Model For Drug Transport Research Articles

Endogenous drug transporters in in vitro and in vivo models for the prediction of drug disposition in man

The epithelial canine and porcine kidney cell lines MDCK, MDCKII and LLC-PK1, respectively are employed to establish recombinant models of drug transport. Endogenous drug carriers in these cells may contribute to the activities of recombinant drug transporters, thus making it difficult to assess their properties. We analysed the expression of endogenous transporters in these cell lines by RT-PCR and by determining drug transporter activities. Concerning drug efflux, multidrug resistance protein 1 (MDR1) and MRP1 mRNAs were found in all lines. MRP2 mRNA was expressed in all cell lines except MDCK. Transepithelial transport of vinblastine and its modulation by a MDR1-specific inhibitor or by the MDR1- and MRP-inhibitor verapamil, indicated that MDCKII cells have, in comparisons to the other cell lines, relatively high levels of functional MDR1 while vinblastine transport in MDCK cells is likely to be mediated more by MRP1. Notably, LLC-PK1 cells displayed little activity attributable to either MDR1 and MRP1, thus making them suitable for the expression of these efflux pumps. Of the drug uptake carriers, OATP-A mRNA was only expressed in MDCK cells. OATP-C mRNA was barely detectable in MDCK cells and absent in MDCKII and LLC-PK1 cells. In agreement with transcriptional profiling, the OATP-mediated uptake of either estradiol-glucuronide or estrone-sulfate was either absent or barely detectable in all cell lines thus implying that they are suitable to establish recombinant models for human OATP’s. Transcriptional profiling was also performed on porcine and canine tissues and revealed that MRP1 was expressed in canine but not in human or porcine liver, whereas surprisingly OATP-C was expressed in canine kidney but only in human and porcine liver. The findings presented are relevant to the use of porcine and canine models for drug disposition.

Combined modeling and experimental approach for the development of dual-release polymer millirods

This paper describes a combined modeling and experimental approach for the design and development of a polymer device to provide local drug therapy to thermally ablated solid tumors. The polymer device, in the shape of cylindrical millirod, will be implanted via image-guided procedures into the center of the ablated tumor. Drug released from the millirod aims to eliminate residual cancer cells at the boundary of the normal and ablated tissue following thermal ablation to provide an effective treatment of the total tumor volume. The design of the millirod release kinetics is based on a mathematical model of drug transport in the ablated tumor and the surrounding normal tissue. The optimal release kinetics consists of a dual-release process—a burst release followed by sustained release—to provide the most optimal drug pharmacokinetics at the ablation boundary. Model analysis leads to a quantitative correlation of burst dose and release rates to the ablation size and the drug concentration at the ablation boundary. A three-layer polymer millirod is produced by a dip-coating method, and in vitro study demonstrates the dual-release kinetics in which a burst release occurs within 2 h followed by a sustained release over 7 –10 days. Independent control of the burst and sustained release rates is achieved by varying the structural composition of the outer and middle layers of the millirods, respectively. Results from this study provide the rational basis and experimental feasibility of dual-release millirods for further efficacy studies in solid tumors.

Computational approaches to the prediction of the blood–brain distribution

This review attempts to summarise present knowledge related to the theoretical modelling of drug transport across the blood–brain barrier. Several computational protocols are described ranging from quantum mechanics-based approaches through molecular mechanics-related techniques to simple and fast procedures based on only the 2-D graph of the investigated structures. Amazingly, few descriptors have been shown to influence the derived relationships in a significant manner and a cornerstone in most of the described models are terms describing hydrogen bonding. A very quick quantitative assessment of the brain partitioning of a compound has also been devised using the following two rules: If N+O (the number of nitrogen and oxygen atoms) in a molecule is less than or equal to five, it has a high chance of entering the brain. The second rule predicts that if log P−( N+O) is positive then log BB is positive.

Conditionally immortalized cell lines as a new in vitro model for the study of barrier functions.

