In Vitro–In Vivo Extrapolation Method to Predict Human Renal Clearance of Drugs

Renal clearance is a key determinant of the elimination of drugs. To date, only few in vitro–in vivo extrapolation (IVIVE) approaches have been described to predict the renal organ clearance as the net result of glomerular filtration, tubular secretion, and tubular reabsorption. In this study, we measured in LLC-PK₁ cells the transport of 20 compounds that cover all four classes of the Biopharmaceutical Drug Disposition System. These data were incorporated into a novel kidney model to predict all renal clearance processes in human. We showed that filtration and secretion were main contributors to the renal organ clearance for all compounds, whereas reabsorption was predominant for compounds assigned to classes 1 and 2. Our results suggest that anionic drugs were not significantly secreted in LLC-PK₁ cells, resulting in under-predicted clearances. When all study compounds were included a high overall correlation between the reported and predicted renal organ clearances was obtained (R² = 0.83). The prediction accuracy in terms of percentage within twofold and threefold error was 70% and 95%, respectively. In conclusion, our novel IVIVE method allowed to predict the human renal organ clearance and the contribution of each underlying process.     

Use of In Vitro�CIn Vivo Correlation to Predict the Pharmacokinetics of Several Products Containing a BCS Class 1 Drug in Extended Release Matrices

Purpose

To determine if an IVIVC model can predict PK profiles of varying formulations of a BCS Class 1 drug that is a salt of a weak base.

Method

An IVIVC model (Level A) was created by correlating deconvoluted in vivo absorption data obtained from oral administration of 50?mg, 100?mg, and 200?mg fast and slow extended release formulations with in vitro percent dissolved using residual regression analysis. The model was then used to predict the in vivo profile of five test products that varied in formulation characteristics.

Results

The model passed internal validation for predicted Cmax and AUC. For external validation, in vitro data of five different test formulations was utilized. The model passed external validation for two test formulations that were different but belonging to the same release mechanism as that of the reference formulation. Three formulations failed external validation because they belonged to either a mixed or different release mechanism. The model and results were further confirmed using GatstroPlus? simulation software.

Conclusions

These observations indicate that an IVIVC model for a BCS class I drug may be applicable to varying formulations if the principle of the drug release is similar.     

The Irrelevance of In Vitro Dissolution in Setting Product Specifications for Drugs Like Dextromethorphan That are Subject to Lysosomal Trapping

The purpose of the present study was to develop a physiologically based pharmacokinetic model for dextromethorphan (DEX) and its metabolites in extensive and poor metabolizers. The model was used to study the influence of dissolution rates on the sensitivity of maximum plasma concentration and area under the concentration-time curve for immediate release formulations. Simulation of in vitro cellular transwell permeability was used to confirm lysosomal trapping. GastroPlus? was used to build a mechanistic absorption and physiologically based pharmacokinetic model of DEX. The model simulations were conducted with and without lysosomal trapping. The simulated results matched well with observed data only when lysosomal trapping was included. The model shows that DEX is rapidly absorbed into the enterocytes, but DEX and its metabolites only appear slowly in the portal vein and plasma, presumably due to lysosomal trapping. For this class of drug, the rate of in vitro and in vivo dissolution is not a sensitive factor in determining bioequivalence. This study shows that dissolution and the rate of absorption into the enterocytes are clinically irrelevant for the performance of DEX immediate release product. An understanding of the entire underlying mechanistic processes of drug disposition is needed to define clinically relevant product specifications for DEX.     

