Exploring the Predictive Power of the In Situ Perfusion Technique towards Drug Absorption: Theory, Practice, and Applications.

Considering the broad applications and popularity, the in situ perfusion technique is an established and interesting approach to evaluate the absorption mechanisms of drug molecules in specific regions of the intestinal tract. Compared to perfusion studies in humans, this surrogate model shows several familiar characteristics making it interesting to apply this technique in rats in the non-clinical stage of drug product development. The differences in gastrointestinal (GI) anatomy and physiology between rats and humans are thoroughly discussed in the present review. Moreover, an in-depth overview of the Doluisio (i.e., closed-loop) versus the single-pass intestinal perfusion (i.e., open-loop) technique is shown. Finally, applications and future perspectives of the technique are presented.

Effect of Common Excipients on Intestinal Drug Absorption in Wistar Rats.

The aim of the present paper is to study the effect of common excipients on the permeability of atenolol (as drug absorbed mainly by passive diffusion) and rhodamine (as P-glycoprotein substrate). The apparent permeability was measured by an in situ perfusion method in Wistar rats using the closed loop Doluisio's method. Permeability values were characterized in the absence and presence of 18 commonly used excipients. Excipient concentrations were selected based on the amounts in oral immediate release dosage forms, which failed the test during the human bioequivalence studies. Atenolol was studied with and without excipients in the whole small intestine, whereas rhodamine was tested in three different intestinal segments to account for the differential expression of P-glycoprotein, and it was further on tested in the ileum, in the presence of excipients. Atenolol presented higher permeability values when it was administered with colloidal silica, croscarmellose, hydroxypropyl methylcellulose (HPMC), magnesium stearate, MgCO3, poly(ethylene glycol) 400, poly(vinylpyrrolidone), sorbitol, starch, and TiO2 rhodamine showed higher permeability values when it was administered with croscarmellose and HPMC. On the one hand, the mechanisms of action were not discernible with the proposed experiments. On the other hand, commercial formulations do not present a single excipient but several, which can counteract their effects. The in situ perfusion technique can be useful for a preliminary screening and risk analysis, while the in vivo pharmacokinetic results would be needed to define conclusive effects.

New Insights of Oral Colonic Drug Delivery Systems for Inflammatory Bowel Disease Therapy.

Colonic Drug Delivery Systems (CDDS) are especially advantageous for local treatment of inflammatory bowel diseases (IBD). Site-targeted drug release allows to obtain a high drug concentration in injured tissues and less systemic adverse effects, as consequence of less/null drug absorption in small intestine. This review focused on the reported contributions in the last four years to improve the effectiveness of treatments of inflammatory bowel diseases. The work concludes that there has been an increase in the development of CDDS in which pH, specific enzymes, reactive oxygen species (ROS), or a combination of all of these triggers the release. These delivery systems demonstrated a therapeutic improvement with fewer adverse effects. Future perspectives to the treatment of this disease include the elucidation of molecular basis of IBD diseases in order to design more specific treatments, and the performance of more in vivo assays to validate the specificity and stability of the obtained systems.

Intestinal Permeability Study of Clinically Relevant Formulations of Silibinin in Caco-2 Cell Monolayers.

An ever-growing number of preclinical studies have investigated the tumoricidal activity of the milk thistle flavonolignan silibinin. The clinical value of silibinin as a bona fide anti-cancer therapy, however, remains uncertain with respect to its bioavailability and blood⁻brain barrier (BBB) permeability. To shed some light on the absorption and bioavailability of silibinin, we utilized the Caco-2 cell monolayer model of human intestinal absorption to evaluate the permeation properties of three different formulations of silibinin: silibinin-meglumine, a water-soluble form of silibinin complexed with the amino-sugar meglumine; silibinin-phosphatidylcholine, the phytolipid delivery system Siliphos; and Eurosil85/Euromed, a milk thistle extract that is the active component of the nutraceutical Legasil with enhanced bioavailability. Our approach predicted differential mechanisms of transport and blood⁻brain barrier permeabilities between the silibinin formulations tested. Our assessment might provide valuable information about an idoneous silibinin formulation capable of reaching target cancer tissues and accounting for the observed clinical effects of silibinin, including a recently reported meaningful central nervous system activity against brain metastases.

