496 research outputs found
sj-docx-1-nah-10.1177_02601060231166821 - Supplemental material for Change in carbohydrate intake one year after Roux-en-Y gastric bypass: A prospective study
No full textSupplemental material, sj-docx-1-nah-10.1177_02601060231166821 for Change in carbohydrate intake one year after Roux-en-Y gastric bypass: A prospective study by Joke Vliebergh, Ina Gesquiere, Veerle Foulon, Patrick Augustijns, Matthias Lannoo, Ellen Deleus, Ann Meulemans, Chantal Mathieu, Ann Mertens, Christophe Matthys, Bart Van der Schueren and Roman Vangoitsenhoven in Nutrition and Health</p
Biorelevante bepaling van de oplosbaarheid van geneesmiddelen
No full textIntestinal solubility and permeability are importantbiopharmaceutical parameters determining absorption of systemically actingdrugs. When a drug is characterized by low solubility and/or low permeability, oralbioavailability can be seriously impaired. To improve the efficiency of theselection of drug candidates, solubility and permeability are screened for inan early phase of the drug discovery and development process. Up to date,however, plain aqueous buffer systems are often being used during these screeningprocedures. These buffers are far from physiologically relevant and one mayquestion the relevance of this approach.In recentyears, a lot of attention has been paid to the effect of intraluminalconditions (e.g. pH and bile secretions) on intestinal absorption. This has resultedin the definition of media simulating the fasted and fed state upper gastrointestinaltract (fasted state simulated intestinal fluid, FaSSIF, and fed state simulatedintestinal fluid, FeSSIF, respectively). Although these biorelevant media wereproposed on the basis of intraluminal composition and characteristics (pH, bilesecretion, osmolality, buffer capacity), lipolytic products present after foodintake (digestion of food derived lipids) were not incorporated into thesemedia. Furthermore, although these media may be used in in vitro model systems (especially to determine solubility anddissolution), they have not yet found their way into high throughput screeningmodels. Practicalities along with low-cost efficiency and the absence of firm in vitro-in vivo correlations have delayed their routine integration intodrug discovery.This research work focused on the characterization of human duodenalfluids and the development of alternative media which can more easily beincorporated into screening procedures estimating the intraluminal solubilityof drug candidates during drug discovery and early drug develoment.To attainthis goal, 3 different study approaches were set up. In a first study, humanintestinal fluids (HIF) were characterized on a time-after-food basis in 3nutritional states (fasted, fed and fat-enriched fed state). A second studyevaluated the solubilizing capacity of these HIF for 5 poorly water solublemodel drugs and compared it to the solubility of these drugs in existingsimulated intestinal fluids (SIF). The third study explored d-α-tocopherylpolyethyleneglycol 1000 succinate (TPGS) containing media as alternative solventsystems for intraluminal solubility estimation; 17 model drugs were selectedand HIF were used as reference. Evaluation was based on the concept offunctional biorelevance meaning that HIF and SIF should have comparablesolubilizing capacity rather than an identical match of composition (compositionalbiorelevance).In thefirst study, a thorough characterization of fasted and fed (standard fed andfat-enriched fed) HIF was accomplished. Composition and characteristics of HIFwere determined with respect to pH, bile salts, phospholipids, lipolyticproducts (tri-, di-, monoglycerides and free fatty acids), surface tension andosmolality. For all of these intraluminal parameters, a substantial inter- andintra-subject variability was observed. Although variability might be avaluable source of information for the scientist (depending on the objective ofthe study), in our study, it also led to several consequences. Partly due to thehigh variability, it was not evident to distinguish trends based on individualtime-based evolution of the intraluminal parameters. Therefore, average profilesmight give more insight; for instance, for total lipolytic products, on thebasis of the median profile, after reaching a maximum 30 minutespostprandially, a gradual decrease was seen until a steady-state was noticed120 minutes after food intake. The minimal differences between fasted, fed andfat-enriched fed state HIF can be seen as another consequence of the highvariability. In general, and based on average concentration profiles, a fedintraluminal state corresponds to higher concentrations of bile salts,phospholipids, total lipolytic products, osmolality and surface tension, and toa (slightly) lower pH as compared to the fasted state. Considering the biggerintestinal volume in the fed and fat-enriched fed state, absolute amounts werehigher in the fed state (standard fed and fat-enriched fed state) as comparedto the fasted state. The concentration-time-profiles of the intraluminalparameters were not substantially different in the fed as compared to thefat-enriched fed state.The second study explored the solubilizing capacity of HIF and SIFfor 5 poorly water soluble drugs (danazol, diazepam, indomethacin, ketoconazoleand nifedipine) representing acidic, basic and neutral drugs. For this study,HIF described in study 1 were used as solvent systems; solubility of the drugsin SIF was assessed in FaSSIF and FeSSIF, in FeSSIF version 2 (FeSSIF v2), andin early, middle and late FeSSIF. The composition of these new versions ofFeSSIF was altered compared to the original FeSSIF in order to implement theeffect of lipolytic products and to concede to the dynamic intraluminalenvironment, especially after food intake. In correspondence with the aforementionedstudy on the composition of HIF, a high inter- and intra-subject variabilitywas seen for the solubilizing capacity of HIF. As concentrations and notabsolute amounts of biorelevant components affect solubilizing capacity, thesame implications as for study 1 are relevant here. First, based on individualprofiles, time-dependent trends are not clear. Second, differences between the3 nutritional states were not very straightforward. Only for the earlypostprandial phase (30 to 60 minutes after food intake), a difference insolubilizing capacity between the standard fed state and the fat-enriched fedstate, and between a fed and the fasted state, respectively, was found. Examinationof the effect of intraluminal parameters on solubility of the 5 model drugs,showed a clear correlation between solubility of indomethacin and pH. For theother drugs, a combination of parameters determined the solubility in HIF. Whenthe solubilizing capacity of HIF and SIF was compared, in general FaSSIF andFeSSIF underestimated the solubility of the model drugs in HIF; especially theearly postprandial phase was underestimated by FeSSIF. The second generationFeSSIF, FeSSIF v2, did not result in much improvement. On the contrary, earlyFeSSIF overestimated the solubility obtained in the first 90 minutes after foodintake.These 2studies documented that pooling of HIF fractions, on subject or on time basis,may potentially lead to loss of valuable information. Nevertheless, dependingon the goal, pooling may be the most interesting approach, for example tovalidate in-vitro set-ups.The thirdstudy explored whether TPGS containing media may be used to estimate intraluminalsolubility. The