The Effect of Drug Content Reduction on the In Vitro and In Vivo Properties of Levonorgestrel-Releasing Intravaginal Rings

Intravaginal rings (IVRs) are an option for continuous administration of drugs in women. However, a considerable amount of excess drug often remains in the ring upon removal. The current study focuses on comparing 2 IVRs releasing levonorgestrel (LNG). Both formulations were designed to release 40 μg of LNG daily, however, with a significant difference in the total amount of drug (10.6 vs. 176.9 mg). Numerical simulations and in vitro release rate testing were utilized in designing the IVRs and confirming the similarity of drug release. Moreover, a pharmacokinetic (PK) study was performed in 13 healthy Japanese women to investigate both formulations during the intended wearing period of 28 days. The primary PK metrics was the average concentration of LNG in plasma at defined time points under stable conditions. Statistical evaluation of the ratio of the main PK metrics indicated values almost in the bioequivalence range. Furthermore, drug content determinations for used and unused IVRs were analyzed for confirming the expected drug delivery in vivo. In summary, it was shown that with proper design, even major differences in the total drug content of IVR formulations might not result in significant effects in the in vitro and in vivo release properties.     

Dissolution Methods to Increasing Discriminatory Power of In Vitro Dissolution Testing for Ibuprofen Free Acid and Its Salts

The predictive capacity of in vitro dissolution tests using the Biopharmaceutics Classification System (BCS)–based experimental setup to anticipate in vivo bioequivalence outcomes for BCS class 2 weak acids has been questioned. In this work, the effect of buffer concentration media was investigated as a possible approach to ensuring the discriminative capacity of the in vitro dissolution methods. The case example used to test this approach was ibuprofen, formulated as either the free acid or in various salt forms. By matching the concentration of buffers commonly used to prepare media which aim to simulate the intestinal conditions with that of bicarbonate buffer, which is the predominant buffer species in vivo, to arrive at the same surface pH (pH₀), the discriminative power of the in vitro dissolution tests was improved. To simulate the in vivo results even better, a pretreatment at acidic pH was added to the dissolution test simulating the gastric conditions to create a 2-stage test. With the 2-stage test, it was possible to account for differences in disintegration in a more physiologically relevant way and thus to better reflect the in vivo performance of the various formulations.     

In Vitro Assessment of Supersaturation/Precipitation and Biological Membrane Permeation of Poorly Water-Soluble Drugs: A Case Study With Albendazole and Ketoconazole

This study aimed to elucidate the relationship between supersaturation and precipitation and the effect of a supersaturated state on drug membrane permeation. Stock solutions of albendazole (ALB) and ketoconazole (KTZ) dissolved in dimethyl sulfoxide (0.1-50 mg/mL) were diluted 100-fold with buffer solution (pH 6.8, 37°C). In the case of ALB, a supersaturated state and immediate precipitation were observed at 10 μg/mL or less and 20 μg/mL or higher, respectively. When KTZ was used, at an initial concentration of 200 μg/mL or higher, precipitation was observed, although the dissolved concentration remained at approximately 120 μg/mL for at least 30 min. These dissolved concentrations of ALB and KTZ related to approximately 10-fold and 14-fold over the saturated solubility from respective bulk powder. An in vitro permeation study implied that the rate of drug permeation across a biological membrane increased with increasing supersaturation. These results suggested favorable strategies for development of a supersaturable formulation could depend on the precipitation properties of the drug. Immediate- and controlled-release forms might be suitable for supersaturable formulations for KTZ and ALB, respectively.     

Formulation Development,Optimization, and In Vitro–In Vivo Characterization of Natamycin-Loaded PEGylated Nano-Lipid Carriers for Ocular Applications

The present study aimed at formulating and optimizing natamycin (NT)-loaded polyethylene glycosylated nano-lipid carriers (NT-PEG-NLCs) using Box-Behnken design and investigating their potential in ocular applications. Response surface methodology computations and plots for optimization were performed using Design-Expert^® software to obtain optimum values for response variables based on the criteria of desirability. Optimized NT-PEG-NLCs had predicted values for the dependent variables which are not significantly different from the experimental values. NT-PEG-NLCs were characterized for their physicochemical parameters; NT's rate of permeation and flux across rabbit cornea was evaluated, in vitro, and ocular tissue distribution was assessed in rabbits, in vivo. NT-PEG-NLCs were found to have optimum particle size (<300 nm), narrow polydispersity index, and high NT entrapment and NT content. In vitro transcorneal permeability and flux of NT from NT-PEG-NLCs was significantly higher than that of Natacyn^®. NT-PEG-NLC (0.3%) showed improved delivery of NT across the intact cornea and provided concentrations statistically similar to the marketed suspension (5%) in inner ocular tissues, in vivo, indicating that it could be a potential alternative to the conventional suspension during the course of fungal keratitis therapy.     

