Optimization of Self-Double Emulsifying Drug Delivery System Using Design of Experiments for Enhanced Oral Bioavailability of Gentamicin: In-vitro, Ex-vivo and In-vivo Studies.

Water-in-oil-in-water (w/o/w) double emulsions have shown excellent capability in augmenting the enteral bioavailability of BCS class III drugs, besides being effective controlled-release formulations. However, the problem of thermodynamic instability has restrained their industrial applicability. The self-double emulsifying drug delivery system (SDEDDS) is one of several approaches used to improve the stability of double emulsions. SDEDDS is a mixture of primary emulsion and secondary surfactant that can spontaneously emulsify into double emulsions in an external aqueous environment with mild agitation. Here, we prepared SDEDDS of gentamicin sulfate by response surface methodology. Selected optimized formulations (ODS1 and ODS2) were evaluated for zeta potential (Y1), optical clarity (Y2), release at 420 min (Y3), emulsion stability index (Y4) and self-emulsification time (Y5). For ODS1, Y1=-35.45 (±1.06)mV, Y2=53.19 (±0.35)%, Y3=75.79 (±0.60)%, Y4=93.97(±0.15)% and Y5=0.631 (±0.014)min, whereas for ODS2, Y1=-35.70 (±0.56)mV, Y2=48.09 (±0.64)%, Y3=76.61 (±0.99)%, Y4=93.00(±0.94)% and Y5=0.687(±0.02)min. Furthermore, ex-vivo studies on intestinal permeability revealed that SDEDDS improved membrane permeability compared to drug solution. Histopathology investigations revealed that SDEDDS promoted permeation without causing significant local membrane distortion. In addition, in-vivo studies revealed a 2.84 -fold increase in AUC0-∞ of optimized SDEDD compared to pure drug oral solution.

Serum Stabilities and Antiviral Activities of Chemically Modified Peptides Against Dengue Serotypes 1-4.

Dengue presents a major public health concern in over 100 countries due to the absence of an effective vaccine and antiviral therapy against all four dengue virus (DENV) serotypes. Several antiviral peptides were previously reported to inhibit at least three or all four DENV serotypes. Chemical modifications such as d-amino acid substitutions, polyethylene glycol (PEG)ylation, and cyclization could be applied to peptides to improve their biological activities and stability in serum. The PEGylated peptide 3 (PEG-P3) was identified to be the most promising antiviral candidate as it demonstrated good inhibitory effects against all four DENV serotypes during the pre- and post-infection stages, Based on the RP-HPLC and LC/MS analysis, peptide 4 was identified to be more stable in human serum than peptide 3, with 78.9 % and 41.6 % of the peptides remaining after 72 h of incubation in human serum, respectively. Both peptides were also able to retain their antiviral activities against specific DENV serotypes after 72 h incubation in human serum. PEG-P3 was found to be more stable than the unmodified peptide 3 with 89.4 % of PEG-P3 remaining in the human serum after 72 h of incubation. PEG-P3 was able to retain its inhibitory effects against DENV-1 to 4 after 72 h of incubation in human serum. This study provided insights into the antiviral activities and stabilities of the unmodified and chemically modified peptides in human serum.

In-vitro dynamic dissolution/bioconversion/permeation of fosamprenavir using a novel tool with an artificial biomimetic permeation barrier and microdialysis-sampling.