solubilizing capacity of phosphate buffer containing differentpercentages of TPGS was examined for 17 model drugs with differentphysicochemical characteristics and was compared to the solubilizing capacityof early postprandial HIF, and SIF. Although direct measurement of theintraluminal solubility by solubility in FaSSIF and FeSSIF demonstrated anunderestimation, the predictive power of FaSSIF and FeSSIF was quite goodtaking into account the obtained relationship between solubility in FaSSIF andFeSSIF as compared to the experimental solubility in HIF. Based on the sameapproach, a similar predictive power could be accomplished by 0.1% and 2% TPGSfor the fasted and the fed state, respectively. Bearing in mind the simplicity,lower cost and stability of these TPGS containing media, 0.1% and 2% TPGS mightbe useful media for the estimation of intraluminal solubility of drugcandidates.In conclusion, we have demonstrated substantial inter- and intra-subjectvariability in time-dependent composition and solubilizing capacity of HIFwhich may have an impact on intestinal drug absorption and might be an one ofthe underlying reasons for the variable drug plasma-time profiles oftenobserved. The solubilizing capacity of HIF may satisfactorily be simulated byFaSSIF and FeSSIF. A comparable predictive power is attained by 0.1% and 2%TPGS in buffer on condition that solubilities are corrected by implementing therelationship between the solubility in SIF and the experimental solubility inHIF. TPGS containing media may be instrumental for high throughput screeningmodels in drug discovery and early drug development.status: Publishe
Nieuwe in vitro en in vivo modellen om de vaginale opname van microbiciden te bestuderen
No full textNovel in vitro and in vivo model systems to study intravaginal microbicide uptakeAnti-HIV microbicides are compounds that are vaginally or rectally administered prior to intercourse to prevent viral transmission. In the search for an effective product, insufficient drug permeation across the vaginal mucosa may cause failed protection in case of microbicides that are active at the level of the HIV host cells, which are mainly localized in the lamina propria. To evaluate the tissue penetration ability of vaginally applied drugs, we established a simple in vitro set up, consisting of an apical and basal chamber that are separated by a layer of HEC-1A cells. This procedure was validated by means of the model microbicides tenofovir, darunavir, saquinavir and dapivirine. The compounds solubility and tissue permeability were identified as key determinants for vaginal uptake. Saquinavir and dapivirine permeation across HEC-1A was low because of poor permeability and solubility, respectively. In contrast, tenofovir showed the highest transport attributable to its good aqueous solubility. In addition, the impact of formulation factors on the permeation potential was investigated. The inclusion of solubilizing cyclodextrins or polyethylene glycol resulted in an enhanced permeation of the hydrophobic compound dapivirine. An excipient-induced reduction in the permeability may however counteract the solubilizing effect, as demonstrated for the emulsion formulation of darunavir. The optimization of drug uptake by inclusion of formulation excipients must take into account this delicate balance. In a next step, the described in vitro approach was applied for the biopharmaceutical assessment of the diaryltriazine compound series which resulted in the selection of UAMC01398 as the lead microbicide candidate, owing to its relatively high aqueous solubility in addition to its beneficial safety and activity profile. Two stable and safe aqueous-based gel formulations were identified for UAMC01398 including a non-solubilizing gel and a gel containing the solubilizer sulfobutyl ether-ß-cyclodextrin (SBE-ßCD, 5%); in addition, a film delivery system, consisting of the excipients hydroxypropylmethylcellulose and polyethylene glycol 400 with UAMC01398 in the amorphous state, was developed. Compared to the non-solubilizing gel, the SBE-ßCD gel and the film formulation increased the UAMC01398 uptake both in vitro across HEC-1A cell layers and in vivo in rabbits. The developed formulations are suitable for the vaginal delivery of UAMC01398 and allow further in vivo evaluation of the microbicide potential of this compound.The possible use of a supersaturation strategy to overcome solubility issues of hydrophobic microbicides was demonstrated for dapivirine. Despite being in a thermodynamically unstable state, supersaturated dapivirine showed not to precipitate in the formulation vehicle as such and in biorelevant fluids in the presence of several excipients including hydroxypropylmethylcellulose, polyethylene glycol 1000 and cyclodextrins. Dapivirine transport across HEC-1A cell layers was higher for supersaturated gels compared to suspension gels. The supersaturated dapivirine (500 µM) gel containing 2.5% of SBE-ßCD significantly increased vaginal drug uptake in rabbits compared to a non-solubilizing suspension gel and an SBE-ßCD suspension gel. The supersaturation approach thus allows the formulation of hydrophobic microbicides at concentrations above their solubility, thereby enhancing their vaginal permeation.Vaginal uptake may also be influenced by drug transporters expressed in the vaginal epithelium. We confirmed the protein expression of the efflux transporters Pgp, BCRP and MRP-2 in endocervical and vaginal tissue of premenopausal women. Several microbicide candidates including darunavir, saquinavir and maraviroc could be categorized as Pgp and MRP-2 substrates and their disposition may thus be affected by these transporters. The Pgp transporter was observed to significantly reduce the vaginal uptake of the model Pgp substrate talinolol in vivo in rabbits when formulated in a neutral, but not in an acidic gel. Consequently, the expression of efflux transporters may limit the vaginal permeation of their substrates, including certain microbicides. In conclusion, we propose the implementation of in vitro solubility and permeability evaluation, including the assessment of transporter interactions, to estimate the vaginal tissue permeation potential of microbicides prior to progressing into animal or clinical studies. This procedure may contribute to the selection of promising microbicide candidates, as illustrated for the diaryltriazines, and to successful formulation development, as shown for UAMC01398 and dapivirine. The in vitro drug transport across HEC-1A cell layers correlated well with the in vivo vaginal permeation as determined in rabbits. Formulation approaches that install solubilization or supersaturation are indicated to enhance vaginal drug uptake of poorly soluble microbicides.status: Publishe
Top-downproductie van Geneesmiddel Nanokristallen: Stabiliseringvan Nanosuspensies, Miniaturisatie van de Productie van Nanosuspensies en Verdere Omzetting tot Vaste Vormen