Application of an In Vitro Dissolution/Permeation System to Early Screening of Oral Formulations of Poorly Soluble,Weakly Basic Drugs Containing an Acidic pH-Modifier

This study aimed to evaluate the usefulness of the dissolution/permeation system (D/P system) as an in vitro tool for early screening of oral formulations of weakly basic drugs containing an acidic pH-modifier. Dipyridamole, having a prominent pH-dependent solubility, was used as a model drug, and various granules containing different amounts of fumaric acid were prepared. Prepared granules were administered orally to hypochlorhydria model rats. It was confirmed that fumaric acid improved the absorption of dipyridamole depending on its amount in the granules. Separately, dissolution and permeation of dipyridamole were observed in vitro in the D/P system. When using a medium with a low buffer capacity which mimicked the human intestinal fluid, rank order of the permeated amount of dipyridamole from various granules in the D/P system did not correlate with its absorption in hypochlorhydric rats. In contrast, when applying a medium with high buffer capacity, the permeated amount in the D/P system well reflected the effects of fumaric acid on the in vivo absorption of dipyridamole. In conclusion, by setting appropriate experimental protocols according to the properties of test compounds and formulations, D/P system can be a potent in vitro tool to predict in vivo performance of oral formulations.     

InVitro,Ex Vivo,and In Vivo Evaluation of a Dual pH/Redox Responsive Nanoliposomal Sludge for Transdermal Drug Delivery

A dual pH/redox responsive copper-glyglycine-prednisolone succinate–loaded nanoliposomal (NL) sludge was successfully synthesized and optimized using a Box-Behnken design of experiments. Preformulation design variables indicated that relative ratios of phospholipids, considerably influences NL size, thus altering the degree of drug loading in the formulation. In vitro evaluation further confirmed optimum release kinetics of the NL sludge, corresponding closely to ex vivo permeation studies, demonstrating effective transdermal delivery of prednisone succinate (PS) through a pig skin model, which closely resembles human skin anatomy. The pH/redox stimuli responsiveness of the NL sludge further demonstrated superior properties in vivo using a Sprague-Dawley rat model. The NL sludge displayed the greatest release of PS within 24 h of evaluation, falling within the acceptable therapeutic range of PS dose efficiency. In vivo results further displayed the greatest absorption of PS under inflammatory induced conditions, thus confirming the unique pH/redox responsive properties of the NL sludge. It was thus confirmed that the copper-glyglycine-prednisolone succinate–loaded NL sludge has significant potential for application in chronic inflammatory conditions such as tumor necrosis factor receptor–associated periodic syndrome (TRAPS), designed to release an effective dose of corticosteroid, as a transdermal drug delivery formulation, for effective therapeutic efficacy.     

Erratum to: Characterization of Pharmacokinetics in the Göttingen Minipig with Reference Human Drugs: An In Vitro and In Vivo Approach

Pharmaceutical Research -     

Extrapolation of the Hepatic Clearance of Drugs in the Absence of Albumin In Vitro to That in the Presence of Albumin In Vivo: Comparative Assessement of 2 Extrapolation Models Based on the Albumin-Mediated Hepatic Uptake Theory and Limitations and Mechanistic Insights