Conditionally immortalized brain and retinal capillary endothelial and choroid plexus epithelial cell lines were established from a transgenic rat (Tg rat) and mouse (Tg mouse) harboring the temperature-sensitive simian virus 40 (ts SV 40) large T-antigen. These cell lines exhibit temperature-sensitive cell growth due to the expression of ts SV 40 large T-antigen. Mouse brain (TM-BBB) and rat brain (TR-BBB) and rat retinal (TR-iBRB) capillary endothelial cell lines appear to have a spindle-fiber shaped morphology and exhibit the typical endothelial markers, such as von Willebrand factor and acetylated low-density lipoprotein uptake. These cell lines express in vivo influx and efflux transporters, such as P-glycoprotein (P-gp) and GLUT1, which is capable of 3-O-methyl-D-glucose transport. TM-BBB cells are able to undergo efflux transport of cyclosporin A, which is a substrate for P-gp transport activity. They may also express oatp2 and exhibit dehydroepiandrosterone sulfate and digoxin uptake activity. TR-BBB cells express the mRNA of multidrug resistance associated protein 1 (MRP1) and a large neutral amino acid transporter, which consists of LAT1 and 4F2hc. TR-iBRB cells exhibit pH-dependent L-lactic acid transport activity and express the mRNA of monocarboxylate transporter (MCT) 1 and 2. The choroid plexus epithelial cell line (TR-CSFB) has polygonal cell morphology, expresses the typical choroid plexus epithelial cell marker, transthyretin, and has Na+, K+-ATPase located on the apical side. TR-CSFB cells also exhibit amino acid transport activity which has been observed in vivo. These barrier cell lines established from the Tg rat and Tg mouse have in vivo transport functions and are good in vitro models for drug transport to the brain and retina and as a screen for drugs which might be capable of delivery to the brain and retina.

Pharmacokinetics of orally administered camptothecins.

Phase I trials of oral camptothecins, including camptothecin (CPT) and irinotecan (CPT-11), have reported substantial interpatient variability in systemic exposure, which could result in suboptimal antitumor activity in some patients or enhanced risk for toxicity in others. This investigation evaluates the contribution of intestinal absorption and first-pass metabolism in the disposition of oral CPT and CPT-11, respectively. The transport of CPT in Caco-2 cell lines (validated model of intestinal drug transport) was concentration dependent and saturable (Vmax: 34 x 10(-5) cm/sec and Km: 20 microM), and was temperature dependent with an activation energy (Ea) of 11.7 kcal/mole. Cumulatively, this data was indicative of carrier-mediated intestinal transport. In addition, a reduction of transport in the presence of sodium azide plus deoxyglucose suggested ATP dependence. Thus, variable expression and availability of intestinal transporters could contribute to the observed wide variability in the exposure to oral CPT. CPT-11 is hydrolyzed by the ubiquitous enzyme carboxyl esterase to active SN-38, and first-pass metabolism of oral CPT-11 would include both intestinal and hepatic hydrolysis. Incubation of CPT-11 with S9 fractions of human liver and intestinal tissues resulted in variable rates of formation of SN-38. The mean (+/- SD) specific activities (pmoles/min/mg) were: liver (8.57 +/- 10.4, n = 8), duodenum (5.06 +/- 3.7, n = 4), jejunum (6.44 +/- 2.8, n = 5), ileum (4.81 +/- 2.4, n = 5), colon (1.93 +/- 1.5, n = 6), and rectum (0.82, n = 1). Interestingly, there was a decrease in SN-38 formation by tumor tissue compared to matched normal liver and colon tissues. Therefore variable first-pass metabolism could contribute to the substantial differences in the systemic exposures to CPT-11 and SN-38 in patients receiving oral CPT-11.