Fosamprenavir is a phosphate ester prodrug that, upon dissolution, is cleaved to the poorly soluble yet readily absorbable parent drug amprenavir. In this study, a novel cell-free in vitro setup with quasi-continuous monitoring of the dynamic dissolution/bio-conversion/permeation of fosamprenavir was designed and tested. It consists of side-by-side diffusion cells, where the donor and acceptor compartments are separated by the biomimetic barrier PermeaPad®, and sampling from the donor compartment is accomplished via a microdialysis probe. Externally added bovine alkaline phosphatase induced bioconversion in the donor compartment. Microdialysis sampling allowed to follow the enzymatic conversion of fosamprenavir to amprenavir by the bovine alkaline phosphatase in an (almost) real-time manner eliminating the need to remove or inactivate the enzyme. Biomimetic conversion rates in the setup were established by adding appropriate amounts of the alkaline phosphatase. A substantial (6.5-fold) and persistent supersaturation of amprenavir was observed due to bioconversion at lower (500 µM) concentrations, resulting in a substantially increased flux across the biomimetic barrier, nicely reflecting the situation in vivo. At conditions with an almost 10-fold higher dose than the usual human dose, some replicates showed premature precipitation and collapse of supersaturation, while others did not. In conclusion, the proposed novel tool appears very promising in gaining an in-depth mechanistic understanding of the bioconversion/permeation interplay, including transient supersaturation of phosphate-ester prodrugs like fosamprenavir.

Evaluation of a Five-Probe Metabolic Control Cocktail in Long-Term Cocultured Human Hepatocytes.

Hepatocyte cocultures like HepatoPac have become more frequently used for the assessment of the intrinsic clearance of slowly metabolised drugs during drug discovery due to a superiority in enzymatic activity over time compared to liver microsomal fractions and suspended primary hepatocytes. However, the relatively high cost and practical limitations prevent several quality control compounds to be included in studies and the activities of many important metabolic enzymes are consequently often not monitored. In this study, we have evaluated the possibility for a cocktail approach of quality control compounds in the human HepatoPac system to ensure adequate activity of the major metabolising enzymes. Five reference compounds were selected based on their known metabolic substrate profile in order to capture major CYP and non-CYP metabolic pathways in the incubation cocktail. The intrinsic clearance of the reference compounds when incubated as singlets or in a cocktail was compared and no considerable difference was observed. We show here that a cocktail approach of quality control compounds allows for easy and efficient evaluation of the metabolic competency of the hepatic coculture system over an extended incubation period.

Inexpensive and Easy-To-Use Alternative to the Die Holder for Pharmacopoeial Intrinsic Dissolution Tests.

The pharmacopoeial test method "Intrinsic Dissolution" (Ph.Eur. 2.9.29) is used to study the rate of dissolution for powders of active pharmaceutical ingredients normalized by the surface area. Therefore, powders are compacted into a special metal die holder, which is immersed into a dissolution vessel of the dissolution test apparatus (described in Ph.Eur. 2.9.3). However, in some cases, the test cannot be performed because the compacted powder would not stay in the die holder when in contact with the dissolution medium. In this study, we investigated the removable adhesive gum (RAG) as an alternative to the official die holder. Intrinsic dissolution tests were carried out to exemplify the use of the RAG for this purpose. As model substances, acyclovir and its co-crystal with glutaric acid were used. The RAG was validated for compatibility, release of extractables, unspecific adsorption and the ability to block drug release through the covered surfaces. The results showed that the RAG leaked no unwanted substances, showed no adsorption of acyclovir and blocked its release from covered surfaces. The intrinsic dissolution tests revealed, as expected, a constant release of drug with a small standard deviation between replicates. It was possible to distinguish the acyclovir release from the co-crystal and from the pure drug compound. In conclusion, the findings of this study suggest to consider removable adhesive gum as an easy-to-use and inexpensive alternative to the compendial die holder in intrinsic dissolution tests.

When Interactions Between Bile Salts and Cyclodextrin Cause a Negative Food Effect: Dynamic Dissolution/Permeation Studies with Itraconazole (Sporanox®) and Biomimetic Media.

