Characterization of luminal contents from the fasted human proximal colon.

To treat colonic diseases more effectively, improved therapies are urgently needed. In this respect, delivering drugs locally to the colon is a key strategy to achieve higher local drug concentrations while minimizing systemic side effects. Understanding the luminal environment is crucial to efficiently develop such targeted therapies and to predict drug disposition in the colon. In this clinical study, we collected colonic contents from an undisturbed fasted proximal colon via colonoscopy and characterized their composition with regard to drug disposition. Colonic pH, osmolality, protein content, bile salts, lipids, phospholipids and short-chain fatty acids were investigated in 10 healthy volunteers (8 male and 2 female, age 19-25). The unique environment of the proximal colon was reflected in the composition of the sampled luminal fluids and the effect of the microbiota could be observed on the pH (median 6.55), the composition of bile salts (majority deconjugated and secondary), and the abundance of short-chain fatty acids. At the same time, an increase in phospholipid concentration, osmolality and total protein content compared to reported ileal values was seen, likely resulting from desiccation. Lipids could only be found in low quantities and mainly in the form of cholesterol and free fatty acids, showing almost complete digestion and absorption by the time luminal contents reach the colon. All characteristics also displayed the considerable intersubject variability found in different regions of the gastrointestinal tract. This study contributes to an improved understanding of the luminal conditions in the proximal colon and facilitates the development of new predictive tools to study colonic drug absorption.

Investigating the Mechanisms behind the Positive Food Effect of Abiraterone Acetate: In Vitro and Rat In Situ Studies.

The anticancer agent abiraterone suffers from an extensive positive food effect after oral intake of the prodrug abiraterone acetate (Zytiga). The underlying processes determining postprandial abiraterone absorption were investigated in this study. The impact of lipids and lipid digestion products on (i) the solubility of abiraterone acetate and abiraterone, (ii) the conversion of abiraterone acetate to abiraterone, and (iii) the passive permeation of abiraterone was determined in vitro. The interaction of abiraterone acetate and abiraterone with vesicles and colloidal structures in the simulated fed state media containing undigested lipids and lipid digestion products enhanced the solubility of both compounds but limited the esterase-mediated hydrolysis of abiraterone acetate and the potential of abiraterone to permeate. Rat in situ intestinal perfusion experiments with a suspension of abiraterone acetate in static fed state simulated media identified abiraterone concentrations in the perfusate as the main driving force for absorption. However, experiments with ongoing lipolysis in the perfusate highlighted the importance of including lipid digestion as a dynamic process when studying postprandial abiraterone absorption. Future research may employ the in situ perfusion model to study postprandial drug absorption from a dynamic lipolysis-mediated intestinal environment to provide reference data for the optimisation of relevant in vitro models to evaluate food effects.

Integration of advanced methods and models to study drug absorption and related processes: An UNGAP perspective.

This collection of contributions from the European Network on Understanding Gastrointestinal Absorption-related Processes (UNGAP) community assembly aims to provide information on some of the current and newer methods employed to study the behaviour of medicines. It is the product of interactions in the immediate pre-Covid period when UNGAP members were able to meet and set up workshops and to discuss progress across the disciplines. UNGAP activities are divided into work packages that cover special treatment populations, absorption processes in different regions of the gut, the development of advanced formulations and the integration of food and pharmaceutical scientists in the food-drug interface. This involves both new and established technical approaches in which we have attempted to define best practice and highlight areas where further research is needed. Over the last months we have been able to reflect on some of the key innovative approaches which we were tasked with mapping, including theoretical, in silico, in vitro, in vivo and ex vivo, preclinical and clinical approaches. This is the product of some of us in a snapshot of where UNGAP has travelled and what aspects of innovative technologies are important. It is not a comprehensive review of all methods used in research to study drug dissolution and absorption, but provides an ample panorama of current and advanced methods generally and potentially useful in this area. This collection starts from a consideration of advances in a priori approaches: an understanding of the molecular properties of the compound to predict biological characteristics relevant to absorption. The next four sections discuss a major activity in the UNGAP initiative, the pursuit of more representative conditions to study lumenal dissolution of drug formulations developed independently by academic teams. They are important because they illustrate examples of in vitro simulation systems that have begun to provide a useful understanding of formulation behaviour in the upper GI tract for industry. The Leuven team highlights the importance of the physiology of the digestive tract, as they describe the relevance of gastric and intestinal fluids on the behaviour of drugs along the tract. This provides the introduction to microdosing as an early tool to study drug disposition. Microdosing in oncology is starting to use gamma-emitting tracers, which provides a link through SPECT to the next section on nuclear medicine. The last two papers link the modelling approaches used by the pharmaceutical industry, in silico to Pop-PK linking to Darwich and Aarons, who provide discussion on pharmacometric modelling, completing the loop of molecule to man.

