In Vitro–In Vivo Correlation on Delivery of Drug Candidates to Articular Cartilage

PURPOSE: In the treatment of osteoarthritis (OA), some of the therapeutic approaches require delivery of drug(s) to the diseased cartilage. Presence of adequate drug levels in the cartilage is one of the important criteria in selection and ranking of lead compounds. The purpose of this study was to investigate the correlation in cartilage compound levels between in vitro experiments and in vivo animal studies. MATERIALS AND METHODS: Bovine cartilage samples were incubated with test compounds of various concentrations in a culture medium, in the absence or presence of 25 mg/ml of serum albumin which served as an artificial synovial fluid (SF). The test compounds were also dosed to rabbits, the animal model used for efficacy studies, over a six-week treatment period. Test article concentrations in plasma, SF, and cartilage were determined by LC/MS/MS analysis. RESULTS AND CONCLUSIONS: A correlation in cartilage drug concentration was observed between in vitro and in vivo studies. Plasma protein binding and the test article's affinity to cartilage were the major determining factors for drug delivery to cartilage in vivo.     

The Combination of GIS and Biphasic to Better Predict In Vivo Dissolution of BCS Class IIb Drugs,Ketoconazole and Raloxifene

The formulation developments and the in vivo assessment of Biopharmaceutical Classification System (BCS) class II drugs are challenging due to their low solubility and high permeability in the human gastrointestinal (GI) tract. Since the GI environment influences the drug dissolution of BCS class II drugs, the human GI characteristics should be incorporated into the in vitro dissolution system to predict bioperformance of BCS class II drugs. An absorptive compartment may be important in dissolution apparatus for BCS class II drugs, especially for bases (BCS IIb) because of high permeability, precipitation, and supersaturation. Thus, the in vitro dissolution system with an absorptive compartment may help predicting the in vivo phenomena of BCS class II drugs better than compendial dissolution apparatuses. In this study, an absorptive compartment (a biphasic device) was introduced to a gastrointestinal simulator. This addition was evaluated if this in vitro system could improve the prediction of in vivo dissolution for BCS class IIb drugs, ketoconazole and raloxifene, and subsequent absorption. The gastrointestinal simulator is a practical in vivo predictive tool and exhibited an improved in vivo prediction utilizing the biphasic format and thus a better tool for evaluating the bioperformance of BCS class IIb drugs than compendial apparatuses.     

Influence of Dissolution Media and Presence of Alcohol on the In Vitro Performance of Pharmaceutical Products Containing an Insoluble Drug

The purpose of this investigation is to determine how the dissolution media may influence the release rate of an insoluble drug in in vitro conditions. Some oral dosage forms containing ibuprofen, a molecule that shows pH-dependent solubility, are tested. They are evaluated in different media to simulate the gastrointestinal transit at paddle rotation speeds of 50 and 100 rpm. Moreover, the potential effect of different ethanol concentrations on drug release is tested. The dissolution profiles of the tablets show a similar behavior in water (pH 1.0) and phosphate buffer (pH 4.5) where the 2 doses are not completely dissolved. The soft capsules show a different behavior: a certain amount of ibuprofen, which is in solution inside the capsule, reprecipitates in water and in the pH 4.5 buffer. Instead, ibuprofen dissolves rapidly in the pH 6.8 buffer from all the formulations. In the water-ethanol solutions, the dissolution curves show a valuable increase in the drug dissolved at higher ethanol concentrations.     

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

Pharmaceutical Research -     

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

Pharmaceutical Research - This study aims to expand our understanding of the mechanisms of drug absorption, distribution, metabolism and excretion in the Göttingen minipig to aid a...     

Evaluation of the in Vivo Disintegration of Solid Dosage Forms of a Bile Acid Sequestrant in Dogs Using γ-Scintigraphy and Correlation to in Vitro Disintegration