No full textIn de inleiding (Hoofdstuk I) werd, naast een algemene inleiding over nanokristallen als een formuleringsstrategie, een overzicht gegeven van de beschikbare literatuur betreffende de subtopics die in dit werk bestudeerd werden. Vervolgens werden de doelstellingen geformuleerd in Hoofdstuk II. Deze doelstellingen zijn de volgende: (i) evaluatie van de invloed van de stabilisator, diens concentratie en geneesmiddeleigenschappen op de stabilisering van nanosuspensies (Hoofdstuk III), (ii) de studie van de miniaturisatie van de productie van nanosuspensies en hun fysico-chemische evaluatie (Hoofdstuk IV), en (iii) karakterisering van de verdere transformatie van nanosuspensies in vaste vormen. Betreffende laatsgenoemde doelstelling werd de invloed van drogen zonder toevoegen van additionele matrixvormers bekeken (Hoofdstuk V), de invloed van additionele matrixvormers bij vriesdrogen (Hoofdstuk VI & VII) en bij sproeidrogen (Hoofdstuk IX). Het onderzoek betreffende de stabilisering van nanosuspensies werd gepresenteerd in Hoofdstuk III. Dit hoofdstuk omvat een screening gebruikmakend van 13 verschillende stabilisatoren, toegepast in 3 concentraties op 9 structureel verschillende geneesmiddelen. Algemeen werd gezien dat hogere concentraties aan stabilisator een positief effect hadden op de slaagkans om nanosuspensies te produceren en de daaropvolgende stabilisering van de nanosuspensies. De semi-synthetische polymeren (HPMC, MC, HEC, HPC, NaCMC, NaAlg) vertoonden een eerder zwak potentieel (gemiddelde slaagkans 10%), hetgeen deels te verklaren was door de viscositeits-gelimiteerde concentratie waarbij ze toepasbaar zijn. De lineaire synthetische polymeren PVP30 en PVP90 vertoonden betere stabilisering indien toegepast aan hogere concentraties (een slaagkans van 56% voor PVP30 aan een 100 wt% concentratie, relatief ten opzichte van het geneesmiddel). Dit effect was nog sterker uitgesproken voor de synthetische copolymeren P188 and K-IR (beide 67% slaagkans aan een 100 wt% concentratie, relatief ten opzichte van het geneesmiddel). De surfactanten P80 en TPGS, tenslotte, hadden het beste stabilisatiepotentieel. Waar P80 toegepast in concentraties van 25 en 100 wt% een slaagkans van 89% gaf, bleek TPGS bruikbaar voor alle geneesmiddelen in deze concentraties. Het systeem met 25 wt% werd daarom geselecteerd als modelsysteem in de daaropvolgende Hoofdstukken IV, V en VIII. Onderzoek met betrekking tot de miniaturisatie van de productie van nanosuspensies is het onderwerp van Hoodstuk IV. Gebruik makend van een microtiterplaat-design was het productieproces te miniaturiseren tot hoeveelheden overeenkomend met 10 mg geneesmiddel. Alhoewel dit formaat zich leent tot relatief vlotte implementatie in een geautomatiseerde omgeving kan vervorming en/of erosie van de microtiterplaat het praktisch gebruik van dit design beperken. Ter omzeiling hiervan werd het malen in glazen vials in een kogelmolen geëvalueerd als alternatief. Ook hier kon het productieproces tot 10 mg geminiaturiseerd worden metvergelijkbare resultaten voor de bekomen gemiddelde deeltjesgrootte als bij het microtiterplaat-design. Aangezien een geminiaturiseerd productieproces gepaard moet gaan met een miniaturisatie van de karakterisering van de nanosuspensies, werd de haalbaarheid om evaluaties uit te voeren op hoeveelheden nanosuspensie overeenkomend met 1 mg geneesmiddel onderzocht. De bepaling van het geneesmiddelgehalte was precies en accuraat, hetgeen de haalbaarheid van de transfer van minitieuze hoeveelheden mogelijk maakt. DLS werd geïdentificeerd als de preferentiële techniek voor deeltjesgroottebepaling op geminiaturiseerde wijze. Staalvoorbereiding bleek bepalend te zijn voor morfologie evaluatie (SEM): het staal uitsmeren op de houder gaf hierbij de beste resultaten. Thermische analyse (DSC) kon uitgevoerd worden gebruik makend van verschillende staalvoorbereidingstechnieken, alhoewel de toegepaste voorbereidingstechniek een duidelijke invloed had op het smeltgedrag van de stabilisator. Verdere karakterisering van de vaste toestand met X-stralenpoederdiffractie in capillairen was mogelijk. Betere resultaten werden hierbij bekomen wanneer de nanosuspensie werd gevriesdroogd. Ook het direct meten in de gesuspendeerde toestand was mogelijk maar gaf minder intense diffractiepieken. Laatstgenoemde kan echter gebruikt worden ter identificatie van de vaste toestand van het geneesmiddel na malen. Samenvattend kan gesteld worden dat zowel de productie als de fysicochemische karakterisering van hoeveelheden nanosuspensie overeenkomend met 10 mg geneesmiddel mogelijk bleek. Deze aanpak kan toegepast worden voor screening-onderzoek in parallelle designs, een bruikbaar hulpmiddel tijdens preklinische ontwikkeling. Na onderzoek betreffende de stabilisering en miniaturisatie van nanosuspensies werd verdere omzetting van de nanosuspensies in vaste vormen bestudeerd in de daaropvolgende hoofdstukken. Essentieel hierbij is het behoud van het aantrekkelijke, snelle dissolutiegedrag van de systemen na redispergeren van de gedroogde producten. De fysische instabiliteit van de nanosuspensies tijdens korte-termijn stabiliteitsstudies voor de TPGS-gestabiliseerde nanosuspensies bleek een verder argument voor omzetting van de nanosuspensies in vaste vormen (Hoofdstuk V). In dit hoofdstuk werden vriesdrogen en sproeidrogen van de nanosuspensies geëvalueerd zonder toevoegen van additionele matrixvormers. Beide processen resulteerden in geagglomereerde poeders. Het geneesmiddel eerder dan het droogproces werd geidentificeerd als zijnde doorslaggevend voor de dissolutiekarakteristieken van de uiteindelijke poeders. Na evaluatie van de oppervlakte-hydrofobiciteit van de geneesmiddelen werd geconcludeerd dat geneesmiddelen met een meer hydrofoob oppervlak (waarvoor de TPGS adsorptie hoger was dan 5 mg TPGS/m2), resulteerden in agglomeraten waarvoor desintegratie de snelheidsbepalende stap werd in het totaal dissolutieproces. Eénzelfde trend werd gevonden voor componenten met een logP-waarde hoger dan 4. Er dient echter op gewezen te worden dat uitzonderingen bestaan in de correlatie tussen logP en oppervlaktehydrofobiciteit, hetgeen de voorspellende waarde van logP in vraag stelt. Deze data suggereren dat de noodzaak tot toevoeging van additionele matrixormers voorafgaand aan de droogstap verregaand bepaald wordt door de hydrofobiciteit van het geneesmiddel. De studie van matrixvormers is het onderwerp van de daaropvolgende hoofdstukken. In Hoofdstuk VI, wordt een studie besproken betreffende het vriesdrogen van nanosuspensies van loviride met sucrose als matrixvormer. Malen in de maalkamer van een kogelmolen bleek succesvol voor de productie van de nanosuspensies op laboratoriumschaal. In aanwezigheid van Tween® 80 en poloxamer 188 als stabilisatoren resulteerde het proces na 4 uur malen in nanosuspensies met een gemiddelde diameter van 264 ± 14 nm en een distributiebreedte van 59 ±6 nm. Co-vriesdrogen van de nanosuspensies met sucrose gaf nanopoeders die resuspendeerbaar waren en homogeen betreffende het gehalte aan loviride. Loviride, sucrose en poloxamer 188 waren kristallijn in de nanopoeders. Een kristallijne sucrosematrix werd gevormd binnen de 24 uur na vriesdrogen. Het voordeel van het co-vriesdrogen van de nanosuspensies met sucrose was drievoudig: (i) door de vorming van de kristallijne sucrosematrix na vriesdrogen is het mogelijk een vast poeder te bekomen, hetgeen aantrekkelijk is met betrekking tot verdere verwerking tot een finale doseervorm, (ii) sucrose had een beschermend effect op de agglomeratie van de nanodeeltjes tijdens vriesdrogen en (iii) de snel oplossende sucrosematrix resulteerde in een product met snelle desintegratie dat de aantrekkelijke invloed van het verhoogde specifieke oppervlakte van de nanokristallen op de dissolutie-eigenschappen kon behouden. Dit laatste werd aangetoond door de verbeterde dissolutieprofielen en een verhoogd cumulatief transport doorheen Caco-2 monolagen. De evaluatie van sucrose en microkristallijne cellulose (MCC) als matrixvormers voor vriesdrogen van itraconazole