The extrapolation of hepatic clearance (CL) from data determined in an in vitro assay in the absence of albumin (ALB) to that in the presence of ALB in liver in vivo was often inaccurate using traditional in vitro-to-in vivo extrapolation (IVIVE) methods for drugs binding to the ALB. It is recognized that considering an ALB-facilitated hepatic uptake phenomenon in the IVIVE can improve the extrapolation. Therefore, the present study provides a comparison of 2 existing models that account for the ALB-facilitated hepatic uptake phenomenon in the IVIVE of CL. These models assume an interaction of the ALB-bound drug complex with the hepatocyte membrane that enhanced the dissociation of the drug from ALB to result in increased unbound intracellular drug levels available for metabolism or transporter-mediated elimination. One model refers to the old facilitated-dissociation model (FDM), which is based on a binding isotherm and necessitates knowing the specific input parameters of the interaction (i.e., relative capacity of the interaction, dissociation constant, number of binding sites, and ALB concentration). The other model is based on the same theory but is recent and more speculative although it presumes that each interaction between the ALB-drug complex and the hepatocyte surface would at all times enhance and deliver the dissociated bound drug moiety into the hepatocytes and therefore, has the advantage to use less binding information. Consequently, this second model simply consists of adjusting the unbound fraction determined in plasma in vitro of each drug (fu_p-adjusted) with the real differential of ALB concentration between the plasma and liver in vivo to estimate the corresponding differential of ALB-drug complex also assumed available to deliver the unbound drug moiety for hepatic uptake in vivo versus in vitro. Application of these 2 models (FDM and fu_p-adjusted) significantly improved the IVIVEs of CL of drugs, and hence, the next step was to compare these 2 models with the same data set. Recently published data on the hepatic uptake of 2 organic anions, namely 1-anilino-8-naphthalene sulfonate and pitavastatin, provide all binding information. As expected, the results indicate that these 2 models are conceptually and mathematically equivalent as well as they successfully predicted the experimentally determined ratios of the unbound intrinsic CL (CL_int) in the presence of ALB in vivo to that in the absence of ALB in vitro. However, the 2 models were equivalent particularly for pitavastatin because its ALB-drug complex showed a relevant capacity of interaction and dissociation with the hepatocyte membrane. Conversely, for 1-anilino-8-naphthalene sulfonate, the model of fu_p-adjusted overestimated the ratio of unbound CL_int by contrast to the FDM model because its ALB-drug complex demonstrated a significantly lower capacity of interaction with the membrane. The rational is simply because the model of fu_p-adjusted presumably assumed an important facilitated-uptake phenomenon for each drug, whereas the FDM model was derived from binding data specific to each drug. Overall, these 2 models are complementary, and all contribute toward achieving the same objective of quantifying the ALB-facilitated uptake phenomenon; however, the FDM model is more specific, but its application necessitates collecting more binding data compared with the model of fu_p-adjusted that can be used prospectively to predict the maximal effect of the facilitated-hepatic uptake in IVIVE.     

Gastrointestinal Responsive Polymeric Nanoparticles for Oral Delivery of Insulin: Optimized Preparation,Characterization, and In Vivo Evaluation

Gastrointestinal responsive polymeric nanospheres (NPs) based on hydroxypropyl methylcellulose phthalate were prepared using spontaneous emulsification solvent diffusion method for improved oral administration of insulin. The NPs prepared under optimized conditions have an encapsulation efficiency of 90% and a particle size of about 200 nm. In vitro drug release experiments demonstrated that the NPs exhibited a gradient release profile of loaded drug when the pH value gradually increased from 3.0 to 7.4. Enzyme resistance experiments showed that under simulated gastrointestinal conditions, the NPs protected more than 60% of the drug from being degraded by trypsin. The oral hypoglycemic experiments revealed that insulin-loaded NPs could significantly reduce blood glucose levels in diabetic rats with a relative bioavailability of 8.6%. Ex vivo imaging investigation of rat tissues showed that the drug-loaded NPs could promote the absorption of insulin in the ileum and colon. The work described here suggests that the gastrointestinal responsive polymeric NPs may be promising candidates for improving gastrointestinal tract delivery of hydrophilic biomacromolecules. Accordingly, the results indicated that hydroxypropyl methylcellulose phthalate NPs with gastrointestinal stimuli responsiveness could be a promising candidate for oral insulin delivery.     