A Mathematical Model of Drug Transport in Human Breast Cancer

A mathematical model of drug transport in tissue has been developed on the basis of a clinical study of patients with breast cancer, treated with the drug doxorubicin and of drug transport experiments using cultured human breast cancer cells. The clinical study revealed doxorubicin gradients in tumor islets of densely packed cancer cells. The mathematical model allows simultaneous drug transport through the cellular network (transcellular pathway), through the intercellular interstitium (paracellular pathway), and across the boundary between the two networks. The effective diffusion coefficient of the interstitial network is found to be much higher than that of the cellular network, in spite of the fact that the interstitium thickness is only 20–40 nm. The model simulations can be made to fit the results of the clinical study. A long-continued simulation (40 days) of drug transport into a spherical islet with a radius of 150 μm, after a bolus injection of doxorubicin, reveals that the maximum average drug concentration at the islet centre is only reached after 224 h, while it decreases by a factor 15 from the boundary to the centre of the islet. The area under the curve in a plot of the average drug concentration versus time only decreases by 10% from the boundary to the centre of the islet.

Dissection of drug‐binding‐induced conformational changes in P‐glycoprotein

P-glycoprotein (Pgp) is a membrane-transport ATPase. It uses energy from ATP hydrolysis to transport pleiotropic cytotoxic drugs from inside to outside of cells. Thus, elevated expression of Pgp in cancer cells causes multidrug resistance. It is now known that the conformational state of Pgp changes during its catalytic cycle. However, how ATP hydrolysis relates to drug binding by Pgp is yet to be determined. In this study, we used limited trypsin digestion of Pgp in isolated inside-out membrane vesicles to investigate the effects of drugs on Pgp conformation and to determine the drug-bound conformational states of Pgp in the catalytic cycle. We found that (a) binding of vinblastine or verapamil alone can cause a conformational change in Pgp, but the change induced by the drug binding is different from that induced by nucleotide binding, (b) there may be at least two binding sites for Pgp substrates, one for drugs such as vinblastine and verapamil and the other for drugs such as colchicine and adriamycin, (c) the conformation of Pgp bound by ATP and vinblastine is different from the conformation bound by either one alone, and (d) the ADP-bound Pgp does not bind vinblastine. Based on these observations and our previous studies, we propose a model for drug binding and transport in the catalytic cycle of Pgp.

Mathematical modelling of drug transport in emulsion systems.

Two mathematical models for the prediction of drug transport in triphasic (oil, water and micellar) emulsion systems as a function of micellar concentration have been developed and these models were evaluated by comparing experimental and simulated data. Fick's first law was used to derive a transport model for hydrophilic drugs, assuming that the oil/water (o/w) partitioning process was fast compared with membrane transport and therefore drug transport was limited by the membrane. Consecutive rate equations were used to model transport of hydrophobic drugs in emulsion systems assuming that the o/w interface acts as a barrier to drug transport. Benzoic acid and phenol were selected as hydrophilic model drugs. Phenylazoaniline and benzocaine were selected as hydrophobic model drugs. Transport studies at pH 3.0 and 7.0 were conducted using side-by-side diffusion cells. According to the hydrophilic model, an increase in micellar concentration is expected to decrease drug transport rates. The effective permeability coefficients (Peff) of drugs were calculated using an equation relating Peff and the total apparent volume of drug distribution (determined experimentally using drug/membrane permeability and partition coefficient values). The hydrophobic model was fitted to the experimental data for the cumulative amount of model drug in the receiver cells using a weighted least-squares estimation program (PCNONLIN). The oil/continuous phase partitioning rates (k1) and the membrane transport rates (k2) were estimated. The goodness of fit was assessed from the correlation coefficients of plots of predicted versus experimental data. The predicted data were consistent with the experimental data for both the hydrophilic and hydrophobic models.

Human P-glycoprotein exhibits reduced affinity for substrates during a catalytic transition state.