Insight into the Colonic Disposition of Sulindac in Humans.

NSAIDs such as celecoxib and sulindac play a critical role in the treatment of colorectal cancer, yet it is not understood how sufficiently high concentrations are reached in colonic tissue. We previously demonstrated that an incomplete small intestinal absorption of celecoxib enables gut driven drug accumulation in caecal tissue, which is most likely needed for inducing remission. However, a multistage dissolution experiment suggested a more extensive absorption of sulindac relative to celecoxib, though still incomplete. To study whether caecal accumulation of sulindac is solely plasma driven or also gut driven, we performed an exploratory clinical study in healthy volunteers. After intake of a tablet of sulindac (200 mg; Arthrocine), two colonoscopies (1.0-2.5 h, and 6.0-7.5 h after drug intake) were performed to assess concentrations of sulindac and metabolites in plasma, caecal tissue and caecal contents. We observed that sulindac, even without the use of a colon-targeted delivery strategy, can arrive at the colonic lumen due to incomplete absorption and biliary excretion, and that the microbiota can catalyse the production of sulindac sulfide, which then accumulates in a high and local manner in the colonic tissue. These data can be relevant for drug development in the treatment of colorectal adenomas and cancer.

Insight into the colonic disposition of celecoxib in humans.

Although the effect of NSAIDs such as celecoxib on the progression of colorectal polyps has been established, it is currently unknown how sufficiently high concentrations of celecoxib are reached in colonic tissue. Indeed, the lipophilic and poorly soluble celecoxib is orally dosed as an immediate release capsule without any colon-targeting delivery strategy. In the present study, we aimed to distinguish between plasma and gut driven caecal tissue accumulation of celecoxib in healthy volunteers. After developing a protocol to reliably collect colonic biopsies and contents, the disposition of celecoxib was assessed in plasma, caecal tissue and caecal contents collected after intake of a celecoxib capsule (200 mg; Celebrex®) with 240 mL of tap water. During a first colonoscopy (1.0-2.5 h after drug intake), plasma concentrations of celecoxib and its carboxy metabolite were increasing, while caecal tissue concentrations were relatively low. As no celecoxib was present in caecal contents, tissue accumulation was clearly plasma driven. During a second colonoscopy (6.0-7.5 h after drug intake), tissue concentrations of the drug and its metabolite were substantially higher despite decreasing plasma concentrations. As a high amount of celecoxib was found in the caecal contents, the increased tissue accumulation most likely resulted from direct uptake of celecoxib from the gut. These data demonstrate that incomplete small intestinal absorption of the poorly soluble drug celecoxib enables gut driven drug accumulation in caecal tissue, which is, most likely, critical for the role of this NSAID in the prevention of colorectal cancer.

Assessment of Passive Intestinal Permeability Using an Artificial Membrane Insert System.

Despite reasonable predictive power of current cell-based and cell-free absorption models for the assessment of intestinal drug permeability, high costs and lengthy preparation steps hamper their use. The use of a simple artificial membrane (without any lipids present) as intestinal barrier substitute would overcome these hurdles. In the present study, a set of 14 poorly water-soluble drugs, dissolved in 2 different media (fasted state simulated/human intestinal fluids [FaSSIF/FaHIF]), were applied to the donor compartment of an artificial membrane insert system (AMI-system) containing a regenerated cellulose membrane. Furthermore, to investigate the predictive capacity of the AMI-system as substitute for the well-established Caco-2 system to assess intestinal permeability, the same set of 14 drugs dissolved in FaHIF were applied to the donor compartment of a Caco-2 system. For 14 drugs, covering a broad range of physicochemical parameters, a reasonable correlation between both absorption systems was observed, characterized by a Pearson correlation coefficient r of 0.95 (FaHIF). Using the AMI-system, an excellent predictive capacity of FaSSIF as surrogate medium for FaHIF was demonstrated (r = 0.96). Based on the acquired data, the AMI-system appears to be a time- and cost-effective tool for the early-stage estimation of passive intestinal permeability for poorly water-soluble drugs.

Rapid conversion of the ester prodrug abiraterone acetate results in intestinal supersaturation and enhanced absorption of abiraterone: in vitro, rat in situ and human in vivo studies.