nanosuspensies werd besproken in Hoofdstuk VII. Itraconazole nanosuspensies, gestabiliseerd met 25 wt% TPGS (337 ± 74 nm, DLS) vertoonden volledige dissolutie binnen enkele minuten. Aangezien deze dissolutiekarakteristieken niet konden behouden worden na vriesdrogen van de nanosuspensies wegens agglomeratie van de nanodeeltjes, werden twee matrixvormers geëvalueerd in hoeveelheden van 50, 100 and 200 wt% (relatief ten opzichte van het gewicht van itraconazole). Voor de traditionele matrixvormer sucrose werd opgemerkt dat het gebruik van hogere hoeveelheden sucrose onverwachts resulteerde in een vertraging van de vrijgave [tijd voor 63 % vrijgave, Td = 42.0 ± 6.9 min (0 %), 22.7 ± 8.7 min (50 %), 40.1 ± 8.3 min (100 %), 209.2 ± 178.9 min (200 %)]. Alhoewel hogere hoeveelheden sucrose een duidelijk cryoprotectief effect hadden op de agglomeratie van de nanokristallen, trad een duidelijke agglomeratie op tijdens de laatste fase van de droogstap. Ten gevolge hiervan werd een negatief eindeffect geobserveerd van het toepassen van grotere hoeveelheden sucrose. Voor MCC werd gezien dat hogere hoeveelheden MCC resulteerden in versnelde vrijgave [Td = 42.0 ± 6.9 min (0 %), 10.5 ± 0.7 min (50 %), 6.4 ± 1.2 min (100 %), 3.1 ± 0.5 min (200 %)]. Twee fasen konden onderscheiden worden in de profielen: een initiële fase van burst release, veroorzaakt door de individueel gedispergeerde nanokristallen en een fase van vertraagde vrijgave die kan verklaard worden door een geagglomereerde geneesmiddelfractie. Deze resultaten onderstrepen duidelijk de waarde van MCC als een alternatieve matrixvormer voor het vriesdrogen van itraconazole nanosuspensies. Een verdere studie van alternatieve matrixvormers, nu voor sproeidroog-toepassingen, was het onderwerp van Hoofdstuk VIII. Vier alternatieve matrixvormers werden hiertoe geëvalueerd, voor drie verschillende modelgeneesmiddelen (cinnarizine, itraconazole en phenylbutazone). Op vlak van hun potentieel om de hoge dissolutiesnelheden van de nanosuspensies te behouden werd de volgende volgorde gezien voor de matrixvormers: Fujicalin® < Avicel®PH101 < Aerosil®200 < Inutec®SP1. Alhoewel het sproeidrogen van een zuivere Fujicalin®-suspensie leidde tot een verregaand opbreken van de granulaten was het niet mogelijk voordeel te putten uit het grote specifieke oppervlak van de primaire partikels waaruit deze granulaten zijn opgebouwd. Voor Avicel®PH101 werden variabele resultaten bekomen. In tegenstelling tot Fujicalin®, resulteerde het grote specifieke oppervlak van Aerosil®200 wel in goede matrixvormer-eigenschappen. Tenslotte werden voor Inutec®SP1 de beste resultaten bekomen. Dispersie van itraconazole in de poeders, zoals geëvalueerd met behulp van Cl-mapping (X-ray microanalyse) van tabletten van de poeders, ondersteunde de hypothese dat betere geneesmiddeldispersie in de poeders resulteert in verbeterde dissolutie, met uitzondering van Avicel®PH101.status: Publishe
Karakterisering van het intraluminale gedrag van geneesmiddelen
No full textOral intake is the preferred route of administration for systemically acting drugs. Upon intake of a dosage form, various simultaneously ongoing events in the gastrointestinal tract, including drug transit, release, dissolution and transepithelial transport, result in the appearance of a drug into the blood circulation. In addition to possible hepatic first-pass extraction, the rate and extent of intestinal absorption determine the initial phase of the plasma concentration-time profile of the drug and have a large impact on its bioavailability. Various factors, including drug properties, formulation factors and gastrointestinal variables, may all affect the intestinal absorption process. In recent years, a lot of attention has been paid to the effect of intraluminal conditions (e.g. pH and bile secretions) on intestinal absorption. This resulted in the definition of media simulating intestinal fluids which may be used as biorelevant media in in vitro model systems for intestinal absorption. However, various aspects remain to be further investigated. Especially with respect to intraluminal conditions after intake of an oral dosage form, e.g. intraluminal concentrations of drugs and excipients, the gastrointestinal tract remains to a large extent a 'black box'.
The main objectives of this dissertation research were (1) to explore intraluminal conditions (e.g. drug concentrations) after oral drug intake and (2) to evaluate the impact of these conditions on transepithelial transport.
To accomplish these objectives, an intubation approach was evaluated to aspirate and characterize intraluminal fluids from the upper gastrointestinal tract after intake of a real dosage form in man. This approach was applied to study the intraluminal behavior of the drugs theophylline, amprenavir and the prodrug fosamprenavir. The effect of intraluminal conditions on transepithelial transport was investigated in the human Caco-2 cell culture system.
In a first study, the feasibility of the protocol was explored by monitoring intraluminal concentrations of theophylline in aspirated duodenal and jejunal fluid, after intake of an immediate or slow release formulation. The observed concentration-time profiles after intake of the immediate release dosage form were in agreement with fast dissolution and absorption of theophylline, which could be expected as theophylline belongs to class I of the Biopharmaceutical Classificarion System (BCS). Administration of the slow release dosage form resulted in a gradual appearance of theophylline and lower intestinal concentrations. This study illustrated, for the first time, the assessment of intraluminal drug concentration-time profiles; these profiles may assist in the evaluation of formulations in the upper gastrointestinal tract.
A second study aimed at investigating the intraluminal behavior of a solubilizing formulation (Agenerase®) of the poorly water-soluble drug amprenavir (BCS class II). Concentrations of amprenavir and the solubilizing excipient TPGS were determined in duodenal and jejunal fluid. The presence of TPGS resulted in high intraluminal amprenavir concentrations (mM-range), an illustration of solubilization in vivo.
Subsequently, the effect of these intraluminal conditions on the transepithelial transport of amprenavir was studied in vitro. We showed that solubilization of amprenavir resulted in a large increase of the amprenavir flux across Caco-2 monolayers. In addition, the interaction between amprenavir and the efflux carrier P-gp was reduced in the presence of intestinal fluids and completely inhibited in the presence of TPGS. These results suggest a limited effect of P-gp on amprenavir absorption in vivo. The study illustrated the importance of integrating biorelevant conditions in the in vitro assessment of transepithelial transport.
An alternative for solubilizing formulations to increase the absorption of poorly water-soluble drugs, is the development of prodrugs with improved solubility. In this respect, a phosphate ester prodrug of amprenavir, fosamprenavir, has been commercialized. In order to increase insight into the mechanism behind intestinal absorption from a phosphate ester prodrug, we illustrated in vitro the creation of a supersaturated amprenavir solution in intestinal fluid after dephosphorylation of fosamprenavir. The absorptive flux across Caco-2 monolayers was significantly enhanced when starting from a supersaturated solution. The study showed the importance of supersaturation in real intestinal media.