Mass Transport Analysis of the Enhanced Buffer Capacity of the Bicarbonate-CO2 Buffer in a Phase-Heterogenous System: Physiological and Pharmaceutical Significance.

The bicarbonate buffer capacity is usually considered in a phase-homogeneous system, at equilibrium, with no CO2 transfer between the liquid buffer phase and another phase. However, typically, an in vitro bicarbonate buffer-based system is a phase-heterogeneous system, as it entails continuously sparging (bubbling) the dissolution medium with CO2 in a gas mixture, at constant ratio, to maintain a constant partial pressure of CO2 (g) and CO2(aq) molarity at a prescribed value, with CO2 diffusing freely between the gas and the aqueous phases. The human gastrointestinal tract is also a phase-heterogeneous system, with CO2 diffusing across the mucosal membrane into the mesenteric arterial blood, which serves as a sink for CO2 from the intestinal lumen. In this report, a mass transport analysis of the apparent buffer capacity of a phase-heterogeneous bicarbonate-CO2 system is developed. It is shown that, most significantly, a phase-heterogeneous bicarbonate-CO2 system can have a much higher buffer capacity than a phase-homogeneous system such that the buffer capacity is dependent on the bicarbonate concentration. It is double that of a phase-homogeneous system at the pH = p Ka for a monoprotic buffer at the same concentration. This buffer capacity enhancement increases hyperbolically with pH above the p Ka, thus providing a much stronger buffering to keep the pH in the physiologically neutral range. The buffer capacity will be dependent on the bicarbonate molarity (which in vivo will depend on the bicarbonate secretion rate) and not the pH of the luminal fluid. Further, there is no conjugate acid accumulation as a result of bicarbonate neutralization, since the resulting carbonic acid (H2CO3) rapidly dehydrates producing CO2 and H2O. The mass transport analysis developed in this report is further supported by in vitro experimental results. This enhanced bicarbonate buffer capacity in a phase-heterogeneous system is of physiological significance as well as significant for the dissolution and absorption of ionizable drugs.

In Vitro Dissolution as a Tool for Formulation Selection: Telmisartan Two-Step IVIVC.

The purpose of this investigation was to develop an exploratory two-step level A IVIVC for three telmisartan oral immediate release formulations, the reference product Micardis, and two generic formulations (X1 and X2). Correlation was validated with a third test formulation, Y1. Experimental solubility and permeability data were obtained to confirm that telmisartan is a class II compound under the Biopharmaceutic Classification System. Bioequivalence (BE) studies plasma profiles were combined using a previously published reference scaling procedure. X2 demonstrated in vivo BE, while X1 and Y1 failed to show BE due to the lower boundary of the 90% confidence interval for Cmax being outside the acceptance limits. Average plasma profiles were deconvoluted by the Loo-Riegelman method to obtain the oral fractions absorbed ( fa). Fractions dissolved ( fdiss) were obtained in several conditions in USP II and USP IV apparatus, and later, the results were compared in order to find the most biopredictive model, calculating the f2 similarity factor. The apparatus and conditions showing the same rank order than in vivo data were selected for further refinement of conditions. A Levy plot was constructed to estimate the time scaling factor and to make both processes, dissolution and absorption, superimposable. The in vitro dissolution experiment that reflected more accurately the in vivo behavior of the different formulations of telmisartan employed the USP IV dissolution apparatus and a dissolution environment with a flow rate of 8 mL/min and a three-step pH change, from 1.2 to 4.5 and 6.8, with a 0.05% of Tween 80. Thus, these conditions gave rise to a biopredictive dissolution test. This new model is able to predict the formulation differences in dissolution that were previously observed in vivo, which could be used as a risk-analysis tool for formulation selection in future bioequivalence trials.

Linking the Gastrointestinal Behavior of Ibuprofen with the Systemic Exposure between and within Humans-Part 1: Fasted State Conditions.