The marketed oral solution of itraconazole (Sporanox®) contains 40% (259.2 mM) of 2-hydroxypropyl-ß-cyclodextrin (HP-ß-CD). The obvious role of HP-ß-CD is to solubilize itraconazole and to overcome its poor aqueous solubility that restricts its absorption. In this study, we investigated the biorelevance of in vitro experiments by the influence of biomimetic media (containing bile salts and phospholipids) on the predicted itraconazole absorption from the commercial HP-ß-CD-based Sporanox® solution. We performed phase-solubility studies of itraconazole and dynamic 2-step-dissolution/permeation studies using a biomimetic artificial barrier, Sporanox® solution, and fasted state simulated intestinal fluid (FaSSIF_V1). Both FaSSIF_V1 and HP-ß-CD increased the apparent solubility of itraconazole when used individually. In combination, their solubility-enhancing effects were not additive probably due to the competition of bile salts with itraconazole for the hydrophobic cavity of HP-ß-CD. Our combined dissolution/permeation experiments indicated the occurrence of a transient supersaturation from Sporanox® upon two-step dissolution. Through systematic variation of bile salt concentrations in the biomimetic media, it was observed that the extent and the duration of supersaturation depend on the concentrations of bile salts: supersaturation was rather stable in the absence of bile salts and phospholipids. The higher the bile salt concentration, the faster the collapse of the transient supersaturation occurred, an effect which is nicely mirrored by reduced in vitro permeation across the barrier. This is an indication of a negative food effect, which in fact correlates well with what earlier had been observed in clinical studies for Sporanox® solution. In essence, we could demonstrate that in vitro two-stage dissolution/permeation experiments using an artificial barrier and selected biomimetic media may predict the negative effects of the latter on cyclodextrin-based drug formulations like Sporanox® Oral Solution and, at the same time, provide a deeper mechanistic insight.

Development of In Vitro Dissolution Testing Methods to Simulate Fed Conditions for Immediate Release Solid Oral Dosage Forms.

In vitro dissolution testing is widely used to mimic and predict in vivo performance of oral drug products in the gastrointestinal (GI) tract. This literature review assesses the current in vitro dissolution methodologies being employed to simulate and predict in vivo drug dissolution under fasted and fed conditions, with emphasis on immediate release (IR) solid oral dosage forms. Notable human GI physiological conditions under fasted and fed states have been reviewed and summarized. Literature results showed that dissolution media, mechanical forces, and transit times are key dissolution test parameters for simulating specific postprandial conditions. A number of biorelevant systems, including the fed stomach model (FSM), GastroDuo device, dynamic gastric model (DGM), simulated gastrointestinal tract models (TIM), and the human gastric simulator (HGS), have been developed to mimic the postprandial state of the stomach. While these models have assisted in expanding physiological relevance of in vitro dissolution tests, in general, these models lack the ability to fully replicate physiological conditions/processes. Furthermore, the translatability of in vitro data to an in vivo system remains challenging. Additionally, physiologically based pharmacokinetic (PBPK) modeling has been employed to evaluate the effect of food on drug bioavailability and bioequivalence. Here, we assess the current status of in vitro dissolution methodologies and absorption PBPK modeling approaches to identify knowledge gaps and facilitate further development of in vitro dissolution methods that factor in fasted and fed states. Prediction of in vivo drug performance under fasted and fed conditions via in vitro dissolution testing and modeling may potentially help efforts in harmonizing global regulatory recommendations regarding in vivo fasted and fed bioequivalence studies for solid oral IR products.

Comparison of Cellular Monolayers and an Artificial Membrane as Absorptive Membranes in the in vitro Lipolysis-permeation Assay.

Permeation across Caco-2 cells in lipolysis-permeation setups can predict the rank order of in vivo drug exposure obtained with lipid-based formulations (LBFs). However, Caco-2 cells require a long differentiation period and do not capture all characteristics of the human small intestine. We therefore evaluated two in vitro assays with artificial lecithin-in-dodecane (LiDo) membranes and MDCK cells as absorptive membranes in the lipolysis-permeation setup. Fenofibrate-loaded LBFs were used and the results from the two assays compared to literature plasma concentrations in landrace pigs administered orally with the same formulations. Aqueous drug concentrations, supersaturation, and precipitation were determined in the digestion chamber and drug permeation in the receiver chamber. Auxiliary in vitro parameters were assessed, such as permeation of the taurocholate, present in the simulated intestinal fluid used in the assay, and size of colloidal structures in the digestion medium over time. The LiDo membrane gave a similar drug distribution as the Caco-2 cells and accurately reproduced the equivalent rank-order of fenofibrate exposure in plasma. Permeation of fenofibrate across MDCK monolayers did not, however, reflect the in vivo exposure rankings. Taurocholate flux was negligible through either membrane. This process was therefore not considered to significantly affect the in vitro distribution of fenofibrate. We conclude that the artificial LiDo membrane is a promising tool for lipolysis-permeation assays to evaluate LBF performance.