Human P-glycoprotein (Pgp), a plasma membrane protein that confers multidrug resistance, functions as an ATP-dependent drug efflux pump. Pgp contains two ATP binding/utilization sites and exhibits ATPase activity that is stimulated in the presence of substrates and modulating agents. The mechanism of coupling of ATP hydrolysis to drug transport is not known. To understand the role of ATP hydrolysis in drug binding, it is necessary to develop methods for purifying and reconstituting Pgp that retains properties including stimulation of ATPase activity by known substrates to an extent similar to that in the native membrane. In this study, (His)6-tagged Pgp was expressed in Trichoplusia ni (High Five) cells using the recombinant baculovirus system and purified by metal affinity chromatography. Upon reconstitution into phospholipid vesicles, purified Pgp exhibited specific binding to analogues of substrates and ATP in affinity labeling experiments and displayed a high level of drug-stimulated ATPase activity (specific activity ranging from 4.5 to 6.5 micromol min-1 mg-1). The ATPase activity was inhibited by ADP in a competitive manner, and by vanadate and N-ethylmaleimide at low concentrations. Vanadate which is known to inhibit ATPase activity by trapping MgADP at the catalytic site inhibited photoaffinity labeling of Pgp with substrate analogues, [125I]iodoarylazidoprazosin and [3H]azidopine, only under ATP hydrolysis conditions. Because vanadate-trapped Pgp is known to resemble the ADP and phosphate-bound catalytic transition state, our findings indicate that ATP hydrolysis results in a conformation with reduced affinity for substrates. A catalytic transition conformation with reduced affinity would essentially result in substrate dissociation and supports a model for drug transport in which an ATP hydrolysis-induced conformational change leads to drug release toward the extracellular medium.

A mathematical model of polymeric controlled drug release and transport in the brain

A two-dimensional finite-element model was developed to simulate the transport and clearance of drugs released from polymers into the brain. This computer model includes the effects of diffusion and bulk flow of interstitial fluid, metabolism and capillary uptake, and is responsive to various controlled release scenarios and anatomical variation. The capability of this model is illustrated by examining the transport of three compounds of interest for neurological applications.

Effects of cellular pharmacology on drug distribution in tissues

The efficacy of targeted therapeutics such as immunotoxins is directly related to both the extent of distribution achievable and the degree of drug internalization by individual cells in the tissue of interest. The factors that influence the tissue distribution of such drugs include drug transport; receptor/drug binding; and cellular pharmacology, the processing and routing of the drug within cells. To examine the importance of cellular pharmacology, previously treated only superficially, we have developed a mathematical model for drug transport in tissues that includes drug and receptor internalization, recycling, and degradation, as well as drug diffusion in the extracellular space and binding to cell surface receptors. We have applied this "cellular pharmacology model" to a model drug/cell system, specifically, transferrin and the well-defined transferrin cycle in CHO cells. We compare simulation results to models with extracellular diffusion only or diffusion with binding to cell surface receptors and present a parameter sensitivity analysis. The comparison of models illustrates that inclusion of intracellular trafficking significantly increases the total transferrin concentration throughout much of the tissue while decreasing the penetration depth. Increasing receptor affinity or tissue receptor density reduces permeation of extracellular drug while increasing the peak value of the intracellular drug concentration, resulting in "internal trapping" of transferrin near the source; this could account for heterogeneity of drug distributions observed in experimental systems. Other results indicate that the degree of drug internalization is not predicted by the total drug profile. Hence, when intracellular drug is required for a therapeutic effect, the optimal treatment may not result from conditions that produce the maximal total drug distribution. Examination of models that include cellular pharmacology may help guide rational drug design and provide useful information for whole body pharmacokinetic studies.