Purpose. To evaluate the in vivo disintegration behavior of tablets and capsules of a bile acid sequestrant, DMP 504, in beagle dogs and to assess the significance of the in vitro disintegration of the dosage forms on subsequent in vivo behavior in order to draw possible in vitro-in vivo correlations. Methods. Tablet and capsule formulations of a bile acid sequestrant, DMP 504, were formulated with samarium oxide and neutron activated to produce radioactive ¹⁵³Sm to noninvasively evaluate their in vivo behavior in beagle dogs by -scintigraphy. A four-way crossover design was completed (n = 4) in which (a) tablets from two different batches were administered under the fasted condition and manufactured using different lots of drug substance where one batch exhibited relatively faster in vitro disintegration time (30 min) than the other tablet batch, which resulted in slower disintegration (45 min), (b) a capsule formulation was administered to fasted beagles, and (c) the tablet having slower in vitro disintegration was also administered in the fed state, and its in vivo disintegration was compared to that observed in the fasted state. Results. Tablets manufactured using a lot of DMP 504 having relatively fast in vitro disintegration (30 min) resulted in relatively rapid in vivo disintegration time (15 min) in the fasted condition. This in vivo disintegration time was comparable to the in vivo disintegration of the capsules (17 min) even though the in vitro capsule disintegration time was considerably faster (2 min). Tablets prepared using a drug substance that provided a longer in vitro disintegration time (45 min) resulted in a slower in vivo disintegration (63 min). There was no difference observed in the in vivo disintegration behavior in fasted and fed dogs for the tablets that provided slower in vitro disintegration. Conclusion. In vivo disintegration of tablets of the bile acid sequestrant DMP 504 correlated with in vitro disintegration times. -Scintigraphy continues to be a good tool to use during early stages of product development to investigate in vivo performance of dosage forms. The results of this study provided evidence that the physical chemical specifications of the drug substance may not always be indicative of in vitro or in vivo performance of tablet dosage form, even when formulation and process are not changed.     

Methodological Aspects of the Use of a Calibrator in In Vivo Microdialysis–Further Development of the Retrodialysis Method

Purpose. To investigate the performance of two alternative retrodialysis recovery methods and to describe the influence of different recoveries on the reliability in estimating unbound extracellular concentrations of morphine. Methods. Unbound concentrations of morphine in striatum and in blood were determined by microdialysis after a 10 min i.v. infusion in freely moving rats. In vivo recovery of morphine was determined by morphine itself, retrodialysis by drug, and by the calibrator nalorphine, retrodialysis by calibrator. Results. The low calibrator recovery in striatum (8.6%) resulted in large variability in the estimation of unbound extracellular concentrations when retrodialysis by calibrator was used. In blood, where the recovery was higher (36%), the variability was smaller. Also, when retrodialysis by drug was used, the variability remained low. This difference is caused by the propagation of errors in the way retrodialysis recovery is determined. Therefore, calibrator recovery values 20% are preferable in concentration estimations using retrodialysis by calibrator. Conclusions. When no time-dependent change in recovery is observed, retrodialysis by drug determined before the systemic administration is the best method. The calibrator is valuable as a quality control during the experiment.     

Population PKPD Modeling of BACE1 Inhibitor-Induced Reduction in Aβ Levels In Vivo and Correlation to In Vitro Potency in Primary Cortical Neurons from Mouse and Guinea Pig

Purpose

The aims were to quantify the in vivo time-course between the oral dose, the plasma and brain exposure and the inhibitory effect on Amyloid β (Aβ) in brain and cerebrospinal fluid, and to establish the correlation between in vitro and in vivo potency of novel β-secretase (BACE1) inhibitors.

Methods

BACE1-mediated inhibition of Aβ was quantified in in vivo dose- and/or time-response studies and in vitro in SH-SY5Y cells, N2A cells, and primary cortical neurons (PCN). An indirect response model with inhibition on Aβ production rate was used to estimate unbound in vivo IC ₅₀ in a population pharmacokinetic-pharmacodynamic modeling approach.

Results

Estimated in vivo inhibitory potencies varied between 1 and 1,000 nM. The turnover half-life of Aβ40 in brain was predicted to be 0.5 h in mouse and 1 h in guinea pig. An excellent correlation between PCN and in vivo potency was observed. Moreover, a strong correlation in potency was found between human SH-SY5Y cells and mouse PCN, being 4.5-fold larger in SH-SY5Y cells.

Conclusion

The strong in vivo-in vitro correlation increased the confidence in using human cell lines for screening and optimization of BACE1 inhibitors. This can optimize the design and reduce the number of preclinical in vivo effect studies.     