The goal of this project was to explore and to statistically evaluate the responsible gastrointestinal (GI) factors that are significant factors in explaining the systemic exposure of ibuprofen, between and within human subjects. In a previous study, we determined the solution and total concentrations of ibuprofen as a function of time in aspirated GI fluids, after oral administration of an 800 mg IR tablet (reference standard) of ibuprofen to 20 healthy volunteers in fasted state conditions. In addition, we determined luminal pH and motility pressure recordings that were simultaneously monitored along the GI tract. Blood samples were taken to determine ibuprofen plasma levels. In this work, an in-depth statistical and pharmacokinetic analysis was performed to explain which underlying GI variables are determining the systemic concentrations of ibuprofen between (inter-) and within (intra-) subjects. In addition, the obtained plasma profiles were deconvoluted to link the fraction absorbed with the fraction dissolved. Multiple linear regressions were performed to explain and quantitatively express the impact of underlying GI physiology on systemic exposure of the drug (in terms of plasma Cmax/AUC and plasma Tmax). The exploratory analysis of the correlation between plasma Cmax/AUC and the time to the first phase III contractions postdose (TMMC-III) explains ∼40% of the variability in plasma Cmax for all fasted state subjects. We have experimentally shown that the in vivo intestinal dissolution of ibuprofen is dependent upon physiological variables like, in this case, pH and postdose phase III contractions. For the first time, this work presents a thorough statistical analysis explaining how the GI behavior of an ionized drug can explain the systemic exposure of the drug based on the individual profiles of participating subjects. This creates a scientifically based and rational framework that emphasizes the importance of including pH and motility in a predictive in vivo dissolution methodology to forecast the in vivo performance of a drug product. Moreover, as no extensive first-pass metabolism is considered for ibuprofen, this study demonstrates how intraluminal drug behavior is reflecting the systemic exposure of a drug.

Linking the Gastrointestinal Behavior of Ibuprofen with the Systemic Exposure between and within Humans-Part 2: Fed State.

Exploring the intraluminal behavior of an oral drug product in the human gastrointestinal (GI) tract remains challenging. Many in vivo techniques are available to investigate the impact of GI physiology on oral drug behavior in fasting state conditions. However, little is known about the intraluminal behavior of a drug in postprandial conditions. In a previous report, we described the mean solution and total concentrations of ibuprofen after oral administration of an immediate-release (IR) tablet in fed state conditions. In parallel, blood samples were taken to assess systemic concentrations. The purpose of this work was to statistically evaluate the impact of GI physiology (e.g., pH, contractile events) within and between individuals (intra and intersubject variability) for a total of 17 healthy subjects. In addition, a pharmacokinetic (PK) analysis was performed by noncompartmental analysis, and PK parameters were correlated with underlying physiological factors (pH, time to phase III contractions postdose) and study parameters (e.g., ingested amount of calories, coadministered water). Moreover, individual plasma profiles were deconvoluted to assess the fraction absorbed as a function of time, demonstrating the link between intraluminal and systemic behavior of the drug. The results demonstrated that the in vivo dissolution of ibuprofen depends on the present gastric pH and motility events at the time of administration. Both intraluminal factors were responsible for explaining 63% of plasma Cmax variability among all individuals. For the first time, an in-depth analysis was performed on a large data set derived from an aspiration/motility study, quantifying the impact of physiology on systemic behavior of an orally administered drug product in fed state conditions. The data obtained from this study will help us to develop an in vitro biorelevant dissolution approach and optimize in silico tools in order to predict the in vivo performance of orally administered drug products, especially in fed state conditions.

Ionic Hydrogel Based on Chitosan Cross-Linked with 6-Phosphogluconic Trisodium Salt as a Drug Delivery System.

In this work, 6-phosphogluconic trisodium salt (6-PG-Na+) is introduced as a new aqueous and nontoxic cross-linking agent to obtain ionic hydrogels. Here, it is shown the formation of hydrogels based on chitosan cross-linked with 6-PG-Na+. This formulation is obtained by ionic interaction of cationic groups of polymer with anionic groups of the cross-linker. These hydrogels are nontoxic, do not cause dermal irritation, are easy to extend, and have an adequate adhesion force to be applied as polymeric film over the skin. This formulation exhibits a first order release kinetic and can be applied as drug vehicle for topical administration or as wound dressing for wound healing. The primary goal of this communication is to report the identification and utility of 6-phosphogluconic trisodium salt (6-PG-Na+) as a nontoxic cross-linker applicable for cationic polymers.

