Organ perfusion Liver model

Some efforts have been made to determine the effect P-gp has on its substrates by use of in situ perfusion methods, including intestinal perfusion, liver perfusion, kidney perfusion, and brain perfusion. These experiments allow the researcher to study the transport of compounds in a physiologically relevant environment in which the integrity of the organ is preserved with regards to cell polarity and representation of all cell types seen in the organ. Furthermore, the reduction in complexity of in situ models versus in vivo studies facilitates the conduct of complex studies and allows more definitive conclusions to be made regarding the role P-gp may play in disposition. 

Using an erythrocytes-containing medium for perfusion one has to take into account the putative involvement of the erythrocytes themselves with respect to uptake of the candidate compound. Therefore, not only the erythrocyte-free perfusate but also the erythrocytes fraction should be included in the analysis of the candidate compound, separately. In our hands the model of isolated perfused liver is metabolically active for up to 3 hours and during this time no decline in hepatic metabolic activity becomes obvious. However, bile flow declined during the perfusion experiments. Therefore, our total perfusion time of isolated livers in our standard experimental setup is limited to 2 hours. The tissue level of the candidate compound analyzed after 2 hours in the liver is a measure for the total amount of compound in the whole organ. This does not necessarily mean the presence of the candidate compound in hepatocytes but additionally in the capillary and biliary space of the liver. 

In Situ Models (Perfusion) The organ perfusion model most closely mimics in vivo drug absorption, metabolism, and elimination. Among various organ perfusion models, the liver perfusion model is the most studied. This... 

In addition to cell-based models, tissue-based models such as the Ussing chamber technique, the everted gut sac approach, and perfused isolated intestinal segments are also used, but only when it is important to understand the absorption processes in more detail. Unlike Caco-2, tissue-based models have the correct physiological levels of transporters and the presence of an apical mucus layer. Also, in situ and isolated organ perfusion methods exist for the gut, liver, lungs, kidneys, and brain and can provide data not directly obtainable in vitro. The isolated perfused liver is particularly useful since it allows an assessment of first-pass hepatic clearance, the quantitative distribution of metabolites in liver, blood, and bile, the effects of binding to plasma proteins and intracellular sites, and cellular uptake processes. 

Figure 22.1 A. Schema for a physiologically based pharmacokinetic model incorporating absorption in the stomach and intestines and distribntion to various tissues. B. Each organ or tissue type includes representation of perfusion (Q) and drug concentrations entering and leaving the tissue. Fluxes are computed by the product of an appropriate rate law, and permeable surface area accounts for the affinity (e.g., lipophilic drugs absorbing more readily into adipose tissue). Clearance is computed for each tissue based on physiology and is often assumed to be zero for tissues other than the gut, the liver, and the kidneys.

PBPK models have also been used to explain the rate of excretion of inhaled trichloroethylene and its major metabolites (Bogen 1988 Fisher et al. 1989, 1990, 1991 Ikeda et al. 1972 Ramsey and Anderson 1984 Sato et al. 1977). One model was based on the results of trichloroethylene inhalation studies using volunteers who inhaled 100 ppm trichloroethylene for 4 horns (Sato et al. 1977). The model used first-order kinetics to describe the major metabolic pathways for trichloroethylene in vessel-rich tissues (brain, liver, kidney), low perfused muscle tissue, and poorly perfused fat tissue and assumed that the compartments were at equilibrium. A value of 104 L/hour for whole-body metabolic clearance of trichloroethylene was predicted. Another PBPK model was developed to fit human metabolism data to urinary metabolites measured in chronically exposed workers (Bogen 1988). This model assumed that pulmonary uptake is continuous, so that the alveolar concentration is in equilibrium with that in the blood and all tissue compartments, and was an expansion of a model developed to predict the behavior of styrene (another volatile organic compound) in four tissue groups (Ramsey and Andersen 1984). 

Physiologically Based Toxicokinetic (PBTK) models are derived similarly to Physiologically Based Pharmacokinetic (PBPK) models, which have been used for a number of years in the development of medicinal drugs. They describe the rat or man as a set of tissue compartments, i.e., liver, adipose tissues, poorly perfused tissues, and richly perfused tissues along with a description of metabolism in the liver. In case of volatile organic compounds a description of gas exchange at the level of the lung is included, see also Section 4.3.6. 