In addition to the in vitro studies with fosamprenavir, the intraluminal behavior of its immediate release formulation (Telzir®) in fasted and fed state conditions was investigated in vivo. Gastric and duodenal concentrations of (fos)amprenavir were monitored in parallel to plasma concentrations of amprenavir. This allowed linking the pharmacokinetic profile to intraluminal drug behavior. We showed that food-induced or inter-subject variations in the absorption rate of amprenavir were related to the duodenal appearance of fosamprenavir. In the fasted state, the observed intraluminal behavior of fosamprenavir suggested relatively fast release and dissolution in the stomach, corresponding to the in vitro dissolution of fosamprenavir tablets in simulated gastric fluid. In the fed state, however, gastric dissolution was postponed, resulting in a strong delay of amprenavir absorption. The postponed dissolution may be attributed to delayed tablet disintegration in nutritional drink. This food-induced delay in disintegration of an immediate release tablet requires further research.
In addition to the assessment of intraluminal drug and excipient concentrations, other intraluminal variables, including pH, osmolality, inorganic phosphate, bile salts and phospholipids, were monitored in the different studies. The obtained data may be helpful in understanding intraluminal drug and formulation behavior, or in the definition of biorelevant media. For instance, as intestinal dephosphorylation is inhibited by inorganic phosphate and the observed intraluminal phosphate concentration was relatively low (< 1 mM), the use phosphate-buffered media, including FaSSIF, in absorption studies with phosphate ester prodrugs, is not biorelevant.
In conclusion, the proposed intubation approach allows monitoring the site- and time-dependent composition of gastrointestinal fluids. In addition to the assessment of other variables, intraluminal drug concentration time-profiles can be generated. Being the result of different gastrointestinal processes, these intraluminal concentrations may increase insight into drug and formulation behavior in the upper gastrointestinal tract. The impact of this behavior on the pharmacokinetic profile of the drug can be evaluated by parallel assessment of plasma concentrations. In addition, knowledge of intraluminal conditions after oral drug intake may assist in the selection of biorelevant conditions, including media and drug concentrations, for the in vitro determination of solubility, dissolution and permeability.status: Publishe
De eerste horde in orale geneesmiddelendispositie: onderzoek naar de impact van het verblijf in de maag op geneesmiddelengedrag in de mens
No full textReducing the late-stage attrition of potential drug candidates can be considered one of the major challenges for the oral drug development process to date. One of the factors potentially leading to the attrition of a drug candidate at the clinical stages of the drug development process is formulation failure. Formulation failure can be attributed to the fact that selecting the appropriate formulation strategy often occurs on a trial-and-error basis. In order for the drug development process to become more efficient, a shift from a trial-and-error based towards a knowledge-based approach is indispensable. Therefore, an in-depth understanding of the gastrointestinal disposition of an orally administered drug is essential. In the past few years, efforts to characterize the gastrointestinal environment and to understand key gastrointestinal processes affecting oral drug disposition have increased. However, historically, research has mainly focused on elucidating drug disposition at the main site of absorption, i.e. the small intestine. Despite the fact that the stomach is typically the first compartment in which an orally ingested drug resides for a longer period of time, little is known about the impact of this gastric residence on a drug’s disposition further down the gastrointestinal tract and systemically.
Therefore, this dissertation focuses on drug disposition in the stomach and its implications for (variability in) intestinal and systemic drug exposure. For this purpose, small-scale clinical studies were performed with healthy human volunteers, specifically investigating the impact of gastric processes (e.g. disintegration/dissolution, supersaturation/precipitation) on oral drug disposition.
In a first study, we investigated the propensity of diclofenac, a weakly acidic Class II drug in the Biopharmaceutics Classification System (BCS), to supersaturate/precipitate in the stomach of healthy volunteers (n=5) under different test conditions and its implications for intestinal drug disposition. Participants were asked to orally ingest one tablet of Cataflam (50 mg diclofenac potassium) pre-dissolved in 240 mL of tap water either under fasted or fed state conditions, with or without prior use of a proton pump inhibitor (PPI; Nexiam , 40 mg esomeprazole). Under fasted state conditions, brief periods of supersaturation resulted in extensive pH-dependent precipitation of diclofenac in the stomach (r = 0.78), while co-administration of a liquid meal and/or prior PPI use significantly reduced the presence of solid drug material in the stomach. Upon transfer from the stomach to the small intestine, solid drug material was found to rapidly re-dissolve, suggesting that gastric drug precipitation will not affect the extent of systemic drug exposure for this compound. In this study, the occurrence of gastric supersaturation and pH-dependent precipitation of a weakly acidic model compound in man after oral administration of a solution was demonstrated for the first time. Furthermore, it was concluded that intestinal re-dissolution rate most likely determines whether or not the extent of systemic drug exposure will be affected by the presence of solid drug material in the stomach.
Subsequently, we investigated the link between upper gastrointestinal motility, intragastric drug disposition and subsequent variability in systemic drug exposure under fasted state conditions. By combining the well-established intraluminal sampling technique with high-resolution manometry recordings along the upper gastrointestinal tract, the impact of gastrointestinal motility on intraluminal and systemic drug disposition could be studied.
First, we investigated the link between fasted state gastric motility and the intragastric distribution of an orally administered drug in healthy subjects (n=8). After oral administration of one tablet of Gabbroral (250 mg paromomycin, BCS Class III) during either a period of contractile activity (i.e. phase II of the Migrating Motor Complex (MMC)) or contractile quiescence (i.e. MMC phase I), drug concentrations were measured in aspirates collected from different regions of the stomach (i.e. corpus and antrum). Subsequently, (in)homogeneous drug distribution in the stomach could be assessed by comparing regional drug concentrations as a function of time. Regardless of gastric contractile activity at the time of drug intake, similarity in regional drug concentrations displayed marked inter-individual variability and intragastric drug distribution was found to be far from homogeneous in all test conditions, challenging the concept of the stomach as a simple, homogeneous dissolution vessel. However, a clear trend towards better mixing of an orally administered drug with gastric contents was observed in the presence of gastric contractions, resulting in a more homogeneous distribution of the drug throughout the stomach compared to dosing in the absence of gastric contractions.
Secondly, we investigated the possibility of mediating variability in gastric motility at the time of dosing and its implications for variability in systemic drug disposition by orally administering a drug with sparkling instead of tap water. To this end, healthy volunteers (n=6) ingested one tablet of Dafalgan (500 mg paracetamol, BCS Class I/III) with either tap or sparkling water. Drug intake with sparkling water resulted in transient pressure increases along the upper gastrointestinal tract, which were not observed in the control condition. Based on systemic drug disposition parameters (i.e. tmax, AUC0-30 min), drug intake with sparkling water resulted in a trend towards faster and less variable absorption of paracetamol from the gastrointestinal tract, indicating a more uniform intraluminal drug disposition. Gastric drug disposition suggested a faster and more reproducible onset of tablet disintegration when administered with sparkling water, likely due to (i) a direct effect (i.e. in vivo dissolution rate) and/or (ii) an indirect effect (i.e. gastrointestinal motility) of sparkling water. Results from this study indicated that by mediating variability in gastric drug disposition, subsequent variability in systemic drug disposition could be reduced. Furthermore, co-administration of a drug with sparkling water may be a simple way to promote a faster and more reproducible onset of therapeutic effect.