Biorelevant in vitro Tools and in silico Modeling to Assess pH-Dependent Drug-drug Interactions for Salts of Weak Acids: Case Example Potassium Raltegravir.

BACKGROUND: Early assessment of pH-dependent drug-drug-interactions (DDIs) for salts of poorly soluble weakly acidic compounds offers various advantages for patient safety, the pharmaceutical industry, and regulatory bodies. Biorelevant media and tests reflecting physiological changes during acid-reducing agent (ARA) co-administration can be used to explore and predict the extent of the pH effect during therapy with ARAs. METHODS: Solubility, one-stage and two-stage dissolution of tablets containing potassium raltegravir, the marketed salt form of this poorly soluble, weakly acidic drug, was investigated using biorelevant media specially designed to reflect administration without and during ARA co-therapy. The dissolution data were then converted into parameters suitable for input into an in silico model (Simcyp™) and the simulated plasma profiles were compared with available pharmacokinetic (PK) data from the literature. RESULTS: Dissolution of the potassium raltegravir formulation in media reflecting ARA co-administration, and thus elevated gastric pH, was faster and more complete than in experiments reflecting the low gastric pH observed in the absence of ARA co-administration. Simulations using data from dissolution experiments with ARA media appropriately bracketed the in vivo data for ARA co-administration in healthy volunteers. CONCLUSION: Dissolution data from in vitro experiments in biorelevant media reflecting physiological changes due to ARA co-administration provide valuable information about potassium raltegravir's behavior during concomitant ARA therapy. The approach may also be suitable for salts forms of other poorly soluble, weakly acidic drugs.

Analytical Challenges Assessing Protein Aggregation and Fragmentation Under Physiologic Conditions.

Therapeutic proteins are administered by injection or infusion. After administration, the physiologic environment in the desired body compartment - fluid or tissue - can impact protein stability and lead to changes in the safety and/or efficacy profile. For example, protein aggregation and fragmentation are critical quality attributes of the drug product and can occur after administration to patients. In this context, the in vivo stability of therapeutic proteins has gained increasing attention. However, in vivo protein aggregation and fragmentation are difficult to assess and have been rarely investigated. This mini-review summarizes analytical approaches to assess the stability of therapeutic proteins using simulated physiologic conditions. Furthermore, we discuss factors potentially causing in vivo protein aggregation, precipitation, and fragmentation in complex biological fluids. Different analytical approaches are evaluated with respect to their applicability and possible shortcomings when it comes to these degradation events in biological fluids. Tracking protein stability in biological fluids typically requires purifying or labeling the protein of interest to circumvent matrix interference of biological fluids. Improved analytical methods are strongly needed to gain knowledge on in vivo protein aggregation and fragmentation. In vitro models can support the selection of lead candidates and accelerate the pre-clinical development of therapeutic proteins.

Primary Human Hepatocyte Spheroid Model as a 3D In Vitro Platform for Metabolism Studies.

