Multidimensional analysis of human intestinal fluid composition

The oral administration of solid dosage forms is the commonest method to achieve systemic therapy and relies on the drug’s solubility in human intestinal fluid (HIF), a key factor that influences bioavailability and biopharmaceutical classification. However, HIF is difficult to obtain and is known to be variable, which has led to the development of a range of simulated intestinal fluid (SIF) systems to determine drug solubility in vitro. In this study we have applied a novel multidimensional approach to analyse and characterise HIF composition using a published data set in both fasted and fed states with a view to refining the existing SIF approaches. The data set provided 152 and 172 measurements of five variables (total bile salt, phospholipid, total free fatty acid, cholesterol and pH) in time-dependent HIF samples from 20 volunteers in the fasted and fed state, respectively. The variable data sets for both fasted state and fed state are complex, do not follow normal distributions but the amphiphilic variable concentrations are correlated. When plotted 2-dimensionally a generally ellipsoid shaped data cloud with a positive slope is revealed with boundaries that enclose published fasted or fed HIF compositions. The data cloud also encloses the majority of fasted state and fed state SIF recipes and illustrates that the structured nature of design of experiment (DoE) approaches does not optimally cover the variable space and may examine media compositions that are not biorelevant. A principal component analysis in either fasted or fed state in combination with fitting an ellipsoid shape to enclose the data results in 8 points that capture over 95% of the compositional variability of HIF. The variable’s average rate of concentration change in both fasted state and fed state over a short time scale (10 min) is zero and a Euclidean analysis highlights differences between the fasted and fed states and among individual volunteers. The results indicate that a 9-point DoE (8 + 1 central point) could be applied to investigate drug solubility in vitro and provide statistical solubility limits. In addition, a single point could provide a worst-case solubility measurement to define the lowest biopharmaceutical classification boundary or for use during drug development. This study has provided a novel description of HIF composition. The approach could be expanded in multiple ways by incorporation of further data sets to improve the statistical coverage or to cover specific patient groups (e.g., paediatric). Further development might also be possible to analyse information on the time dependent behaviour of HIF and to guide HIF sampling and analysis protocols

Linking the concentrations of itraconazole and 2-hydroxypropyl-β-cyclodextrin in human intestinal fluids after oral intake of Sporanox^®

In a previously performed small-scale clinical study, healthy volunteers were asked to ingest an oral solution of itraconazole (Sporanox®) containing 40% 2-hydroxypropyl-β-cyclodextrin (HP-β-CD) (i) with or (ii) without a standardized volume of water (240 mL) after which gastrointestinal and blood samples were collected. Although omitting water during the administration of Sporanox® resulted in noticeably higher duodenal concentrations of itraconazole, systemic exposure was almost unaffected. It is assumed that this discrepancy can be explained by differences in the extent of entrapment of itraconazole in the duodenum caused by differential complexation depending on the concentration of cyclodextrins. To further substantiate this hypothesis, the quantification of HP-β-CD concentrations in the aspirated intestinal fluids was performed by LC-MS/MS. When comparing the intestinal concentrations of itraconazole and HP-β-CD for one single healthy volunteer (HV02) in both test conditions, an excellent correlation was observed (Spearman's rank coefficient of 0.96). Moreover, the data suggest that, similar to aqueous buffer media, also in human intestinal fluids a non-linear relationship exists between itraconazole solubility and HP-β-CD concentration (Ap-type profile; Spearman's rank coefficient of 0.78), indicating that higher order complexes are formed at higher concentrations of HP-β-CD. This difference in extent of entrapment in the inclusion complexes helps to understand the observed impact of water intake on precipitation and permeation behavior of itraconazole in man. Without water intake, higher HP-β-CD concentrations resulted in less precipitation and increased duodenal concentrations of itraconazole. On the other hand, the stronger interaction at higher HP-β-CD concentrations reduced the free fraction of the drug explaining that increased intraluminal concentrations of itraconazole were not translated into an enhanced uptake. In conclusion, quantifying the concentrations of the solubilizing agent HP-β-CD in human intestinal fluids appeared to be of crucial importance to interpret the intraluminal behavior of an orally administered cyclodextrin-based solution