Biopharmaceutical optimization in neglected diseases for paediatric patients by applying the provisional paediatric biopharmaceutical classification system.

AIMS: Unavailability and lack of appropriate, effective and safe formulations are common problems in paediatric therapeutics. Key factors such as swallowing abilities, organoleptic preferences and dosage requirements determine the need for optimization of formulations. The provisional Biopharmaceutics Classification System (BCS) can be used in paediatric formulation design as a risk analysis and optimization tool. The objective of this study was to classify six neglected tropical disease drugs following a provisional paediatric BCS (pBCS) classification adapted to three paediatric subpopulations (neonates, infants and children). METHODS: Albendazole, benznidazole, ivermectin, nifurtimox, praziquantel and proguanil were selected from the 5th edition of the Model List of Essential Medicines for Children from the World Health Organization. Paediatric drug solubility classification was based on dose number calculation. Provisional permeability classification was based on log P comparison versus metoprolol log P value, assuming passive diffusion absorption mechanisms and no changes in passive membrane permeability between paediatric patients and adults. pBCS classes were estimated for each drug, according to different doses and volumes adapted for each age stage and were compared to the adult classification. RESULTS: All six drugs were classified into provisional pBCS in the three paediatric subpopulations. Three drugs maintained the same classification as for adults, ivermectin and benznidazole changed solubility class from low to high in neonates and proguanil changed from low to high solubility in all age stages. CONCLUSION: Provisional pBCS classification of these six drugs shows potential changes in the limiting factors in oral absorption in paediatrics, depending on age stage, compared to the adult population. This valuable information will aid the optimization of paediatric dosing and formulations and can identify bioinequivalence risks when comparing different formulations and paediatric populations.

Usefulness of Caco-2/HT29-MTX and Caco-2/HT29-MTX/Raji B Coculture Models To Predict Intestinal and Colonic Permeability Compared to Caco-2 Monoculture.

The Caco-2 cellular monolayer is a widely accepted in vitro model to predict human permeability but suffering from several and critical limitations. Therefore, some alternative cell cultures to mimic the human intestinal epithelium, as closely as possible, have been developed to achieve more physiological conditions, as the Caco-2/HT29-MTX coculture and the triple Caco-2/HT29-MTX/Raji B models. In this work the permeability of 12 model drugs of different Biopharmaceutical Classification System (BCS) characteristics, in the coculture and triple coculture models was assessed. Additionally, the utility of both models to classify compounds according to the BCS criteria was scrutinized. The obtained results suggested that the coculture of Caco-2/HT29-MTX and the triple coculture of Caco-2/HT29-MTX/Raji B were useful models to predict intestinal permeability and to classify the drugs in high or low permeability according to BCS. Moreover, to study thoroughly the transport mechanism of a specific drug, using a more complex model than Caco-2 monocultures is more suitable because coculture and triple coculture are more physiological models, so the results obtained with them will be closer to those obtained in the human intestine.

Gated Mesoporous Silica Nanocarriers for a "Two-Step" Targeted System to Colonic Tissue.

Colon targeted drug delivery is highly relevant not only to treat colonic local diseases but also for systemic therapies. Mesoporous silica nanoparticles (MSNs) have been demonstrated as useful systems for controlled drug release given their biocompatibility and the possibility of designing gated systems able to release cargo only upon the presence of certain stimuli. We report herein the preparation of three gated MSNs able to deliver their cargo triggered by different stimuli (redox ambient (S1), enzymatic hydrolysis (S2), and a surfactant or being in contact with cell membrane (S3)) and their performance in solution and in vitro with Caco-2 cells. Safranin O dye was used as a model drug to track cargo fate. Studies of cargo permeability in Caco-2 monolayers demonstrated that intracellular safranin O levels were significantly higher in Caco-2 monolayers when using MSNs compared to those of free dye. Internalization assays indicated that S2 nanoparticles were taken up by cells via endocytosis. S2 nanoparticles were selected for in vivo tests in rats. For in vivo assays, capsules were filled with S2 nanoparticles and coated with Eudragit FS 30 D to target colon. The enteric coated capsule containing the MSNs was able to deliver S2 nanoparticles in colon tissue (first step), and then nanoparticles were able to deliver safranin O inside the colonic cells after the enzymatic stimuli (second step). This resulted in high levels of safranin O in colonic tissue combined with low dye levels in plasma and body tissues. The results suggested that this combination of enzyme-responsive gated MSNs and enteric coated capsules may improve the absorption of drugs in colon to treat local diseases with a reduction of systemic effects.