In a final study, the oral disposition of diclofenac (Cataflam , 50 mg diclofenac potassium) was investigated under ‘real-life’ dosing conditions (i.e. tablet as such), focusing on tablet disintegration, drug dissolution and food-related changes in oral drug disposition. The ability of in vitro tools to accurately predict oral drug disposition was evaluated using the in vivo data as a reference. Tools evaluated comprised (i) the conventional USP II dissolution apparatus, (ii) a modified version of the dynamic open flow through test apparatus, and (iii) the TNO gastro-Intestinal Model equipped with the Advanced Gastric Compartment (TIMagc). Under fasted state conditions, a vast presence of solid drug material was observed in the stomach of healthy volunteers. This observation most likely resulted from incomplete drug dissolution and/or precipitation of drug due to the acidity of the gastric environment under fasted state conditions. Similarly, diclofenac was mainly present as a solid in the fed stomach, an observation that could be explained by the re-acidification of gastric contents by the time tablet disintegration was initiated. In both test conditions, solid drug material was found to rapidly (re-)dissolve upon transfer to the small intestine. Due to a marked delay in the initiation of tablet disintegration under fed state conditions, systemic tmax was significantly delayed compared to drug intake under fasted state conditions. However, the extent of systemic drug exposure did not differ between test conditions. In vitro tools were found capable of predicting in vivo intraluminal (and systemic) disposition of diclofenac to some extent, depending on the degree to which the complexity and the dynamic nature of the processes involved were simulated. While the use of static in vitro tools may suffice for the prediction of pH-dependent intraluminal drug behaviour, the investigation of dynamic processes affecting drug disposition (e.g. gastric emptying, food-dependent changes in pH as a function of time) requires more dynamic in vitro tools. Therefore, apart from the biopharmaceutical properties of the compound under investigation, the dynamic nature of the process(es) to be investigated should be taken into account when selecting an appropriate in vitro tool to predict intraluminal drug disposition in man.
In conclusion, we demonstrated that the stomach should not be considered a mere waiting room for drugs to be transferred to their site of absorption, but rather a preparation room determining both the manner in which and rate at which a drug is presented to the small intestine. Furthermore, given the (time-dependent) variation of many gastric variables, intragastric drug disposition can be an important source of variability in oral drug disposition. Therefore, biorelevant in vitro/in silico tools that take into account a drug’s passage through the stomach are essential to accurately predict (variability in) in vivo drug disposition during early-stage screening of drug candidate formulations.status: Publishe
In vitro bepaling van het modulerend effect van farmaceutische componenten op trilhaarslagfrequentie en transepithelieel transport
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HET ONTRAFELEN VAN HET ABSORPTIEPROCES VAN GENEESMIDDELEN AAN DE HAND VAN HET IN SITU RAT MODEL
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Ontwikkeling en karakterisatie van een high throughput bloed-hersenbarrière permeabiliteitstest voor de selectie van potentiële CZS geneesmiddelen
No full textIn de inleiding (hoofdstuk I) werd een overzicht gegeven van de bloed-he rsenbarrière (BHB) en verschillende in-vivo-en in-vitro-metho den die gebruikt worden in drug discovery om de opname van actieve compo nenten in de hersenen te voorspellen, tesamen met hun voor-en nadelen. In-vivo experimenten bieden de meest betrouwbare referentie-informatie v oor het testen en valideren van andere modellen. Een aantal niet-invasie ve en invasieve technieken zijn beschikbaar voor deze in-vivo meting. Ni et-invasieve technieken (bv. positron emissie tomografie, magnetische re sonantie imaging) zijn externe detectiemethoden en kunnen bijgevolg word en toegepast bij de mens. Zij bieden de mogelijkheid om individueel het tijdsverloop van de opname in de hersenen samen met de plasmafarmacokine tiek te meten. De invasieve technieken kunnen worden ingedeeld volgens m ethoden die de hoeveelheid in de hersenen bepalen en volgens methoden di e de snelheid van de hersenpenetratie vaststellen. Het afgelopen decennium werden verscheidene celcultuur-gebaseerde en nie t niet-celcultuur gebaseerde in-vitro-modellen ontwikkeld voor de voorsp elling van in-vivo opname in de hersenen. Celcultuur-gebaseerde modellen gebruiken zowel cellen van cerebrale oorsprong (bv. capillaire hersen e ndotheelcellen, vereeuwigde hersen endotheelcellen) als cellen van niet- cerebrale oorsprong (bv Madin-Darby Canine Kidney, Caco-2). Niet-celcult uur in-vitro-modellen daarentegen maken gebruik van kunstmatige lipiden om de bloed-hersenbarrière na te bootsen (bv. geïmmobiliseerde kunstmati ge chromatografie, parallelle kunstmatige membraan permeabiliteit assay) . In hoofdstuk II, worden de doelstellingen van de thesis vermeld. Het bel angrijkste doel van dit project is het onderzoek of de parallelle kunstm atige membraan permeabiliteit assay (PAMPA) kan worden gebruikt om passi eve BHB permeatie te voorspellen. Met de snelle ontwikkeling van combinatorische chemie en andere high thr oughput synthese methoden, is de analyse van PAMPA stalen uitgegroeid to t het knelpunt van het vroege drug screening proces. Daarom is gevoeligh eid en high throughput kwantificering van de analysemethode een essentie el aspect van het drug discovery proces geworden. In hoofdstuk III, werd een nieuwe generische en high throughput UPLC/MS/MS methode ontwikkeld en geïmplementeerd voor de analyse van de PAMPA stalen. De nieuwe UPLC/M S/MS-methode bestaat uit twee fasen. In een eerste fase wordt een automa tische en compound specifieke multiple reaction monitoring (MRM) methode ontwikkeld gedurende een isocratische UPLC methode (1,5 min) met inject ies in een gescheiden leiding. In een tweede fase werden de monsters gea nalyseerd met een 1,5 min. generische gradiënt UPLC methode na injectie op een BEH C18 kolom (50 mm x 2,1 mm). Verbindingen werden gedetect eerd met een Micromass Waters Quattro Premier massaspectrometer via posi tieve electrospray ionisatie en gebruik makende van de eerder ontwikkeld e en compound specifieke MRM methoden. De lineariteit voor de validatie& nbsp;verbindingen varieerde van 3,05 nM tot 12.50 μM en de det ectielimieten van alle generisch ontwikkelde methoden liggen tussen 0,61 nM en 12 nM in een waterige buffer. De nieuwe generische methode werd s uccesvol geïmplementeerd en resulteerde in een 4-voudige versnelling van analyse, alsmede een 20-voudige toename in de gevoeligheid ten opzichte van andere beschikbare generische LC/MS-methoden. In hoofdstuk IV werd de effectieve permeabiliteit van structureel divers e, commercieel beschikbare en passief diffunderende verbindingen be paald met vier verschillende PAMPA modellen: 1) een PAMPA-BLM (black lip id membrane) model, 2) een PAMPA-DS (double sink) model, 3) een PAMPA-BB B (bloed-hersen barrière) model en 4) een PAMPA-BHB-UWL (onverstoorbaar water laag) model om het meest discriminerende PAMPA-model voor de voors pelling