3D cultures of primary human hepatocytes (PHH) are emerging as a more in vivo-like culture system than previously available hepatic models. This work describes the characterisation of drug metabolism in 3D PHH spheroids. Spheroids were formed from three different donors of PHH and the expression and activities of important cytochrome P450 enzymes (CYP1A2, 2B6, 2C9, 2D6, and 3A4) were maintained for up to 21 days after seeding. The activity of CYP2B6 and 3A4 decreased, while the activity of CYP2C9 and 2D6 increased over time (P < 0.05). For six test compounds, that are metabolised by multiple enzymes, intrinsic clearance (CLint) values were comparable to standard in vitro hepatic models and successfully predicted in vivo CLint within 3-fold from observed values for low clearance compounds. Remarkably, the metabolic turnover of these low clearance compounds was reproducibly measured using only 1-3 spheroids, each composed of 2000 cells. Importantly, metabolites identified in the spheroid cultures reproduced the major metabolites observed in vivo, both primary and secondary metabolites were captured. In summary, the 3D PHH spheroid model shows promise to be used in drug discovery projects to study drug metabolism, including unknown mechanisms, over an extended period of time.

Proteomics-Informed Identification of Luminal Targets For In Situ Diagnosis of Inflammatory Bowel Disease.

Inflammatory bowel disease (IBD) is a chronic condition resulting in impaired intestinal homeostasis. Current practices for diagnosis of IBD are challenged by invasive, demanding procedures. We hypothesized that proteomics analysis could provide a powerful tool for identifying clinical biomarkers for non-invasive IBD diagnosis. Here, the global intestinal proteomes from commonly used in vitro and in vivo models of IBD were analyzed to identify apical and luminal proteins that can be targeted by orally delivered diagnostic agents. Global proteomics analysis revealed upregulated plasma membrane proteins in intestinal segments of proximal- and distal colon from dextran sulfate sodium-treated mice and also in inflamed human intestinal Caco-2 cells pretreated with pro-inflammatory agents. The upregulated colon proteins in mice were compared to the proteome of the healthy ileum, to ensure targeting of diagnostic agents to the inflamed colon. Promising target proteins for future investigations of non-invasive diagnosis of IBD were found in both systems and included Tgm2/TGM2, Icam1/ICAM1, Ceacam1/CEACAM1, and Anxa1/ANXA1. Ultimately, these findings will guide the selection of appropriate antibodies for surface functionalization of imaging agents aimed to target inflammatory biomarkers in situ.

Nifedipine Release From Extended-Release Solid Oral Formulations Using In Vitro Dissolution Testing Under Simulated Gastrointestinal Compression.

Drug release plays a critical role in defining bioavailability for an extended release solid oral drug products and predictive dissolution tests are desired to establish clinically relevant quality standards for batch release. The objective of this study focuses on exploring the possible impacts of 1 gastrointestinal (GI) parameter for 1 drug: simulated GI contractions on nifedipine release (in 2 extended release solid oral formulations). The 60 mg nifedipine osmotic pump product A, and polymer matrix-based products B and C were examined in the study. An in-house dissolution system was used to simulate various levels of GI contractions on tested samples, and to monitor changes of sample mechanical properties during dissolution testing. The results show that the polymer matrix-based formulation failed to provide controlled release when simulated GI contraction was above 100 g of force. The method may be useful for polymer matrix-based products to assess potential formulation-related interactions with the GI tract during in vivo drug dissolution.

Utilising Magnetically Isolated Lysosomes for Direct Quantification of Intralysosomal Drug Concentrations by LC-MS/MS Analysis: An Investigatory Study With Imipramine.

Lysosomes are acidic intracellular organelles that can extensively sequester basic lipophilic drugs due to pH and membrane partitioning, and therefore may significantly influence subcellular drug concentrations. Current in vitro methods for lysosomal drug sequestration evaluation typically lack the ability to accurately and sensitively quantify drug concentrations directly within the lysosome. In the current study, magnetic lysosomal isolation was used in the lysosome rich rat NR8383 cell line and combined with LC-MS/MS analysis to quantify intralysosomal concentrations and lysosomal partitioning (KpLysosome) values of imipramine. The purity of the isolated lysosomes was validated by enzymatic and electron microscopy analysis. Lysosomal imipramine accumulation was explored using 2 methods: addition of imipramine to cells followed by lysosomal isolation (Method 1), and direct addition of imipramine to isolated lysosomes (Method 2). This work highlighted that both experimental buffers and ATP influence intralysosomal drug concentrations, and non-specific drug binding and re-distribution limits the use of Method 1. Method 2 may benefit future lysosomal drug accumulation studies, as imipramine demonstrated high KpLysosome values (3500), comparable to in silico predictions. This study reports a novel method for the direct quantification of intralysosomal drug concentrations that has the ability to be adapted to other cell types.