Effect of Colonic Lactulose Availability on the Timing of Drug Release Onset in Vivo from a Unique Colon-Specific Drug Delivery System (CODES™)

Purpose. To test the hypothesis that the onset of drug release in vivo from a unique colon-specific drug delivery system (CODES) would depend on the colonic availability rate of lactulose. The site specificity of drug release in canine GI tract was also estimated. Methods. CODES tablets were prepared by tableting the granulation of acetaminophen and lactulose, followed with film coating. The pharmacokinetic performance of different CODES formulations was evaluated in six beagle dogs under fasted conditions. The release of acetaminophen and lactulose was also characterized in vitro. Results. The onset of acetaminophen release in beagle dogs was found to be dependent on the coating level of Eudragit E and lactulose loading in the core tablet. At Eudragit E coating levels of 4%, 8%, and 12% (coating weight gain), the onset of in vivo drug release occurred 5.5 (±1.9) h, 4.8 (±1.0) h. and 7.5 (±1.0) h, respectively, after dosing. A similar trend was observed when the loading of lactulose in the core tablet decreased from 78% to 58% and 38%. However, the rate and extent of acetaminophen absorption did not vary significantly in each situation based on the values of AUC and C_max. Conclusions. The onset of drug release in vivo from CODES tablets is predominantly dependent on colonic availability rate of lactulose because drug release from this system is triggered by localized drop of colonic pH from the fermentation of lactulose.     

A Biopredictive In Vitro Comparison of Oral Locally Acting Mesalazine Formulations by a Novel Dissolution Model for Assessing Intraluminal Drug Release in Individual Subjects

Drug release and availability at the site of action are the major factors determining the clinical response for locally-acting gastrointestinal (GI) drug products. The present work focused on the prediction of site and extent of in vivo mesalazine release after oral administration to a variety of subjects using individualized in vitro drug release experiments. First, experiments mimicking GI passages in average adult subjects were performed. Then, results from a study screening fasted in vivo pH and transit profiles in individual subjects were translated into a novel in vitro dissolution model enabling to mimic individual GI pH-profiles and transit times with physiologically relevant dissolution media. A selection of monolithic and multiparticulate mesalazine formulations with pH-dependent and pH-independent drug release was screened with the novel dissolution model. Results of the study indicate that dosage form performance can be significantly different in individual subjects and highlight the importance of addressing individual physiological parameters relevant to intraluminal drug release when the aim is to predict the in vivo performance of locally-acting mesalazine formulations in individual patients. The novel in vitro dissolution approach thus represents a valuable tool for both improving individual oral therapy with locally-acting GI drug products and assessing bioequivalence of these formulations.     

Drug binding in the undernourished: a study of the binding of propranolol to α 1-acid glycoprotein

Summary The binding of propranolol, a drug commonly used in cardiovascular disorders, to ₁-acid glycoprotein (AGP) was studied in vitro in malnutrition. Compared to normal and hospital controls, the level of AGP was found to be elevated in undernourished subjects with and without infection. In the same patients the free drug percentage was significantly diminished. A significant inverse relationship was observed between the percentage of free drug and the level of AGP. The finding suggests that there may be need for an altered dosage regimen in the undernourished.     

Application of Box–Behnken Design to Optimize the Osmotic Drug Delivery System of Metoprolol Succinate and its In Vivo Evaluation in Beagle Dogs

The objective of this study was to develop osmotic pump tablets of metoprolol succinate (MS) using water-soluble pore formers in the semipermeable membrane in place of orifice drilled on the membrane thereby abolishing the shortcomings associated with laser drilling technique. The formulation was optimized for coating variables such as semipermeable membrane level (X ₁) and pore former level (X ₂) by response surface methodology using Box–Behnken design (BBD) employing design expert software. The developed osmotic pump tablet was found to sustain the drug release up to 20 h at zero-order rate. The pharmacokinetic study in Beagle dogs showed delayed T _max and lower C _max compared to marketed brand TOPROL XL, indicating a slow and more sustained release behavior of MS from the optimized osmotic tablets in comparison with the existing matrix-based marketed dosage form. Thus, a novel approach for the controlled release of MS from osmotic pump tablets has been successfully developed using BBD, which is valuable for the advancement of controlled drug delivery of other water-soluble drugs.     

Investigating the Impact of Drug Crystallinity in Amorphous Tacrolimus Capsules on Pharmacokinetics and Bioequivalence Using Discriminatory In Vitro Dissolution Testing and Physiologically Based Pharmacokinetic Modeling and Simulation