The Effects of Hexetidine (Oraldene™) on the Adherence of Candida Albicans to Human Buccal Epithelial Cells In Vitro and Ex Vivo and on In Vitro Morphogenesis

Purpose. This study reports the effects of hexetidine (Oraldene) on two virulence attributes of Candida albicans, namely,in vitro and ex vivoadherence of yeast cells to buccal epithelial cells (EEC) and in vitro morphogenesis. Methods. The effects of hexetidine treatment of either yeast cells (stationary and exponential phases) or BEC on Candidal adherence, in terms of viable and non-viable adherent yeast cells, were evaluated using an acridine orange stain in conjunction with fluorescence microscopy. Ex vivoanti-adherence effects were determined by rinsing BEC in vivo with hexetidine (0.1%), removal of BEC after defined periods and inclusion in the adherence assay. The effects of hexetidine on morphogenesis were evaluated using light microscopy. Yeast cell viability following exposure to a range of concentration of hexetidine (0.005-0.1 % v/v) for defined periods was determined following serial dilution and enumeration on solid media. Results. Treatment of stationary and exponential phase yeast cells or BEC with hexetidine (0.1%) for a range of times (10–300 s) or, alternatively, with a range of concentrations of hexetidine (0.005–0.1 %) for a fixed time (30s) significantly decreased the resultant Candidal/ epithelial adhesion. No correlations were observed between reduced adherence and either time of treatment or hexetidine concentration. In vivotreatment of BEC with hexetidine (0.1%) for 30s resulted in prolonged and significant reductions in the ex vivo adherence of both viable and non-viable yeast cells for periods of up to (and including) four hours post-rinsing. Treatment of C. albicans blastospores with hexetidine (0.05, 0.1% v/v) for 10s and 30s totally inhibited Candida morphogenesis, whereas treatment with lower antiseptic concentrations significantly reduced the extent of Candida morphogenesis and the rate of hyphal development. The effects of hexetidine on yeast cell viability were both concentration and time-dependent. Conclusions. The reduced adherence of C. albicans to BEC and the modification or inhibition of morphogenesis following exposure to hexetidine suggests a clinical role for hexetidine in the prophylaxis of both superficial candidosis and the systemic complications resulting from invasion of sub-epithelial tissue.     

Proliferation,Metabolic Activity,and Adipogenic Differentiation of Human Preadipocytes Exposed to 2 Surfactants In Vitro

Fat grafting is a pivotal technique for tissue repair. Adipose stromal cells, including preadipocytes, play a major role in the regenerative effects attributed to fat grafting. But the benefits are impaired by the low survival of the graft due to mechanical stress during harvesting, hypoxia, and nutrient deprivation. Nonionic surfactant molecules demonstrated their efficacy in preventing and repairing mechanical damage on the cellular membrane, but it is poorly understood if and how they affect cellular viability, proliferation, and differentiation. We investigated the influence of 2 nonionic surfactants, Kolliphor^®P188 and Kolliphor^®EL, on cultured human preadipocytes. We analyzed their effects on metabolic activity, cell number, adipogenic differentiation, and secretion of growth factors. Kolliphor^®P188 increased metabolic activity, while it did not influence proliferation and differentiation as well as growth factors release. Kolliphor^®EL confirmed its cytotoxic effect at the highest concentrations applied. Contrariwise, treatment with lower concentrations significantly raised metabolic activity, induced adipogenesis, and increased insulin-like growth factor-1 and vascular endothelial growth factor secretion. The effect on differentiation was inhibited by blocking peroxisome proliferator-activated receptor gamma. Our results revealed important effects of surfactants on preadipocytes' survival, proliferation, death, and the interplay with their environment. Particularly Kolliphor^®EL provides modes of action, which could recommend it for novel treatment to improve fat graft viability.     