Growth Hormone Aggregates Activation of Human Dendritic Cells Is Controlled by Rac1 and PI3 Kinase Signaling Pathways.

The presence of protein aggregates in biological products is suggested to promote immunogenicity, leading to the production of anti-drug antibodies with neutralizing capacities. This suggests a CD4+ T-cell dependent adaptive immune response, thus a pivotal role for antigen-presenting cells, such as dendritic cells (DCs). We previously showed that human growth hormone aggregates induced DC maturation, with notably an increase in CXCL10 production. DC phenotypic modifications were sufficient to promote allogeneic CD4+ T-cell proliferation with Th1 polarization. In this work, we identified the main intracellular signaling pathways involved in DC activation by human growth hormone aggregates, showing that aggregates induced p38 mitogen-activated protein kinase, extracellular signal-regulated kinase, and c-Jun N-terminal kinase phosphorylation, as well as nuclear factor κB subunit p65 nuclear translocation. Next, investigating the implication of Rho GTPases and phosphoinositide 3-kinase (PI3K) in activated DC showed that Rac1 and Cdc42 regulated the phosphorylation of MAP kinases, whereas PI3K was only implicated in c-Jun N-terminal kinase phosphorylation. Furthermore, we showed that Rac1 and PI3K pathways, but not Cdc42, regulated the production of CXCL10 via the MAP kinases and nuclear factor κB. Taken together, our results bring new insight on how protein aggregates could induce DC activation, leading to a better understanding of aggregates role in therapeutic proteins immunogenicity.

Development of a Pertactin-Coated Beads Approach for Screening of Functional Monoclonal Antibodies.

Vaccine manufacturers have recently focused on the development of in vitro potency assays to promote 3R's strategy to replace animal testing. To be able to develop an in vitro potency assay, the immunological characteristics of the monoclonal antibodies used in the assay should be well understood as these antibodies likely reflect the biological activity of a vaccine product. The PRN antigen is one of the immunogenic antigens included in many commercialized acellular pertussis vaccines. Development of an in vitro potency assay for PRN is challenging as the biological properties of PRN are not well understood. In addition, binding of Bordetella pertussis to human cells occurs through multiple bacterial molecules, which makes it very challenging to assess if antibodies contribute to prevention of bacterial adhesion. To overcome these challenges, the functionality of several in-house anti-PRN mAbs has been investigated through a novel approach using PRN-coated beads. We were able to consistently quantify the inhibition of PRN-mediated adhesion for each anti-PRN mAb. Application of the protein-coated beads model has not only enabled screening of functional anti-PRN mAbs but can also be expanded for screening of antibodies against other bacterial or viral antigens.

Nonlinear Method to Predict the Distribution of Structurally Diverse Compounds between Blood and Tissue.

In this work, methods for predicting the distribution of compounds between blood and tissue were investigated using nonlinear regression analysis. For the tissue/blood partition coefficient, 282 compounds and 810 activity data for seven tissues were selected. Twenty-four parameters were studied for each state of the compound. A study set with the most compounds and activity data in similar studies was established, and a model with good prediction ability was obtained. A total of 773 data points were randomly divided into two data sets: the training set contained 623 data points (n = 623, r = 0.822, s = 0.438, F = 142.2, and Q = 0.814) and the test set contained 150 data points (n = 150, r = 0.814, and s = 0.334). Furthermore, individual tissue/blood distribution coefficients were also studied in depth to obtain separate models for predicting the distribution coefficient of a drug between blood and a tissue. By applying these models, not only can the tissue/blood distribution coefficient or single tissue/blood distribution coefficient of the seven tissues and organs of the human body be predicted, but the distribution of the drug molecules in different states (neutral, cation, and anion) in the three tissue components can also be predicted.

