Advertisement
Research ArticleArticle

Uptake of Teicoplanin by Isolated Rat Hepatocytes: Comparison with in Vivo Hepatic Distribution

Raquel F. Reinoso, Brian A. Telfer, Beverley S. Brennan and Malcolm Rowland
Drug Metabolism and Disposition April 2001, 29 (4) 453-459;

Abstract

Tissue distribution of teicoplanin, a large glycopeptide antibiotic, is slow but at equilibrium its whole body distribution volume is relatively large (Vss = 1.18–2.78 liter/kg), despite a high binding to plasma albumin. In vivo distribution into liver is extensive. Previous in vitro homogenate studies suggest that teicoplanin binds to cell membranes but only enters some cells. This possibility was investigated with isolated hepatocytes incubated for 4 h with [14C]teicoplanin alone and in the presence of additional teicoplanin (1 and 100 μg/ml). Uptake was determined after separating the cells by rapid centrifugation through a dibutyl phthalate layer and assessing viability by the trypan blue exclusion test. Teicoplanin cell uptake curves, initially rapid followed by slower distribution (which agrees with in vivo findings), were adequately described by a closed two-compartment model. Whereas entry into hepatocytes was independent of drug concentration, binding to the cell exterior membrane was concentration-dependent. The equilibrium distribution ratio (Kpuc ± S.D.; 42 ± 10) was somewhat smaller than estimated in vivo (106 ± 9), but similar to that reported previously in vitro using liver homogenates (54 ± 11). Also, the estimated membrane permeability-surface area product was larger in vitro than in vivo (PSu ± S.D.; 5.5 ± 2.9 versus 0.74 ± 0.10 ml/min per whole liver). The most likely explanation for this difference is that in vivo only a small fraction of the total cell surface area is exposed to the perisinusoidal space, where exchange occurs.

Teicoplanin, a glycopeptide antibiotic chemically related to vancomycin, is a mixture of five major and structurally related components with similar polarities, molecular masses (1564–1908 Da), pharmacokinetics (Bernareggi et al., 1990), and biological activity (Borghi et al., 1984). Noteworthy among its pharmacokinetics features is an unexpected relatively large volume of distribution across mammalian species, including humans (1.18–2.78 l/kg) (Antony et al., 1991), despite its large size and high binding to plasma albumin (88–97%) (Assandri and Bernareggi, 1987; Dykhuizen et al., 1995).

After intravenous administration to rats of either single (Bernareggi et al., 1986) or multiple doses (Zanolo et al., 1991), teicoplanin distribution into the tissues occurs first rapidly and then more slowly such that distribution equilibrium is reached only after a relatively long time (>24 h). At equilibrium, the liver is a major tissue of distribution, with the highest tissue-plasma concentration ratio.

Previously, we determined the potential tissue affinity of teicoplanin in vitro after adding drug to homogenates of many rat tissues (Reinoso et al., 1998). In vitro binding data overestimated drug distribution in vivo, suggesting that in vivo teicoplanin can access some but not all of the tissue binding components. In the specific case of erythrocytes, teicoplanin binds to the cell membrane but does not enter the cells. To ascertain whether this is also the case with the liver, the present study was aimed at investigating the permeation properties of teicoplanin in isolated hepatocytes, and correlating these results with those reported in vivo.

Experimental Procedures

Materials.

[14C]Teicoplanin (s.a. 10.4 μCi/mg) was kindly supplied by Lepetit Research Center, Gruppo Lepetit S.p.A. (Milan, Italy). [3H]Inulin (s.a. 0.63 mCi/mg) was obtained from Amersham Pharmacia Biotech (Piscataway, NJ). [3H]Water (s.a. 25 mCi/ml) and [14C]sucrose (s.a. 516 mCi/mmol) from PerkinElmer Life Sciences (Boston, MA) and Sigma Chemical Co. (St. Louis, MO), respectively. All other chemicals were of analytical grade and commercially available.

Isolation of Hepatocytes.

Male Sprague-Dawley rats (240–260 g) served as liver donors. The procedure followed to isolate hepatocytes has been described in detail previously (Hayes et al., 1993). It is based on the collagenase perfusion protocol of Berry and Friend (1969) and the two-step end of lobe technique reported by Strom et al. (1982). After isolation, cells were resuspended in a suitable volume of Williams' Medium E (WME1) buffered at pH 7.4 with 0.25 mM HEPES and kept at 4°C until use. Cell viability for each experiment was determined by the trypan blue exclusion test (Baur et al., 1975); a minimum initial cell viability of 85% was required for subsequent use.

Uptake Experiments.