The aim of this study was to evaluate the intestinal disposition of abiraterone acetate, an ester prodrug of the anticancer agent abiraterone. Stability of the prodrug and solubility and dissolution characteristics of both abiraterone and abiraterone acetate were monitored in vitro. Moreover, the in vivo intraluminal concentrations of abiraterone and abiraterone acetate upon intake of one tablet of 250 mg abiraterone acetate were assessed in healthy volunteers. The intestinal absorption resulting from the intraluminal behavior of the ester prodrug was determined using the rat in situ intestinal perfusion technique with mesenteric blood sampling. Simulated and aspirated human intestinal fluids of the fasted state were used as solvent systems. Upon incubation of abiraterone acetate in human intestinal fluids in vitro, rapid hydrolysis of the prodrug was observed, generating abiraterone concentrations largely exceeding the apparent solubility of abiraterone, suggesting the existence of intestinal supersaturation. These findings were confirmed in vivo, by intraluminal sampling of duodenal fluids upon oral intake of an abiraterone acetate tablet by healthy volunteers. Rat in situ intestinal perfusion experiments performed with suspensions of abiraterone and abiraterone acetate in human intestinal fluids of the fasted state revealed significantly higher flux values upon perfusion with the prodrug than with abiraterone. Moreover, rat in situ intestinal perfusion with abiraterone acetate suspensions in simulated fluids of the fasted state in presence or absence of esterases demonstrated that increased hydrolytic activity of the perfusion medium was beneficial to the intestinal absorption of abiraterone. In conclusion, the rapid hydrolysis of abiraterone acetate in the intraluminal environment appears to result in fast and extensive generation of abiraterone supersaturation, creating a strong driving force for abiraterone absorption.

Vaginal expression of efflux transporters and the potential impact on the disposition of microbicides in vitro and in rabbits.

In order to reach sufficiently high tissue concentrations and thus be effective, vaginally applied anti-HIV microbicides that are active at the level of the immune cells must permeate across the cervicovaginal mucosal layer. Cellular efflux transporters, such as Pgp, BCRP, and MRP-2, have been demonstrated to greatly affect drug disposition at different sites in the body including the intestine and the blood-brain barrier; their possible role on drug uptake from the female genital tract, however, has not been elucidated yet. In the present study, the protein expression of Pgp, BCRP, and MRP-2 in endocervical and vaginal tissue of premenopausal women was confirmed by Western blot analysis. To enable the assessment of transporter effects in vitro, the identification of an appropriate cervicovaginal cell line was pursued. The cervical SiHa cell line was observed to express mRNA of the 3 studied transporters, but only MRP-2 was found to be active. Consequently, the established Caco-2 cell line was utilized as an alternative in which the interaction of 10 microbicide candidates with the efflux transporters was studied. Darunavir, saquinavir, and maraviroc were identified as Pgp and MRP-2 substrates. The impact of Pgp on in vivo drug disposition was further examined for the model Pgp substrate talinolol in rabbits. Its vaginal uptake was significantly reduced by Pgp-mediated efflux when formulated in a neutral but not in an acidic gel. Our findings indicate the expression of a functional Pgp transporter in the vaginal mucosa that may severely reduce the vaginal uptake of Pgp substrates, including certain microbicide candidates, especially in women with an increased vaginal pH.

Development and characterization of a solid dispersion film for the vaginal application of the anti-HIV microbicide UAMC01398.

The purpose of this work was to design and evaluate a vaginal film delivery system for UAMC01398, a novel non-nucleoside reverse transcriptase inhibitor currently under investigation for use as an anti-HIV microbicide. UAMC01398 (1mg) films consisting of hydroxypropylmethylcellulose (HPMC) and polyethylene glycol 400 (PEG400) in different ratios were prepared by solvent evaporation. Based on its flexibility, softness and translucent appearance, the 30% PEG400 and 70% HPMC containing film was selected for further assessment. The vaginal film formulation was fast-dissolving (<10 min in 1 mL of vaginal fluid simulant), stable up to at least one month and safe toward epithelial cells and lactobacilli. Furthermore, formulating UAMC01398 into the film dosage form did not influence its antiviral activity. Powder X-ray diffraction revealed the amorphous nature of the UAMC01398 film, resulting in enhanced compound permeation across the epithelial HEC-1A cell layer, presumably owing to the induction of supersaturation. The in vivo vaginal tissue uptake of UAMC01398 in rabbits, as measured by systemic concentrations, was increased compared to the previously established non-solubilizing gel (significant difference) and sulfobutyl ether-ß-cyclodextrin (5%) containing gel. To conclude, we identified a film formulation suitable for the vaginal delivery of UAMC01398.