Acoustic Droplet Ejection and Open Port Interface for Rapid Analysis of Metabolic Stability Assays.

In vitro absorption, distribution, metabolism and elimination (ADME) assays are widely used for profiling compounds in pharmaceutical drug discovery programs. Many compounds are screened in metabolic stability assays, using liver microsomes as a model of intrinsic hepatic clearance. Analysis of metabolic stability assays has relied on high throughput LC-MS/MS techniques to keep up with automated assays and compound profiling needs. An experimental alternative to sample analysis via fast chromatography employs an open port interface (OPI) which dilutes and directs acoustically-ejected droplets from microtiter plates to a conventional electrospray ion source for ionization and introduction into a mass spectrometer. Metabolic stability assays of 37 commercial drug compounds using in human, dog, rat and mouse liver microsomes (LMs), were analyzed by LC-MS/MS and an experimental breadboard version of an ADE-OPI-MS/MS system. Results from the experiments comparing intrinsic clearance (CLint) generated with ADE-OPI-MS/MS vs fast LC-MS/MS for all compounds showed ≥86% of CLint values were within a factor of two with R2 ≥ 0.86 using 25 nL and 5 nL sample ejection volumes on the ADE-OPI-MS/MS instrument. Throughput with the experimental ADE-OPI-MS/MS system used in this study was more than ten-fold faster than analysis by the fast LC-MS/MS at 1.3 s/sample versus 17.2 s/sample, respectively.

Simultaneous Analysis of Dissolution and Permeation Profiles of Nanosized and Microsized Formulations of Indomethacin Using the In Vitro Dissolution Absorption System 2.

The in vitro dissolution absorption system 2 (IDAS2), a recent invention comprised a conventional dissolution vessel containing 2 permeation chambers with Caco-2 cell monolayers mounted with their apical side facing the dissolution media, permits simultaneous measurement of dissolution and permeation of drugs from intact clinical dosage forms. The objectives of this study were (1) to assess the utility of IDAS2 in the determination of the effect of particle size on in vitro performance of indomethacin and (2) to find out whether the behavior in IDAS2 of 2 indomethacin products differing in particle size is correlated with their in vivo behavior. Indomethacin dissolution and permeation across Caco-2 cell monolayers were simultaneously measured in IDAS2; the dissolution and permeation profiles were simultaneously modeled using a simple two-compartment model. Compared to microsized indomethacin, the nanosized formulation increased the dissolution rate constant by fivefold, whereas moderately increasing the permeation rate constant and the kinetic solubility. As a result, the drug amount permeated across the Caco-2 cell monolayers doubled in the nanosized versus microsized formulation. The in vitro results showed a good correlation with in vivo human oral pharmacokinetic parameters, thus emphasizing the physiological relevance of IDAS2 data in predicting in vivo absorption.

Relative Bioavailability Risk Assessment: A Systematic Approach to Assessing In Vivo Risk Associated With CM&C-Related Changes.

Relative bioavailability (RBA) studies are often carried out to bridge changes made between drug products used for clinical studies. In this work, we describe the development of a risk assessment (RA) tool that comprehensively and objectively assesses the risk of noncomparable in vivo performance associated with Chemistry, Manufacturing, and Controls (CM&C)-related changes. The RA tool is based on a risk grid that provides a quantitative context to facilitate discussions to determine the need for an in vivo RBA study. Relevant regulatory guidances and the required in vitro and in silico absorption modeling data, on which the RA is based, are discussed. In addition, an analysis of previously executed RBA studies at Eli Lilly and Company over a period of several years is presented. The risk grid incorporates individual risk factors for a given study and provides a recommendation on the risk associated with bypassing an RBA study. The outcome of an RA results in 1 of 3 possible risk zones; lower tier risk, intermediate tier risk, and upper tier risk. In cases where the outcome from the RA falls into the intermediate tier risk zone, further in depth data analysis is required.