Uptake studies were carried out with [14C]teicoplanin (0.2 μCi; 19.23 μg) and [3H]inulin (0.4 μCi) in the absence and presence of additional teicoplanin (final concentrations in the cell suspension, 1 or 100 μg/ml). Hepatocyte suspension (4 × 106 cells/ml) and WME were preincubated for 5 to 10 min at room temperature. Once both of them were at the same temperature, WME was spiked with the compounds and added to the cell suspension (1:1). The resultant hepatocyte suspension (12 ml) was incubated at 37°C with gentle shaking and the viability determined routinely throughout the experiment. At preset time intervals, ranging between 0 and 4 h, two aliquots of the cell suspension were removed: one aliquot (100 μl) was used to determine the concentration of compounds in the cell suspension and the other to measure teicoplanin uptake by hepatocytes (500 μl). Uptake was determined after separating the hepatocytes from the incubation medium by centrifugation (9000 rpm, 2 min) of the cells through a dibutyl phthalate layer (density = 1.04). This yielded two layers separated by the water-impermeable oil; the lower layer contained the drug taken up by the hepatocytes and the upper layer the extracellular drug. After sampling the latter (200 μl), the supernatant was discarded and the oil surface washed with distilled water. The oil phase was then discarded and the tip of the tubes, which contained the cells as a pellet, was frozen in liquid nitrogen and cut. Radioactivity was assayed in the three samples taken (cell suspension, WME, and cell pellet). Parallel experiments in which hepatocytes were absent were performed, since binding of teicoplanin to glass and plastic surfaces has been reported (White and Reeves, 1991).

Intracellular Volume Measurement and Correction for Adhering Fluid.

Determination of the intracellular volume was performed in parallel experiments by adding [3H]water (0.4 μCi) and [14C]sucrose (0.2 μCi) to the hepatocyte suspension. Following separation by the dibutyl phthalate method as described above, samples of cell and medium were analyzed for radioactivity. The intracellular volume was estimated by subtracting the extracellular volume (given by [14C]sucrose) from the total aqueous volume determined by using [3H]water (Raghoebar et al., 1987).

Concentrations of hepatocyte-associated teicoplanin were calculated by dividing the amount of [14C]teicoplanin taken up into the cells by the intracellular volume. This amount was corrected for drug in the extracellular fluid trapped between cells (approximately 3% of the drug present in cells), the volume of which was determined separately using [3H]inulin, an extracellular marker. Data were expressed as concentration normalized for the dose administered (1/ml).

Analytical Method.

Radioactivity in the samples was determined by liquid scintillation counting (Wallac 1409) after the addition of 8 ml of scintillation fluid (Optiphase HiSafe II, LKB Wallac, Turku, Finland). Before counting, samples were digested overnight in 1 ml of Soluene-450 (Packard Instrument Company, Meriden, CT) and then mixed with 2 M HCl (0.1 ml) and isopropanol (1 ml) before being bleached with 30% H2O2 (0.3 ml). Quenching was corrected automatically by an external standard method. Results are expressed as disintegrations per minute.

Data Analysis.

Hepatocyte studies

Taking into account the negligible extent of metabolism reported for teicoplanin (Bernareggi et al., 1992), the distribution of compound between WME and hepatocytes was described by a closed two-compartment model.

Assuming that only the unbound drug is able to pass across the cell membrane and binding to the cell surfaces is so fast as to be at steady state with respect to movement across the cell membrane, following the addition of teicoplanin to WME, changes in the amounts of drug within the cell and WME compartments can be expressed by eqs. 1 and 2:dqcdt=Vc·dCcdt=PSu(fum·Cm−fuc·Cc) Equation 1dqmdt=Vm·dCmdt=PSu(fuc·Cc−fum·Cm) Equation 2where the subscripts c and m refer to the cell and WME compartments, respectively, q is the amount of teicoplanin,C is the total drug concentration, V is the volume of the corresponding compartment, PSu the permeability-surface area product of the diffusing (unbound) compound across the cell membrane, and fu the drug fraction unbound. Since albumin, which is the plasma binding protein for teicoplanin, was not present in the cell suspension, fum was assumed to be equal to 1. Therefore, Cm is the unbound concentration in the medium. The reciprocal of fuc is the equilibrium cell-to-unbound drug in medium concentration ratio, Kpuc.

The model was fitted to the two measured concentrations of teicoplanin, total concentration in hepatocytes (Cc*), and in WME (Cm). Teicoplanin concentration associated with hepatocyte comprised both drug within the hepatocyte and that bound to the cell exterior membrane:Cc*=qc+qcmVc Equation 3where qcm is the amount of teicoplanin bound to the cell membrane, which is directly proportional to the unbound concentration of teicoplanin in the WME compartment:qcm=Kvitro·fum·Cm Equation 4where Kvitro is the proportionality constant between qcm and unbound drug concentration in the medium (fum·Cm). In this model, the initial concentration in the WME compartment is given byDose/(Vm + fum·Kvitro), whereDose is the total amount of teicoplanin added to the suspension.