An analysis of drug and solute transport through membranes based on potential barriers

Modelling of drug or solute transport in membranes including those used in transdermal systems is often based on Fickian diffusion analysis. Such transport requires knowledge of the size exclusion characteristics and the membrane reflection coefficient as functions of the physiological conditions of the system. We offer a modified analysis with which one can calculate drug reflection coefficients in polymer membranes used in transdermal delivery. In our analysis, the solute molecule is visualized as traveling across successive potential barriers created by steric effects, electrostatic interactions or other forces from charged or uncharged molecules. The membrane is assumed to present only one potential barrier since the random network need not possess any anisotropy. A two-dimensional network with vanishing thickness allows the approximation of one-directional transport. The result is the formulation of a potential barrier model that can be used to analyze various solute transport situations. Various experimental results from our laboratory and other investigators are analyzed using this theory. For the time being the theoretical model applies to relatively low molecular weight solutes.

A predictive algorithm for skin permeability: the effects of molecular size and hydrogen bond activity.

To develop a predictive algorithm of nonelectrolyte transport through skin based upon a partitioning-diffusion model. Drug permeability is described by a partitioning-diffusion equation. Through free-energy relationships, partitioning is related to the drug's molecular volume (MV), and hydrogen bond donor (Hd) and acceptor (Ha) activity. Diffusion is related to the drug's MV using a theory of diffusion through lipid lamellae based on free-volume fluctuations within the lipid domain. These two explicit descriptions are combined to give an equation describing permeability in terms of the permeant's physical properties. The aqueous permeability coefficients of 37 nonelectrolytes through human epidermis were evaluated as a function of these physical properties using a multiple regression analysis. The results of the regression analysis show that 94% of the variability in the data can be explained by a model which includes only the permeant's MV, Hd and Ha. These results further provide an algorithm to predict skin permeability based upon the values of these parameters. In addition, the relative contribution of various chemical functional groups (e.g., -COOH) is derived, and can be used to predict skin transport from drug structure alone. A biophysically relevant model of drug transport through human skin is derived based solely on the physical properties of the drug. The model provides an algorithm to predict permeability from the drug's structure and/or physical properties. Moreover, the model is applicable to a number of lipid barrier membranes, suggesting a common transport mechanism in all.

Two-layer membrane model for iontophoretic drug transport through excised rat skin.

Iontophoretic and passive transport of an ionized drug (sulfisoxazole) across excised rat skin was studied using a two-chamber cell with four electrodes under successive experimental conditions: without electrical current (stage-I) and with electrical current (stage-II). Two iontophoretic/diffusion models, i.e. a one-layer membrane model and a two-layer membrane model, in which a difference in the electrical potential gradient was taken into account between the stratum corneum and epidermis/dermis layer, were constructed to describe the non-steady-state drug permeation process during ionotrophoresis. The observed iontophoretic lag-time was two times greater than the calculated value based on the one-layer membrane model. According to the two-layer membrane model, the calculated ionotophoretic lag-time agreed with the observed value. It was revealed by model adaptation to the observed data that the stratum corneum fraction of the electro-chemical potential difference across the whole skin caused by the iontophoresis was around 90%. This result was consistent with the observation that the direct current resistance of whole skin was seven times greater than that of stripped skin.

Caco-2 cell monolayers as a model for drug transport across the intestinal mucosa.

Human colon adenocarcinoma (Caco-2) cells, when grown on semipermeable filters, spontaneously differentiate in culture to form confluent monolayers which both structurally and functionally resemble the small intestinal epithelium. Because of this property they show promise as a simple, in vitro model for the study of drug absorption and metabolism during absorption in the intestinal mucosa. In the present study, the transport of several model solutes across Caco-2 cell monolayers grown in the Transwell diffusion cell system was examined. Maximum transport rates were found for the actively transported substance glucose and the lipophilic solutes testosterone and salicyclic acid. Slower rates were observed for urea, hippurate, and saliylate anions and were correlated with the apparent partition coefficient of the solute. These results are similar to what is found with the same compounds in other, in vivo absorption model systems. It is concluded that the Caco-2 cell system may give useful predictions concerning the oral absorption potential of new drug substances.

Random Brick Model for Drug Transport across Stratum Corneum

A model for drug permeation across the stratum corneum is developed to predict the effect of the physical configuration and chemical composition of the stratum corneum on the skin permeability of drugs. The experimental data of percutaneous absorption across different skin specimens are well interpreted by the present model.