Addition of Cationic Surfactants to Lipid-Based Formulations of Poorly Water-Soluble Acidic Drugs Alters the Phase Distribution and the Solid-State Form of the Precipitate Upon In Vitro Lipolysis

It has been previously shown that the interaction of some weakly basic drugs with oppositely charged fatty acids during digestion can influence the solid-state form of the drug if it precipitates. The present study hypothesized the opposite effect for weakly acidic drugs. Tolfenamic acid (TA) and an oppositely charged cationic surfactant, didodecyldimethylammonium bromide (DDAB) were combined in a model medium chain lipid formulation. The phase distribution upon in vitro lipolysis was determined using HPLC and the solid-state form of precipitated TA was determined using X-ray diffraction and crossed polarized light microscopy. TA precipitated in a different polymorphic crystalline form to the starting reference material in the absence of DDAB but precipitated in an amorphous form when DDAB was included in the same formulation. The solubility of TA upon dispersion and digestion of the formulation was considerably higher in the presence of DDAB. The findings point to ionic interactions between TA and DDAB as the reason for the improved drug solubility throughout digestion, and precipitation of drug in an amorphous salt form, analogous to what has been observed in the past for some poorly water-soluble weakly basic drugs with anionic co-formers.     

Integration of Precipitation Kinetics From an In Vitro,Multicompartment Transfer System and Mechanistic Oral Absorption Modeling for Pharmacokinetic Prediction of Weakly Basic Drugs

Solubility, dissolution, and precipitation in the gastrointestinal tract can be critical for the oral bioavailability of weakly basic drugs. To understand the dissolution and precipitation during the transfer out of the stomach into the intestine, a multicompartment transfer system was developed by modifying a conventional dissolution system. This transfer system included gastric, intestinal, sink and supersaturation, and reservoir compartments. Simulated gastric fluid and fasted state simulated intestinal fluid were used in the gastric and intestinal compartment, respectively, to mimic fasted condition. The new transfer system was evaluated based on 2 model weak bases, dipyridamole and ketoconazole. Traditional 2-stage dissolution using 250 mL of simulated gastric fluid media, followed by 250 mL of fasted state simulated intestinal fluid, was used as a reference methodology to compare dissolution and precipitation results. An in silico model was built using R software suite to simulate the in vitro time-dependent dissolution and precipitation process when formulations were tested using the transfer system. The precipitation rate estimated from the in vitro data was then used as the input for absorption and pharmacokinetic predictions using GastroPlus. The resultant simulated plasma concentration profiles were generally in good agreement with the observed clinical data, supporting the translatability of the transfer system in vitro precipitation kinetics to in vivo.     

Oral Delivery of Highly Lipophilic,Poorly Water-Soluble Drugs: Self-Emulsifying Drug Delivery Systems to Improve Oral Absorption and Enable High-Dose Toxicology Studies of a Cholesteryl Ester Transfer Protein Inhibitor in Preclinical Species

BMS-A is an inhibitor of cholesteryl ester transfer protein and is a highly lipophilic compound (clogP 10.5) with poor aqueous solubility (<0.0001 mg/mL at pH 6.5). The compound exhibits low oral exposure when dosed as cosolvent solution formulations. The purpose of this study was to evaluate lipid-based formulations for enabling high-dose toxicology studies and enhancing toxicology margins of BMS-A in preclinical studies in nonrodent species. The solubility of BMS-A was screened in lipid and cosolvent/surfactant excipients, and prototype formulations were developed. In vitro tests showed that fine/microemulsions were formed after aqueous dilution of lipid formulations, and BMS-A was transferred from oil phase to aqueous phase with enhanced solubility following lipid digestion. When dosed in dogs at 200 mg/kg, a Gelucire-based formulation exhibited more than 10-fold higher exposure compared to the solution formulation and was thus selected for toxicology studies in dogs. For monkeys, an olive oil formulation was developed, and the exposure was about 7-fold higher than that from the solution. In summary, lipid-based drug delivery could be applied in early stages of drug discovery to enhance oral exposure and enable preclinical toxicology studies of highly lipophilic compounds, while facilitating the candidate selection of a molecule which is more specifically designed for bioperformance in a lipid-based drug delivery strategy.