van de BHB permeabiliteit te bepalen. Zowel het PAMPA-BBB als he t PAMPA-BLM model identificeerden de verbindingen die de BHB passer en (BBB +) en die slecht doordringen in de hersenen (BBB-). Daaruitvolge nd konden, voor deze modellen, BBB+ en BBB- classificatie limieten gedef inieerd worden op basis van de bekomen effectieve permeabiliteiten. De PAMPA-modellen werden vervolgens toegepast op 14 structureel diverse interne J&J verbindingen met gekende LogBB waarden. Op basis van de ze LogBB waarden werden BBB classificaties vastgesteld (BBB +: LogBB&nbs p;≥ 0; BBB-: LogBB < 0). PAMPA-BLM resulteerde in drie vals p ositieve identificaties, terwijl de PAMPA-BBB slechts één verb inding onjuist classificeerde. Daarenboven werd voor alle 14 verbin dingen de efflux ratio bepaald door middel van een Caco-2 experiment. De vals positieve verbindingen, in beide modellen, vertoonden een verhoogd e efflux ratio waardoor de onjuiste classificatie kon verklaard worden. Voor deze beperkte set van verbindingen, kan dus zowel het PAMPA-BLM mod el als het PAMPA-BBB-model worden gebruikt om de BHB permeabiliteit te v oorspellen in combinatie met de efflux ratio. De vorige bevindingen werden verder uitgebreid onderzocht in hoofdstuk V . In deze studie werd onderzocht of algoritmen, die de opname in de hers enen (LogBB) van 88 kandidaat-geneesmiddelen voorspellen, kunnen verbete ren met de integratie van PAMPA gegevens (Peff, membraan flux en retenti e). Meer bepaald werd er onderzocht of de experimenteel bepaalde LogBB w aarden konden voorspeld worden met data van de vier PAMPA modellen en fy sisochemische en structurele verbinding parameters. De verzamelde g egevens werden statistisch geanalyseerd met een gedeeltelijk kleinste kw adraten analyse en verschillende regresiemodellen voor LogBB. Additionee l werden, voor een beperkt aantal verbindingen, plasma-proteinebinding ( PPB) en Caco-2 experimenten uitgevoerd om respectievelijk de ongebonden fractie in het plasma en de efflux ratio te bepalen. Deze gegevens werd gecombineerd met de PAMPA data in een poging om de voorspelling van de i n-vivo penetratie in de hersenen te verbeteren. De bekomen resulaten toonden aan dat de PAMPA-BLM parameters (Peff, flux ) de belangrijkste bijdrage leveren bij de voorspelling van de LogBB waa rden van de 88 J&J verbindingen. De geoptimaliseerde vergelijking met de PAMPA-BLM eigenschappen, bekomen via meervoudige lineaire regressie (ML R), vertoont een verbeterde voorspelling van LogBB ten opzichte van de v ergelijking zonder de PAMPA-BLM data. Niettegenstaande de verbeterde voo rspelling, zijn de coëfficiënten van de PAMPA-BLM parameters eerder klei n. Naast deze bevindingen werd ook aangetoond dat PAMPA-BLM eigenschappen L ogBB beter voorspellen in vergelijking met de andere PAMPA modellen. Daa renboven werd de voorspelling van LogBB significant verbeterd wanneer PA MPA-BLM data gecombineerd werden met plasma-proteinebinding gegevens. De combinatie van de efflux ratio met PAMPA-BLM of PAMPA-BBB Peff waarden, resulteerde in een verbeterde, maar nog steeds onvolledig classificatie (80%) van de BHB doorlaatbare [BBB + (LogBB ≥ 0)] en on doordringbare [BBB-(LogBB < 0)] verbindingen.Concluderend kan gesteld worden dat LogBB niet betrouwbaar wordt voorspeld met enkel PAMP A gegevens. Echter, de combinatie van de PAMPA-BLM data met plasma-prote inebinding en/of efflux data resulteerde in een betere voorspelling van de opname van verbindingen in de hersenen.status: Publishe
Absorptie karakterisering: de in situ intestinale perfusietechniek in de muis
No full textTo investigate the mechanisms by which a drug is absorbed from the intestinal lumen into the blood, many techniques have been developed over the years. In early drug discovery, the absorption properties of new chemical entities are usually assessed by high-throughput techniques, e.g., automated Caco-2 screening. In the later drug discovery stages, when drug properties are being optimized and/or when detailed insight into the mechanisms underlying oral drug disposition isrequired, Caco-2 cells or more advanced absorption models (Ussing Chambers or intestinal perfusion systems) are being used. The Caco-2 model has many advantages, including the fact that (1) it is from human origin, (2) it is suitable for high-throughput screening, (3) it allows retrieving absorption mechanisms as it expresses a wide variety of uptake and efflux transporters, and (4) it provides a good prediction ofthe fraction absorbed (Fa) for passively transported drugs in humans. Unfortunately, this model has also some drawbacks: (1) the expression of CYP3A4 (which is the most abundant phase I drug-metabolizing enzyme present in the human small intestine) is very low to non-existent, whichmay result in overprediction of drug transport, (2) there is no mucus layer which protects the cells and forms an extra barrier for compounds to reach the cells, (3) pregnane X receptor (PXR)-mediated drug-drug interactions cannot be studied since this nuclear receptor is not expressed, (4) there are interlaboratory differences in enzyme and drug transporter expression levels. Another absorption model which can be considered in late discovery and early development stages is the in situintestinal perfusion with mesenteric blood collection. Of all presentlyavailable models, it is the closest one to the in vivo situation becauseof (1) an intact intestinal mucosa, nerve system and blood flow; (2) thepresence of sink conditions; and (3) the expression of all relevant enzymes and transporters. The rat is the standard animal used in this technique. Recently, the in situ intestinal perfusion technique with mesenteric blood sampling was successfully downsized from the rat to themouse in our laboratory, which opens new opportunities in pharmaceuticalresearch. First, the use of knockout mice allows studying the involvement of one specific drug transporter or enzyme in the absorptionof a drug. This may not be possible with the use of chemical inhibitorsdue to a lack of specificity. Second, transgenic (humanized) mice, which carry human genes in their genome, may be invaluable in order to eliminate species differences between rodents and humans.The aim of this dissertation research was to evaluate the use of the mouse in the in situ intestinal perfusion technique with mesenteric blood sampling. We investigated the use of wild-type, knockout and humanized mice in different studies, of which the specific objectives were to:1),,Validate the use of P-gp knockout mice in the in situ intestinal perfusion technique, and explore the role of P-gp in the intestinal permeability for darunavir.2),,Investigate the use of the mouse in situ intestinal perfusion technique to elucidate the role of both efflux transporters (P-gp) and metabolism (P450) towards the intestinal absorption of HIV protease inhibitors.3),,Evaluate the use of PXR/CYP3A4-humanized mice for studying drug-druginteractions involving intestinal P-glycoprotein. The effect of rifampicin (PXR activator) was investigated on the intestinal permeability for darunavir.4),,Explore a novel combination of human intestinal fluids (HIF) with the mouse in situ intestinal perfusion technique to assess food effects. We used indinavir as model compound.HIV protease inhibitors (PIs) were selected as model compounds in our studies because of their interesting pharmacokinetic properties. The drugs of this class are characterized