Caco-2 system. The functional expression of similar carriers was also demonstrated in rat ileum in the Ussing chamber system. Interaction studies with fruit extracts in the Ussing chamber system, as well as in the in-situ intestinal perfusion study, revealed a 2- to 4-fold increase in the absorptive transport of sulfasalazine. These results indicate that food components in the intestinal lumen can have a significant impact on the intestinal absorption characteristics of sulfasalazine by modulating the biochemical barrier function of the intestinal mucosa.

Animals↗
PubMed2004

Chiral capillary electrophoretic method for quantification of apomorphine.

A new method for chiral determination of apomorphine enantiomers was developed and validated. Seven different neutral and charged cyclodextrins were tested for enantioselectivity on R,S-apomorphine. Sulfobutylether-beta-cyclodextrin was found to offer the best resolution, but with this system, four peaks were detected from a solution of the two enantiomers, which was suggested to be the result of different forms of the complex between the selector and apomorphine. A complexation constant was estimated for a complex of 1:1 ratio for the second and the fourth peak, whereas the other two peaks were fitted to a model ratio of 1:2 (analyte-selector). To avoid this phenomenon, hydroxypropyl-beta-cyclodextrin was then chosen as the chiral selector. An optimisation study was performed on three factors: concentration of the chiral selector, pH of the buffer, and applied voltage. Optimum conditions were: 14 mM of hydroxypropyl-beta-cyclodextrin, pH 3.0, and 16 kV. UV detection was at 200 nm. The method was validated at the chosen conditions, offering a limit of detection of 0.2 microM and a limit of quantification of 0.5 microM. The validated method was applied for the determination of R,S-apomorphine in a transport study with an in vitro cell culture model of the intestinal mucosa (Caco-2).

Apomorphine↗
PubMed2004

HPLC with programmed wavelength fluorescence detection for the simultaneous determination of marker compounds of integrity and P-gp functionality in the Caco-2 intestinal absorption model.

A high sensitivity reversed-phase HPLC method is presented for the simultaneous determination of marker compounds of paracellular transport (atenolol), transcellular transport (propranolol) and P-gp functionality (talinolol) in the Caco-2 system. The Caco-2 system is presently commonly accepted as an in vitro cell culture model of the intestinal mucosa. A programmed wavelength fluorescence detection method was used to optimise the response of the marker compounds. This marker compound mixture and the corresponding HPLC assay can be used for in house validation of the Caco-2 system or to evaluate simultaneously the effect of test compounds or absorption enhancing strategies on monolayer integrity and P-gp functionality. The method can easily be adapted to determine the concentration of atenolol, propranolol and talinolol in blood, thus allowing to use the same compounds in the in situ rat perfusion system with blood sampling from the mesenteric vein.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2004

Usefulness of a novel Caco-2 cell perfusion system. I. In vitro prediction of the absorption potential of passively diffused compounds.

A simple, reliable, and user friendly system was established to cultivate Caco-2 cell monolayer for epithelial transport studies. After an initial growth period of 1 week in a CO(2) incubator, Caco-2 cells were cultivated in an automated continuous perfusion system (Minucells and Minutissue, Germany). Medium was constantly renewed at the apical and basal side of the monolayers, which resulted in a continuous supply of nutrients as well as in a continuous removal of metabolite wastes. The monolayers obtained with the new perfusion culture system were evaluated to estimate the passive transport properties of a series of model compounds. The results produced were compared to those of monolayers obtained with the standard 21-day system. The integrity of cell monolayers was checked by measuring transepithelial electrical resistance (TEER) and by the transport of the paracellular leakage marker sodium fluorescein. The results of confocal microscopy as well as TEER measurements indicated the formation of a monolayer on various support filters. The growth and differentiation of Caco-2 cells were highly dependent upon the individual support filters and extracellular matrix proteins used for Caco-2 attachment. The permeability coefficients of several model compounds across Caco-2 cells obtained with the perfusion system were approximately two-fold higher than those obtained using the traditional 21-day Snapwell-based cultures. A good correlation was found between the transport of passively diffused drugs across Caco-2 monolayers differentiated in the perfusion system and the transport according to the standard method. The rank ordering of high permeable model compounds tested through Caco-2 monolayers, differentiated in a perfusion system, was similar to the standard 21-day culture method.

Biological Transport↗
PubMed2003

Metabolism of stevioside in pigs and intestinal absorption characteristics of stevioside, rebaudioside A and steviol.

Stevioside orally administered to pigs was completely converted into steviol by the bacteria of the colon. However, no stevioside or steviol could be detected in the blood of the animals, even not after converting steviol into the (7-methoxycoumarin-4-yl)methyl ester of steviol, a very sensitive fluorescent derivative with a detection limit of about 50 pg. The intestinal transport characteristics of stevioside, rebaudioside A and steviol were also studied in the Caco-2 system. Only a minor fraction of stevioside and rebaudioside A was transported through the Caco-2 cell layer giving a Papp value of 0.16x10(-6) and 0.11x10(-6) cm/s, respectively. The Papp value for the absorptive transport of steviol was about 38.6x10(-6) cm/s while the Papp value for the secretory transport of steviol was only about 5.32x10(-6) cm/s suggesting carrier-mediated transport. The discrepancy between the relatively high absorptive transport of steviol and the lack of steviol in the blood may be explained by the fact that in the Caco-2 study, steviol is applied as a solution facilitating the uptake, whereas in the colon steviol probably is adsorbed to the compounds present in the colon of which the contents is being concentrated by withdrawal of water.

Algorithms↗
PubMed2003

P-glycoprotein attenuating effect of human intestinal fluid.