Delivering a drug in amorphous form in a formulated product is a strategy used to enhance the apparent solubility of a drug substance and its oral bioavailability. Drug crystallization in such products may occur during the manufacturing process or on storage, reducing the solubility advantage of the amorphous drug. However, the impact of partial drug crystallization in the drug product on the resulting bioavailability and pharmacokinetics is unknown. In this study, dissolution testing of commercial tacrolimus capsules (which are formulated to contain amorphous drug), both fresh and those containing different amounts of crystalline drug, was conducted using both United States Pharmacopeia and noncompendial dissolution tests with different dissolution media and volumes. A physiologically based pharmacokinetic (PBPK) absorption model was developed to predict the impact of crystallinity extent on the oral absorption of the products and to evaluate the discriminatory ability of the different dissolution methods. Virtual bioequivalence simulations between partially crystallized tacrolimus capsules versus fresh Prograf or generic tacrolimus capsules were performed using the PBPK model and in vitro dissolution data of the various fresh and partially crystallized capsules under United States Pharmacopeia and noncompendial dissolution conditions. The results suggest that compendial dissolution tests may not be sufficiently discriminatory with respect to the presence of crystallinity in an amorphous formulation. Nonsink dissolution tests using lower dissolution volumes generate more discriminatory profiles that predict different pharmacokinetics of tacrolimus capsules containing different extents of drug crystallinity. In conclusion, the PBPK modeling approach can be used to assess the impact of partial drug crystallinity in the formulated product and to guide the development of appropriate dissolution methods.     

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.     

Prodrug Approach for αΙΙbβ3-Peptidomimetic Antagonists to Enhance Their Transport in Monolayers of a Human Intestinal Cell Line (Caco-2): Comparison of In Vitro and In Vivo Data

Purpose. Different lipophilic derivatives of a potent _b3- antagonist with benzamidino-oxazolidinone structure were investigated with respect to transport and metabolism properties to evaluate their potential as prodrugs with improved absorption behavior. Methods. Intestinal transport and metabolism of the compounds were studied in Caco-2 monolayers under in vitro conditions and quantitated by a reversed-phase HPLC- method. Peroral bioavailability in cynomolgus monkeys and inhibition of platelet aggregation (guinea pig) were compared to in vitro permeability coefficients. Results. N-alkoxycarbonyl- and N-benzoyl- derivatization of the benzamidine-parent drug increased the apparent permeabilities across Caco-2 monolayers by a factor of 25-100 fold. Most prodrugs were transported mainly by passive diffusion, whereas the methoxycarbonyl-derivative EMD 122347 displayed directional transport from basolateral (BL) to apical (AP). This polarized efflux was concentration dependent (saturable kinetics with K_m = 207 M, V_max = 0.275 nmol cm^-2 min^-1) and could be reduced in the presence of verapamil (300 M), an inhibitor of p-glycoprotein. Cell mediated cleavage of the prodrugs was low and showed only slight differences to hydrolysis in buffer solution, indicating a predominantly non enzymatic cleavage. Both peroral bioavailability (monkey) and the inhibition of ex-vivo platelet aggregation (guinea pig) gave the same rank order as the permeability coefficients obtained in Caco-2 monolayers. Conclusions. Alkoxycarbonylamidine- and benzoylamidine promoieties of a RGD mimetic _b3-antagonist considerably increased both effect bioavailabilities in animal experiments as well as in-vitro permeability in cell monolayers, demonstrating the potential of this approach to enhance transport of peptidomimetic drugs.     

Magnetic Resonance Microscopy for Assessment of Morphological Changes in Hydrating Hydroxypropylmethylcellulose Matrix Tablets In Situ–Is it Possible to Detect Phenomena Related to Drug Dissolution Within the Hydrated Matrices?

Purpose

So far, the hydrated part of the HPMC matrix has commonly been denoted as a “gel” or “pseudogel” layer. No MRI-based results have been published regarding observation of internal phenomena related to drug dissolution inside swelling polymeric matrices during hydration. The purpose of the study was to detect such phenomena.

Methods

Multiparametric, spatially and temporally resolved T₂ MR relaxometry, in situ, was applied to study formation of the hydration progress in HPMC matrix tablets loaded with L-dopa and ketoprofen using a 11.7 T MRI system. Two spin-echo based pulse sequences were used, one of them specifically designed to study short T₂ signals.

Results

Two components in the T₂ decay envelope were estimated and spatial distributions of their parameters, i.e. amplitudes and T₂ values, were obtained. Based on the data, different region formation patterns (i.e. multilayer structure) were registered depending on drug presence and solubility. Inside the matrix with incorporated sparingly soluble drug a specific layer formation due to drug dissolution was detected, whereas a matrix with very slightly soluble drug does not form distinct external “gel-like” layer.

Conclusions

We have introduced a new paradigm in the characterization of hydrating matrices using ¹H MRI methods. It reflects molecular mobility and concentration of water inside the hydrated matrix. For the first time, drug dissolution related phenomena, i.e. particular front and region formation, were observed by MRI methods.