Structured solubility behaviour in bioequivalent fasted simulated intestinal fluids

Drug solubility in intestinal fluid is a key parameter controlling absorption after the administration of a solid oral dosage form. To measure solubility in vitro simulated intestinal fluids have been developed, but there are multiple recipes and the optimum is unknown. This situation creates difficulties during drug discovery and development research. A recent study characterised sampled fasted intestinal fluids using a multidimensional approach to derive nine bioequivalent fasted intestinal media that covered over 90% of the compositional variability. These media have been applied in this study to examine the equilibrium solubility of twenty one exemplar drugs (naproxen, indomethacin, phenytoin, zafirlukast, piroxicam, ibuprofen, mefenamic acid, furosemide, aprepitant, carvedilol, tadalafil, dipyridamole, posaconazole, atazanavir, fenofibrate, felodipine, griseofulvin, probucol, paracetamol, acyclovir and carbamazepine) to determine if consistent solubility behaviour was present. The bioequivalent media provide in the majority of cases structured solubility behaviour that is consistent with physicochemical properties and previous solubility studies. For the acidic drugs (pKa 8), basic and neutral drugs solubility is controlled by a combination of media pH and total amphiphile concentration (TAC), a consistent solubility behaviour is evident but with variation related to individual drug interactions within the media. The lowest and highest pH × TAC media identify the lowest and highest solubility in over 78% of cases. A subset of the latter category consisting of neutral and drugs non-ionised in the media pH range have been identified with a very narrow solubility range, indicating that the impact of the simulated intestinal media on their solubility is minimal. Two drugs probucol and atazanavir exhibit unusual behaviour. The study indicates that the use of two appropriate bioequivalent fasted intestinal media from the nine will identify in vitro the maximum and minimum solubility boundaries for drugs and due to the media derivation this is probably applicable in vivo. These media could be applied during discovery and development activities to provide a solubility range, which would assist placement of the drug within the BCS/DCS and rationalise drug and formulation decisions

Fasted intestinal solubility limits and distributions applied to the biopharmaceutics and developability classification systems

After oral administration, a drug’s solubility in intestinal fluid is an important parameter influencing bioavailability and if the value is known it can be applied to estimate multiple biopharmaceutical parameters including the solubility limited absorbable dose. Current in vitro measurements may utilise fasted human intestinal fluid (HIF) or simulated intestinal fluid (SIF) to provide an intestinal solubility value. This single point value is limited since its position in relation to the fasted intestinal solubility envelope is unknown. In this study we have applied a nine point fasted equilibrium solubility determination in SIF, based on a multi-dimensional analysis of fasted human intestinal fluid composition, to seven drugs that were previously utilised to investigate the developability classification system (ibuprofen, mefenamic acid, furosemide, dipyridamole, griseofulvin, paracetamol and acyclovir). The resulting fasted equilibrium solubility envelope encompasses literature solubility values in both HIF and SIF indicating that it measures the same solubility space as current approaches with solubility behaviour consistent with previous SIF design of experiment studies. In addition, it identifies that three drugs (griseofulvin, paracetamol and acyclovir) have a very narrow solubility range, a feature that single point solubility approaches would miss. The measured mid-point solubility value is statistically equivalent to the value determined with the original fasted simulated intestinal fluid recipe, further indicating similarity and that existing literature results could be utilised as a direct comparison. Since the multi-dimensional approach covered greater than ninety percent of the variability in fasted intestinal fluid composition, the measured maximum and minimum equilibrium solubility values should represent the extremes of fasted intestinal solubility and provide a range. The seven drugs all display different solubility ranges and behaviours, a result also consistent with previous studies. The dose/solubility ratio for each measurement point can be plotted using the developability classification system to highlight individual drug behaviours. The lowest solubility represents a worst-case scenario which may be useful in risk-based quality by design biopharmaceutical calculations than the mid-point value. The method also permits a dose/solubility ratio frequency distribution determination for the solubility envelope which permits further risk-based refinement, especially where the drug crosses a classification boundary. This novel approach therefore provides greater in vitro detail with respect to possible biopharmaceutical performance in vivo and an improved ability to apply risk-based analysis to biopharmaceutical performance. Further studies will be required to expand the number of drugs measured and link the in vitro measurements to in vivo results