Drug precipitation-permeation interplay: supersaturation in an absorptive environment.

PURPOSE: The present study investigated the interplay between supersaturation, absorption, precipitation, and excipient-mediated precipitation inhibition by comparing classic precipitation assessment in a non-absorption environment with precipitation/permeation assessment in an absorption environment. Loviride and HPMC-E5 were selected as poorly soluble model drug and precipitation inhibitor, respectively. METHOD: To investigate supersaturation in an absorptive environment, supersaturation was induced at different degrees (DS), using a solvent shift method, in shaken Caco-2 Transwell® inserts containing fasted state simulated intestinal fluid (FaSSIF); to simulate a non-absorption environment, the inserts were parafilm-sealed and did not contain a cell monolayer. Donor and acceptor compartments were sampled as a function of time to determine precipitation kinetics and transport, respectively. RESULTS: In absence of precipitation, loviride transport increased proportionally with the initial DS; however, precipitation limited the supersaturation-induced transport enhancement. Loviride precipitation was found to be less extensive in an absorption environment compared to a non-absorption environment. As a result, the optimal DS obtained in a non-absorption environment (highest amount maintained in solution) did not correlate with the highest transport in an absorption environment. In addition, the impact of HPMC-E5 on loviride transport was inferior to its precipitation inhibitory capacity observed in a non-absorption environment. CONCLUSION: For the first time, the present study explicitly demonstrated that implementation of permeation in precipitation assays is critical to predict the impact of supersaturation, precipitation, and precipitation inhibition on the absorption of poorly soluble drugs.

Supersaturation in human gastric fluids.

PURPOSE: The current study reports on supersaturation, precipitation and excipient mediated precipitation inhibition of five poorly soluble drugs (loviride, glibenclamide, itraconazole, danazol, and etravirine) in human and simulated gastric fluids. METHOD: Upon induction of supersaturation in human gastric fluids (HGFs), simulated gastric fluid (SGF), and fasted state simulated gastric fluid (FaSSGF) using a solvent shift method, supersaturation and precipitation were assessed as a function of time. In addition, the precipitation inhibitory capacity of three polymers (Eudragit® E PO, HPMC-E5, and PVP K25) was investigated. RESULTS: Supersaturation in human gastric fluids was observed for all model compounds, but proved to be relatively unstable (fast precipitation), except for itraconazole. Only modest excipient-mediated stabilizing effects on supersaturation were observed using HPMC-E5 and Eudragit® E PO whereas PVP K25 exerted no effect. In contrast to SGF, the observed precipitation behavior in FaSSGF was similar to the behavior in human gastric fluids. CONCLUSION: The present study demonstrates that supersaturation stability of drugs in human gastric fluids is in general inferior to supersaturation stability in intestinal fluids. As the potential for excipient mediated precipitation inhibition in gastric fluids was only limited, our data suggest that supersaturation should preferably be targeted to the intestine.

CADA, a potential anti-HIV microbicide that specifically targets the cellular CD4 receptor.

The cyclotriazadisulfonamide (CADA) compounds are a new class of specific CD4-targeted HIV entry inhibitors. The in vitro anti-HIV activity of CADA was shown to correlate with its ability to specifically downmodulate cell surface expression of the CD4 receptor in human cells. Here, we evaluated its potential as an anti-HIV microbicide. CADA exerted a clear CD4 receptor downregulating effect in dendritic cells (DC) and subsequently inhibited HIV-1(BaL) replication in DC/T cell co-cultures. The compound proved to be active against a variety of clinical isolates belonging to the HIV-1 subtypes A, B, C, D, F, G, H, AE and O. Furthermore, it prevented human T cells from being infected with the laboratory-adapted strains X4 HIV-1(NL4.3) and R5 SIV(mac251). Flow cytometric analysis demonstrated a significant and dose-dependent downregulation of CD4 on macaque PBMCs. In addition, the compound exerted a marked anti-SIV(mac251) activity in these cells from simian origin. The combination of CADA with cellulose acetate phthalate (CAP) resulted in a synergistic inhibition of HIV-1 and SIV infection. Finally, gel formulated CADA proved to preserve the CD4 downmodulating and antiviral activity of this compound when formulated as a microbicide gel. Thus, our data suggest that CADA may have potential as a broad-spectrum anti-HIV microbicide drug candidate. The preservation of the activity of gel formulated CADA will make it now feasible for testing this unique entry inhibitor in non-human primates, not only as a single drug but also in a synergistic conjunction with other anti-HIV compounds.