Low Buffer Capacity and Alternating Motility along the Human Gastrointestinal Tract: Implications for in Vivo Dissolution and Absorption of Ionizable Drugs.

In this study, we determined the pH and buffer capacity of human gastrointestinal (GI) fluids (aspirated from the stomach, duodenum, proximal jejunum, and mid/distal jejunum) as a function of time, from 37 healthy subjects after oral administration of an 800 mg immediate-release tablet of ibuprofen (reference listed drug; RLD) under typical prescribed bioequivalence (BE) study protocol conditions in both fasted and fed states (simulated by ingestion of a liquid meal). Simultaneously, motility was continuously monitored using water-perfused manometry. The time to appearance of phase III contractions (i.e., housekeeper wave) was monitored following administration of the ibuprofen tablet. Our results clearly demonstrated the dynamic change in pH as a function of time and, most significantly, the extremely low buffer capacity along the GI tract. The buffer capacity on average was 2.26 µmol/mL/ΔpH in fasted state (range: 0.26 and 6.32 µmol/mL/ΔpH) and 2.66 µmol/mL/ΔpH in fed state (range: 0.78 and 5.98 µmol/mL/ΔpH) throughout the entire upper GI tract (stomach, duodenum, and proximal and mid/distal jejunum). The implication of this very low buffer capacity of the human GI tract is profound for the oral delivery of both acidic and basic active pharmaceutical ingredients (APIs). An in vivo predictive dissolution method would require not only a bicarbonate buffer but also, more significantly, a low buffer capacity of dissolution media to reflect in vivo dissolution conditions.

Closed-Loop Doluisio (Colon, Small Intestine) and Single-Pass Intestinal Perfusion (Colon, Jejunum) in Rat-Biophysical Model and Predictions Based on Caco-2.

PURPOSE: The effective rat intestinal permeability (P eff ) was deconvolved using a biophysical model based on parameterized paracellular, aqueous boundary layer, transcellular permeabilities, and the villus-fold surface area expansion factor. METHODS: Four types of rat intestinal perfusion data were considered: single-pass intestinal perfusion (SPIP) in the jejunum (n = 40), and colon (n = 15), closed-loop (Doluisio type) in the small intestine (n = 78), and colon (n = 74). Moreover, in vitro Caco-2 permeability values were used to predict rat in vivo values in the rat data studied. RESULTS: Comparable number of molecules permeate via paracellular water channels as by the lipoidal transcellular route in the SPIP method, although in the closed-loop method, the paracellular route appears dominant in the colon. The aqueous boundary layer thickness in the small intestine is comparable to that found in unstirred in vitro monolayer assays; it is thinner in the colon. The mucosal surface area in anaesthetized rats is 0.96-1.4 times the smooth cylinder calculated value in the colon, and it is 3.1-3.6 times in the small intestine. The paracellular permeability of the intestine appeared to be greater in rat than human, with the colon showing more leakiness (higher P para ) than the small intestine. CONCLUSION: Based on log intrinsic permeability values, the correlations between the in vitro and in vivo models ranged from r2 0.82 to 0.92. The SPIP-Doluisio method comparison indicated identical log permeability selectivity trend with negligible bias.

Exploring different strategies for imbalanced ADME data problem: case study on Caco-2 permeability modeling.