The one-compartment model of distribution assumes that an administered drug is homogeneously distributed throughout the tissue fluids of the body. For instance, ethyl alcohol distributes uniformly throughout the body, and therefore any body fluid may be used to assess its concentration. The two-compartment model of distribution involves two or multiple central or peripheral compartments. The central compartment includes the blood and extracellular fluid volumes of the highly perfused organs (i.e., the brain, heart, liver, and kidney, which receive three fourths of the cardiac output) the peripheral compartment consists of relatively less perfused tissues such as muscle, skin, and fat deposits. When distributive equilibrium has occurred completely, the concentration of drug in the body will be uniform. 

Figure 1 Physiological model for sequential intestinal and hepatic first-pass metabolism. Blood flow to the small intestine is functionally divided into mucosal (Qgm) and serosal (Qgs) blood flow. Mucosal blood flow in the lamina propria perfuses the enterocyte epithelium. Portal blood flow (Qpv), which perfuses the liver is comprised of blood leaving the small intestine and other splanchnic organs such as the stomach and spleen. Blood flow leaving the liver (Qhv) represents the sum of hepatic arterial flow (<2ha) and Qpv. First-pass metabolism of an orally administered substrate (S) to product (P) may occur in the enterocyte or hepatocyte.

Figure 2.4 A typical conceptual representation of a PBTK model for a volatile organic chemical A. Each box represents a tissue compartment and arrows depict arterial and venous blood circulation. RAM refers to the rate of the amount metabolized. Vmax and Km refer to the maximal rate of metabolism and the Michaelis-Menten affinity constant, respectively. C is concentration in blood (V ), fat (FA), richly perfused tissues (RA), poorly perfused tissues (PA), liver (LA), and arterial blood (aA). Ql is the blood flow.

The tissue compartments included in the Johanson model are as follows the lungs, presumed to be the only site of uptake, and arterial blood the liver, presumed to be the only organ where transformation of 2-butoxyethanol takes place the gastrointestinal tract in equilibrium with the liver a group of richly perfused tissues a group of poorly perfused tissues a fat compartment and muscles and skin. Support... 

Physiologic parameters used, biochemical constants, and partition coefficients in the model are shown in Table 2-9. Physiologic constants (organ volume, blood flows, etc.) and tissue and blood coefficients were taken from literature sources (Astrand 1983 Fiserova-Bergerova 1983a). Elimination constants for the liver were taken from experiments in the perfused rat liver (Johanson et al. 1986b). Venous equilibrium was assumed, and competitive inhibition between ethanol and 2-butoxyethanol was assumed. 

To study the in vitro behaviour of carrier molecules, cells may be derived from a cell line or from primary cultures of rat or human liver cells. In general, the latter cells better reflect the in vivo situation than the immortalized cells in a cell line culture. It should be noted, however, that during the isolation procedure the enzymes collagenase and pronase may destroy or damage the target receptors. In vitro preparations that approach the in vivo situation best are liver slices, in which the physiological context of the liver is maintained [229], and the model of the isolated perfused rat liver (IPRL) [230, 231]. These techniques allow us to study carrier-cell interactions within the organ, that is, in the presence of all other liver cells and their secreted mediators. 

Willebrand factor [33]. The central compartment in this model represents primarily the vascular space and the interstitial space of well-perfused organs with permeable capillary walls, especially liver and kidneys, while the peripheral compartment comprises the interstitial space of poorly perfused tissues like skin and (inactive) muscle [19]. 

Primary hepatocytes or liver slices can be used to measure metabolism, but only for a short period of time after the liver sample has been removed from the body however, both models have problems associated with their use. In liver slices, cell-to-cell contact and three-dimensional structure are maintained with a full compliment of cell types (including Kupffer cells) primary hepatocytes have lost the orientation, organization, and nonhepatocyte cells which may contribute to the metabolic activity of the whole liver. Liver slices may suffer from the presence of damaged or dead cells, restricted access of culture media to internal cells, thereby reducing oxygen and nutrient supplies, and from a build-up of toxic products that may result in impaired metabolism. Perfusion of tissue in situ can ameliorate these problems, but of necessity such experiments can normally only be carried out in animals, and these will have different metabolic profiles due to differences in enzyme and transporter expression. 

Isolated lung, liver, and kidney perfusions have been recognized for decades as important models for toxicology and pharmacology. Part of their acceptance relates to the ease of harvest because these organs are anatomically structured with closed vascular systems containing easily identifiable arterial inputs and venous outputs, both amenable to catheterizations with minimal expertise in surgery. In contrast, outside the possible exception of ears, skin does not possess such a closed vascular system. 

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