by an extensive first-pass elimination as they are metabolized by CYP3A4 present in the liver and intestine. In addition, these drugs have been shown to be substrates ofefflux transporters present in the intestine, which potentially limit their absorption. The most important and best characterized intestinal efflux transporter is P-glycoprotein (P-gp), present in the apical membrane of enterocytes. Although all PIs were shown to be substrates of P-gp, the exact role of P-gp in limiting their intestinal absorption in vivo remains to be elucidated. As a consequence of their unfavorablepharmacokinetics, PIs are always co-administered with the pharmacokinetic booster ritonavir, which is known to inhibit their CYP3A4-mediated metabolism. In addition, ritonavir is a known P-gp inhibitor, thereby potentially inhibiting their intestinal efflux.In the first part of this doctoral research, we evaluated the use of P-gp knockout mice in the in situ intestinal perfusion technique. We explored the contribution of P-gp to the transport characteristics of darunavir (up to 100 µM) using Caco-2 monolayers and the in situ intestinal perfusion technique using wild-type and mdr1a/1b(#/#) mice. We observed that, in vitro, P-gp has a modulatory effect on the absorption of darunavir, even at a concentration of 100 µM (efflux ratio= 25). Fasted state simulated intestinal fluids (FaSSIF) partially inhibited P-gp functionality, which was further inhibited by including the P-gp inhibitors verapamil, PSC833 (valspodar), GF120918 (elacridar),or ritonavir. Using the in situ intestinal perfusion technique, we demonstrated that co-perfusion with ritonavir resulted in a similar apparent permeability coefficient to that observed using P-gp knockout mice, which was 2.7-fold higher than in control mice. From these data, we conclude that in mice, even at a relevant intraluminal concentration of darunavir, P-gp has a modulatory effect on the absorption of darunavir. However, this P-gp-mediated darunavir transport is inhibitedwhen it is combined with ritonavir.In the next study, we subsequently expanded our focus from darunavir to a broad series of PIs. In addition, we investigated the role of both P-gp and intestinal metabolism on their absorption. We explored the impact of ritonavir on the intestinal absorption of PIs in two models: the Caco-2 system and the in situ intestinal perfusion model using wild-type mice. In the Caco-2 model, the effect of ritonavir on the permeability of the other PIs was significant for saquinavir (2-fold increase) and indinavir (3-fold increase), negligible for darunavir and amprenavir, and nonexistent for nelfinavir, lopinavir, tipranavir, and atazanavir. However, performing the in situ intestinal perfusion technique in mice for three selected PIs showed a significant increase in the intestinal permeability for all: indinavir (3.2-fold), lopinavir (2.3-fold), and darunavir (3-fold). The effect of aminobenzotriazole (anonspecific P450 inhibitor) on lopinavir permeability was comparable with using ritonavir, whereas there was no effect for indinavir and darunavir. We conclude that ritonavir can boost drug absorption by inhibiting P-glycoprotein and/or metabolism, in a compound-specific manner. Because the dual effect of ritonavir on metabolism and transporter inhibition could only be observed in a system co-expressing both, the Caco-2 model might be insufficient when studying drug-drug interactions at the level of the intestinal mucosa.In the third study, we evaluated the use of humanized mice in the in situ intestinal perfusion technique. Humanized mice hold great promise to overcome species differences between rodents and humans. Conventional rodent models are less suitable for predicting drug-drug interactions at the level of the human intestinal mucosa, especially when nuclear receptors like pregnane X receptor (PXR) are involved. Recently, a transgenic mouse model, expressing both human PXR and CYP3A4, was developed and shown to be a better predictor of CYP3A4 induction by xenobiotics in humans compared to wild-type mice. In the present study, we tested the hypothesis that this mouse model can also predict PXR-mediated induction of intestinal P-gp in humans. By use of the in situ intestinal perfusion technique with mesenteric blood sampling, the effect of oral rifampicin treatment on the intestinal permeability for darunavir was investigated. Rifampicin treatment lowered the intestinal permeability for darunavir by 50 % compared to non-treated mice. The P-gp inhibitor GF120918 increased the permeability for darunavir by 400 % in rifampicin-treated mice, while this was only 56 % in mice that were not treated, thus indicating P-gp induction by rifampicin. The non-specific P450 inhibitor aminobenzotriazole (100 µM) did not affect the permeability for darunavir. Quantitative Western blot analysis of the intestinal tissue showed that rifampicin treatment induced intestinal P-gp levels four-fold, while CYP3A4 levels remained unchanged. Oral co-administration of rifampicin with the phytochemical sulforaphane (present in many cruciferous vegetables including broccoli) for three days increased the permeability for darunavir by 50 % compared to rifampicin treatment alone. These data show that PXR/CYP3A4-humanized mice can be used to study the inducing effects of xenobiotics on intestinal P-gp. In addition, food-drug interactions playing at the level of the intestine can be investigated.In the last study, we explore a novel combination of human intestinal fluids (HIF) with the mouse in situ intestinal perfusion technique to assess food effects. Fed state conditions cannot be easily implemented in the presently available permeability tools, including the frequently used Caco-2 system. Therefore, exploring food effects during drug development can be quite challenging. In this study, we investigated the effect of fasted and fed state conditions on the intestinal absorption of the PI indinavir using simulated and human intestinal fluids in the in situ intestinal perfusion technique in mice. Indinavirwas selected as model compound because it is very susceptible to a negative food effect in humans. Although the solubility of indinavir was 6-fold higher in fed state human intestinal fluid (FeHIF) as compared to fasted state HIF (FaHIF), the intestinal permeation of indinavir was 22-fold lower in FeHIF as compared to FaHIF. Dialysis experiments showed that only a small fraction of indinavir is accessiblefor absorption in FeHIF due to micellar entrapment, possibly explaining its low intestinal permeation. These findings could not be confirmed using simulated intestinal fluids. From these data, we conclude that the use of HIF in the in situ intestinal perfusion model is very promising for biorelevant absorption evaluation as it allows to directlyexplore the complex solubility/permeability interplay on drug absorption.In conclusion, the results obtained in this dissertation research using the mouse in situ intestinal perfusion technique with mesenteric blood sampling clearly show that this powerful tool holds great promise in elucidating drug absorption mechanisms, drug-drug interactions and food effects in the most biorelevant way. The possibility to use knockout and knockin (humanized) mice emphasizes the benefit of using the mouse instead of the rat in this model.status: Publishe
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