In many absorption, distribution, metabolism, and excretion (ADME) modeling problems, imbalanced data could negatively affect classification performance of machine learning algorithms. Solutions for handling imbalanced dataset have been proposed, but their application for ADME modeling tasks is underexplored. In this paper, various strategies including cost-sensitive learning and resampling methods were studied to tackle the moderate imbalance problem of a large Caco-2 cell permeability database. Simple physicochemical molecular descriptors were utilized for data modeling. Support vector machine classifiers were constructed and compared using multiple comparison tests. Results showed that the models developed on the basis of resampling strategies displayed better performance than the cost-sensitive classification models, especially in the case of oversampling data where misclassification rates for minority class have values of 0.11 and 0.14 for training and test set, respectively. A consensus model with enhanced applicability domain was subsequently constructed and showed improved performance. This model was used to predict a set of randomly selected high-permeability reference drugs according to the biopharmaceutics classification system. Overall, this study provides a comparison of numerous rebalancing strategies and displays the effectiveness of oversampling methods to deal with imbalanced permeability data problems.

Investigating the Discriminatory Power of BCS-Biowaiver in Vitro Methodology to Detect Bioavailability Differences between Immediate Release Products Containing a Class I Drug.

The purpose of this work is to investigate the discriminatory power of the Biopharmaceutics Classification System (BCS)-biowaiver in vitro methodology, i.e., to investigate if a BCS-biowaiver approach would have detected the Cmax differences observed between two zolpidem tablets and to identify the cause of the in vivo difference. Several dissolution conditions were tested with three zolpidem formulations: the reference (Stilnox), a bioequivalent formulation (BE), and a nonbioequivalent formulation (N-BE). Zolpidem is highly soluble at pH 1.2, 4.5, and 6.8. Its permeability in Caco-2 cells is higher than that of metoprolol and its transport mechanism is passive diffusion. None of the excipients (alone or in combination) showed any effect on permeability. All formulations dissolved more than 85% in 15 min in the paddle apparatus at 50 rpm in all dissolution media. However, at 30 rpm the nonbioequivalent formulation exhibited a slower dissolution rate. A slower gastric emptying rate was also observed in rats for the nonbioequivalent formulation. A slower disintegration and dissolution or a delay in gastric emptying might explain the Cmax infra-bioavailability for a highly permeable drug with short half-life. The BCS-biowaiver approach would have declared bioequivalence, although the in vivo study was not conclusive but detected a 14% mean difference in Cmax that precluded the bioequivalence demonstration. Nonetheless, these findings suggest that a slower dissolution rate is more discriminatory and that rotation speeds higher than 50 rpm should not be used in BCS-biowaivers, even if a coning effect occurs.

In vitro-in vivo correlations: general concepts, methodologies and regulatory applications.

The major objective of in vitro-in vivo correlations is to be able to use in vitro data to predict in vivo performance serving as a surrogate for an in vivo bioavailability test and to support biowaivers. Therefore, the aims of this review are: (i) to clarify the factors involved during bio-predictive dissolution method development; and (ii) the elements that may affect the mathematical analysis in order to exploit all information available. This article covers the basic aspects of dissolution media and apparatus used in the development of in vivo predictive dissolution methods, including the latest proposals in this field as well as the summary of the mathematical methods for establishing the in vitro-in vivo relationship and their scope and limitations. The incorporation of physiological relevant factors in the in vitro dissolution method is essential to get accurate in vivo predictions. Standard quality control dissolution methods do not necessarily reflect the in vivo behavior, so they rarely are useful for predicting in vivo performance. The combination of physiological based dissolution methods with physiological-based pharmacokinetics models incorporating gastrointestinal variables will lead to robust tools for drug and formulation development, nevertheless their regulatory use for biowaiver application still require harmonization of the mathematical methods proposed and more detailed recommendations about the procedures for setting up dissolution specifications.

Permeability Study of Polyphenols Derived from a Phenolic-Enriched Hibiscus sabdariffa Extract by UHPLC-ESI-UHR-Qq-TOF-MS.