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

Funding Information: AI acknowledges the support of projects icp009 (ALKOOL) of PRACE-ICEI (grant agreement 800858) for awarding access to Piz Daint, at the Swiss National Supercomputing Centre (CSCS), Switzerland and BG05M2OP001–1.001–0004 (UNITe) of the Bulgarian Ministry of Education and Science. For further details on points raised in this article, please contact [email protected]. Funding Information: Acknowledgements. JAGH is supported by the Biocenter Finland, the Helsinki Institute of Life Sciences, and the Faculty of Pharmacy, University of Helsinki. Publisher Copyright: © 2021 The AuthorsThis 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.Peer reviewe

Tyrosine Kinase Inhibitors and Proton Pump Inhibitors:An Evaluation of Treatment Options

Tyrosine kinase inhibitors (TKIs) have rapidly become an established factor in oncology, and have been shown to be effective in a wide variety of solid and hematologic malignancies. Use of the oral administration route of TKIs offers flexibility and is convenient for the patient; however, despite these advantages, the oral route of administration also causes a highly relevant new problem. Acid-inhibitory drugs, such as proton pump inhibitors (PPIs), increase the intragastric pH, which may subsequently decrease TKI solubility, bioavailability, and treatment efficacy. Clear and practical advice on how to manage PPI use during TKI therapy is currently not available in the literature. Since PPIs are extensively used during TKI therapy, prescribers are presented with a big dilemma as to whether or not to continue the combined treatment, resulting in patients possibly being deprived of optimal therapy. When all pharmacological characteristics and data of either TKIs and PPIs are considered, practical and safe advice on how to manage this drug combination can be given

Overcoming immune checkpoint blockade resistance in solid tumors with intermittent ITK inhibition

Cytotoxic CD8 + T cell (CTL) exhaustion is driven by chronic antigen stimulation. Reversing CTL exhaustion with immune checkpoint blockade (ICB) has provided clinical benefits in different types of cancer. We, therefore, investigated whether modulating chronic antigen stimulation and T-cell receptor (TCR) signaling with an IL2-inducible T-cell kinase (ITK) inhibitor, could confer ICB responsiveness to ICB resistant solid tumors. In vivo intermittent treatment of 3 ICB-resistant solid tumor (melanoma, mesothelioma or pancreatic cancer) with ITK inhibitor significantly improved ICB therapy. ITK inhibition directly reinvigorate exhausted CTL in vitro as it enhanced cytokine production, decreased inhibitory receptor expression, and downregulated the transcription factor TOX. Our study demonstrates that intermittent ITK inhibition can be used to directly ameliorate CTL exhaustion and enhance immunotherapies even in solid tumors that are ICB resistant.</p

Microenvironment-Dependent Gradient of CTL Exhaustion in the AE17sOVA Murine Mesothelioma Tumor Model

The immune system, and in particular, cytotoxic CD8+ T cells (CTLs), plays a vital part in the prevention and elimination of tumors. In many patients, however, CTL-mediated tumor killing ultimately fails in the clearance of cancer cells resulting in disease progression, in large part due to the progression of effector CTL into exhausted CTL. While there have been major breakthroughs in the development of CTL-mediated “reinvigoration”-driven immunotherapies such as checkpoint blockade therapy, there remains a need to better understand the drivers behind the development of T cell exhaustion. Our study highlights the unique differences in T cell exhaustion development in tumor-specific CTL which arises over time in a mouse model of mesothelioma. Importantly, we also show that peripheral tumor-specific T cells have a unique expression profile compared to exhausted tumor-infiltrating CTL at a late-stage of tumor progression in mice. Together, these data suggest that greater emphasis shoul