PURPOSE: To evaluate the effect of human intestinal fluid (HIF) on P-glycoprotein (P-gp)-mediated efflux. METHODS: HIF was obtained from eight healthy volunteers by duodenal aspiration. HIF was applied at different concentrations (0-75%) to the apical compartment of the Caco-2 system. Cyclosporin A (CsA) was used as a model compound for P-gp mediated efflux. RESULTS: When the bidirectional transport of CsA across Caco-2 monolayers was assessed, a significant polarity in transport could be observed, the absorptive transport being much lower than the secretory transport. Inclusion of HIF resulted in a moderate increase of the absorptive transport, as well as a significant concentration dependent decrease of the secretory transport, without compromising the integrity of the monolayer. Interestingly, a possible gender difference could be detected as inclusion of HIF obtained from female subjects resulted in a decreased absorptive transport of CsA, whereas inclusion of HIF obtained from male subjects resulted in an increased absorptive transport. The P-gp modulating effect of HIF is not caused by a lack of glucose as an energy source for the efflux mechanism when high concentrations of HIF were present in the buffer used. CONCLUSIONS: The results of this study indicate that the contribution of P-gp efflux carriers may be overestimated when using salt buffer solutions as transport media. Additionally, it can be concluded that (presently unidentified) components of HIF may attenuate the P-gp mediated intestinal efflux. The clinical significance of this modulating effect remains to be investigated.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2003

The effect of food components on the absorption of P-gp substrates: a review.

P-glycoprotein (P-gp), a well characterized efflux mechanism which is functionally expressed in the intestinal epithelium, constitutes, along with intestinal metabolism, an important part of the biochemical barrier function of the intestinal mucosa. This efflux carrier may be responsible for limiting the bioavailability of several drugs after oral intake. Recently, increasing attention is being paid to the interaction of dietary components with the intestinal absorption of drugs. This review focuses on the modulating capacity of food components on the intestinal absorption of P-gp substrates. The possible P-gp inhibitory effects of several dietary constituents are discussed. In addition, this review will also focus on the effect of several bioflavonoids on the P-gp-mediated efflux of drugs. As the role of P-gp (and other efflux carriers, including multidrug resistance-associated proteins and breast cancer resistance protein) in limiting the bioavailability of drugs becomes more clear, more research is required firstly to identify the effect of dietary compounds on these efflux carriers and secondly to reveal the clinical relevance of this interaction.

ATP Binding Cassette Transporter, Subfamily B↗
PubMed2002

Apricot extract inhibits the P-gp-mediated efflux of talinolol.

Within the framework of developing strategies to enhance the intestinal absorption of P-glycoprotein (P-gp) substrates, the modulatory effect of a standardized apricot extract on P-gp-related efflux carriers was investigated in the Caco-2 system, Ussing chambers and the rat in situ perfusion model using talinolol as a model substrate. Using the Caco-2 system, polarity in transport of talinolol could be observed, the absorptive transport being much lower than the secretory transport (P(app-abs) = 1.08 +/- 0.29 x 10(-6) cm/s and P(app-secr) = 11.74 +/- 0.80 x 10(-6) cm/s). Inclusion of apricot extract (1%) in the apical medium resulted in a statistically significantly diminished polarity (P(app-abs) = 4.88 +/- 0.96 x 10(-6) cm/s and P(app-secr) = 9.39 +/- 0.58 x 10(-6) cm/s, p < 0.05). In addition, the inhibitory effect of apricot extract on P-gp related efflux mechanisms was shown to be concentration (0% approximately 0.1% < 0.3% < 1%) and pH dependent. Experiments performed with the Ussing chambers resulted in similar observations. In the rat in situ perfusion model, inclusion of apricot extract (1%) in the perfusion medium resulted in a threefold increase of the amount of talinolol appearing in the collected blood compared to the reference condition (23.6 +/- 5.53 pmol/cm. min and 7.13 +/- 1.08 pmol/cm. min, respectively; p < 0.05). Coadministration of this standardized apricot extract might be a safe and useful strategy to enhance the intestinal absorption of P-gp substrates. The nature and structure of the compound(s) responsible for this inhibiting effect on P-gp-related efflux carriers remain to be elucidated, as well as the exact mechanism by which apricot extract exerts its inhibitory function.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2002

Inhibitory effect of fruit extracts on P-glycoprotein-related efflux carriers: an in-vitro screening.

In this study, standardized food extracts were screened for their possible inhibitory effect on the P-glycoprotein (P-gp)-mediated efflux of 3H-ciclosporin A (CsA) using the in-vitro Caco-2 model. CsA is commonly used as a substrate for P-gp-related efflux carriers and is characterized by a polarity in transport, the absorptive transport being much lowerthan the secretorytransport (polarityfactor: PF approximately 7). Of the 68 tested, nine extracts showed a decreased efflux of CsA (< 75% of the reference value) and were retained for further experiments on the bidirectional transport of CsA across Caco-2 monolayers. Results of these experiments showed that strawberry, orange, apricot and mint extract exert an inhibitory effect on intestinal P-gp-related functionality (PF < 4.2). The effect of apricot extract was also studied on the bidirectional transport of talinolol, a specific P-gp substrate; inclusion of 1%, v/v, in the apical compartment of Caco-2 monolayers resulted in a significantly reduced polarity in the transport of talinolol (PF reference = 15.5; PF in the presence of apricot extract = 2.5). This study suggests that co-administration of fruit extracts might be a conceptually safe and useful strategy to enhance the intestinal absorption of P-gp substrates. More research is necessary to characterize the impact of this inhibition on P-gp-related efflux mechanisms in other absorption models (in-vitro and in-vivo) and to identify the compounds that are responsible for this inhibitory effect.

ATP Binding Cassette Transporter, Subfamily B, Mem↗