Previous findings on the capacity of Hibiscus sabdariffa (HS) polyphenols to ameliorate metabolic disturbances justify the necessity of studies oriented to find the potential metabolites responsible for such an effect. The present study examined the intestinal epithelial membrane permeability of polyphenols present in a phenolic-enriched Hibiscus sabdariffa extract (PEHS), free and encapsulated, using the Caco-2 cell line. Additionally, selected polyphenols (quercetin, quercetin-3-glucoside, quercetin-3-glucuronide, and N-feruloyltyramine) were also studied in the same absorption model. The powerful analytical platform used ultra-high-performance liquid chromatography coupled with ultra-high-resolution quadrupole time-of-flight mass spectrometry (UHPLC-ESI-UHR-Qq-TOF-MS), and enabled the characterization of seven new compounds in PEHS. In the permeation study, only a few compounds were able to cross the cell monolayer and the permeability was lower when the extract was in an encapsulated form. Pure compounds showed a moderate absorption in all cases. Nevertheless, these preliminary results may need further research to understand the complete absorption mechanism of Hibiscus polyphenols.

Modified nonsink equation for permeability estimation in cell monolayers: comparison with standard methods.

Cell culture permeability experiments are valuable tools in drug development and candidate selection, but the monolayer preparation protocols and the calculations procedures can affect the permeability estimation. Hence, standardization and method suitability demonstration are necessary steps for using permeability data for regulatory and in vivo prediction purposes. Much attention is usually paid to experimental procedure validation and less to the mathematical analysis of the results although the standard equations used imply several assumptions that many times do not hold. The aim of this study was to use a simulation strategy to explore the performance of a new proposed modified nonsink equation (MNS) for unidirectional apparent permeability estimation in different types of profiles (of cumulative drug amounts versus time) including those in which the initial permeation rate is altered, considering several levels of experimental variability. The second objective was to compare the MNS method with the classical sink and nonsink approaches and finally to explore its usefulness for BCS classification. Real data from permeability experiments representing atypical profiles have been used for fitting with the three approaches, MNS, sink, and nonsink equations, in order to validate the performance of the new proposed model. The results demonstrated that the MNS method is a precise and accurate equation for calculating the apparent unidirectional permeability in any type of profile and different scenarios of variability, in any sink and nonsink conditions, while the standard nonsink equation fails in obtaining good permeability estimations in those situations in which the initial permeation rate is altered. Linear regression models (S and SC) are not valid under nonsink conditions, as expected, as the underlying assumptions (sink conditions) do not hold, but also in situations in which sink conditions are fulfilled but the system variability is high.

In Vivo Predictive Dissolution and Biopharmaceutic-Based In Silico Model to Explain Bioequivalence Results of Valsartan, a Biopharmaceutics Classification System Class IV Drug.

The purpose of this study was to predict the in vivo bioequivalence (BE) outcome of valsartan (VALS, BCS class IV) from three oral-fixed combination products with hydrochlorothiazide (HCTZ, BCS class III) (Co-Diovan® Forte as reference and two generic formulations in development) by conducting in vivo predictive dissolution with a gastrointestinal simulator (GIS) and a physiologically based biopharmaceutic model (PBBM). In the first BE study, the HCTZ failed, but the VALS 90% CI of Cmax and the AUC were within the acceptance limits, while, in the second BE study, the HCTZ 90% CI of Cmax and the AUC were within the acceptance limits, but the VALS failed. As both drugs belong to different BCS classes, their limiting factors for absorption are different. On the other hand, the gastrointestinal variables affected by the formulation excipients have a distinct impact on their in vivo exposures. Dissolution tests of the three products were performed in a GIS, and a PBBM was constructed for VALS by incorporating in the mathematical model of the in vitro-in vivo correlation (IVIVC) the gastrointestinal variables affected by the excipients, namely, VALS permeability and GI transit time. VALS permeability in presence of the formulation excipients was characterized using the in situ perfusion method in rats, and the impact of the excipients on the GI transit times was estimated from the HCTZ's in vivo results. The model was able to fit the in vivo BE results with a good prediction error. This study contributes to the field by showing the usefulness of PBBM in establishing in vitro-in vivo relationships incorporating not only dissolution data but also other gastrointestinal critical variables that affect drug exposure in BCS class IV compounds.