Modeling and Numerical Computation of Drug Transport in Laminates: Model Case Evaluation of Transdermal Delivery System

This study reports on the modeling and numerical computation of drug transport from a laminated system into the skin. The model is based on the diffusion equation, on partitioning of the drug between individual layers of the device, and on continuity conditions for diffusive flux across the material interfaces of the system. Numerical computation of concentration profiles across the system was performed using a sophisticated software package designed for a general class of kinetics-diffusion problems, including time variable diffusion coefficients and interface conditions in case of nonhomogeneous media. This program is demonstrated by a model case evaluation of a transdermal delivery system for nitroglycerin and applies previous data on drug diffusion and partitioning in this system. The layers involved in drug transport are: a polymer film to carry the drug depot; a microporous membrane, the pores of which are filled with nonpolar liquids and solids; an adhesive layer; and a final layer representing the skin. Transport kinetics in the delivery system and the skin are being followed by plots of concentration profiles as a function of application time. Major concentration gradients indicate sites of release control. For practical purposes, the package is intended to serve for preformulation studies of laminated drug delivery systems.

Use of gamma distributed residence times in pharmacokinetics.

Using a nonclassical statistically based pharmacokinetic concept, a theory is presented which can be applied to the analysis of concentration-time data fitted by power functions of time C = At-ae-bt, which is shown to be equivalent to the assumption of gamma distributed residence times of drugs. The shape and scale parameters a and b, respectively, are interpreted physiologically in terms of a recirculatory model. It is shown how the shape parameter a, which is only dependent on the coefficient of variation of residence times, is affected by the processes of drug distribution and elimination. The time course of the blood concentration following multiple doses and continuous infusion is predicted for gamma-like drug disposition curves. The assumption of gamma distributed disposition residence times is theoretically based on a random walk model of circulatory drug transport, and the conditions are investigated under which gamma curves can be empirically fitted to oral concentration-time data. The parameters of concentration-time profiles following solid dosage forms, for example, are explained by the means and coefficients of variation of the disposition residence time and dissolution time distribution, respectively. The advantages of this concept compared to the conventional method of fitting sums of exponentials to the data are described.

Alternative models for describing the bioconcentration of organics in fish

AbstractThe accumulation of organic residues in fish is the result of competing rates of uptake and elimination, which can be modeled by pharmacokinetic techniques. Although first‐order kinetics are usually assumed, they are rarely verified. Models with biphasic, second‐order or Michaelis‐Menten kinetics may prove to be better choices, depending on exposure level and mode of elimination. The well‐known correlation between bioconcentration factor and partition coefficient (P) derives from separate correlations for the uptake and elimination rate constants with P. While such correlations appear to be linear in the range of log P = 2–5 for organics that are not metabolized, they sometimes fail at higher values of log P. A drug transport model is proposed to account for the “non‐ideal” bioconcentration of a variety of organics in fish. According to this model, uptake is a nonlinear function of partition coefficient, water solubility and membrane permeability.

ALTERNATIVE MODELS FOR DESCRIBING THE BIOCONCENTRATION OF ORGANICS IN FISH

The accumulation of organic residues in fish is the result of competing rates of uptake and elimination, which can be modeled by pharmacokinetic techniques. Although first-order kinetics are usually assumed, they are rarely verified. Models with biphasic, second-order or Michaelis-Menten kinetics may prove to be better choices, depending on exposure level and mode of elimination. The well-known correlation between bioconcentration factor and partition coefficient (P) derives from separate correlations for the uptake and elimination rate constants with P. While such correlations appear to be linear in the range of log P = 2–5 for organics that are not metabolized, they sometimes fail at higher values of log P. A drug transport model is proposed to account for the “non-ideal” bioconcentration of a variety of organics in fish. According to this model, uptake is a nonlinear function of partition coefficient, water solubility and membrane permeability.