In vivo distribution into the liver.

Liver and plasma concentration-time data after intravenous administration of teicoplanin to rats (Bernareggi et al., 1986; Zanolo et al., 1991) were used to define the hepatic drug kinetics in vivo. Assuming that distribution of teicoplanin in the liver is limited only by its passage across the hepatocyte membrane, the rate of drug transfer between the interstitial space (space of Disse) and hepatocyte can be expressed by the same equation used to describe the transport of teicoplanin across hepatocytes (eq. 1). In this particular case,Cm and Cc refer to the drug concentrations in the interstitial and cellular spaces of the liver, respectively.

Taking into account that sinusoids within the liver are discontinuous capillaries (Wisse et al., 1985), which permit free access of large molecules up to the size of albumin, unbound drug concentrations in the vascular and interstitial spaces were assumed to be in equilibrium and equal. Another assumption involved in the model was that drug concentrations in the plasma of the hepatic vascular space and systemic circulation are equal.

By analogy to eq. 1, the rate of transfer within the hepatocyte is then described bydqcdt=VcdCcdt=PSu(fup·Cp−fuc·Cc) Equation 5where the fraction of teicoplanin unbound in plasma (fup) was set equal to 0.1 (Wittendorf et al., 1987) and drug concentration in plasma (Cp) was characterized by the polyexponential equation that best fitted the plasma teicoplanin concentration-time data. Namely,Cp=∑i=1nAi e−λi·t Equation 6where Ai is the coefficient associated with the ith exponential term and λi the corresponding disposition rate constant.

The model was fitted to the total teicoplanin concentration measured in the liver. This concentration was expressed in terms of the total hepatic volume (VH) and the amount of compound in the three tissue spaces, vascular (v), interstitial (is), and cellular (c), together with drug bound to the external cell surface. As with the in vitro situation, the last term was given by eq.4, with Kvivo now representing the proportionality constant between drug bound to the cell surface and unbound drug in plasma. Considering the equality assumed between unbound drug concentration in the interstitial and vascular spaces, the total hepatic drug concentration (CH) is given byCH=Vv+Vis·fupfuis+Kvivo·fupCp+(Vc·Cc)VH Equation 7where tissue volumes (Vv = 1.29 ml;Vis = 1.68 ml;Vc = 7.33 ml) were taken from Khor and Mayersohn (1991), considering a liver weight of 10.3 g (250-g rat) (Bernareggi and Rowland, 1991), and fuis (0.18) was calculated according to McNamara et al. (1983), using a plasma-to-interstitial space albumin concentration ratio of 0.5 (Khor and Mayersohn, 1991). Here, as in vitro, fuc = 1/Kpuc. This cell-to-unbound plasma concentration ratio is related to the cell-to-plasma concentration ratio, Kpc, by Kpc = fup·Kpuc.

Fitting procedure and parameter estimation.

Parameters were estimated by fitting the appropriate equations simultaneously to the in vitro hepatocyte and WME concentration-time data to yield estimates of PSu, fuc,Kvitro, and Vc; eqs. 5 and 7 to the plasma and hepatic concentration-time data in vivo, to yield estimates for PSu and fuc using the program SAAM II (SAAM Institute, 1994). A weighing scheme of (observation)−1 was chosen for the analysis of both in vitro and in vivo data.

Other parameters calculated from the model estimates associated with the in vitro data were the sum of the influx (PSu·fum/Vm) and efflux (PSu·fuc/Vc) first-order rate constants, which characterize the net movement of teicoplanin across the cell membrane (kT), the distribution half-life (t1/2; 0.693/kT), as well as the cell-to-media unbound concentration ratio at steady state (Kpuc=1/fuc). The value of Kpuc in vivo was also calculated from the corresponding estimate of fuc.

Results are expressed as mean ± S.E. unless otherwise indicated. Statistical analysis was performed by two-way analysis of variance. The level of significance was set at p< 0.05.

Results

In Vitro Data.

The viability of the hepatocyte suspensions used in the present study was determined by the trypan blue exclusion test; the value obtained immediately after cell isolation ranged from 90 to 92%. By 30 min of incubation, the viability of the cells had dropped to 60 to 78%, and thereafter remained constant during the subsequent 4-h incubation period.

The marker [14C]sucrose was used to estimate the volume of extracellular fluid trapped between the cells. No significant differences were found in this volume throughout the experiment (6.3 ± 0.3 μl per 106hepatocytes; n = 56). The hepatocyte volume, determined from the total aqueous and extracellular volumes, was 3.9 (±0.3;n = 42) μl per 106 hepatocytes.

To determine the importance of the binding of teicoplanin to the glass surfaces of the equipment, additional experiments without hepatocytes were carried out. These studies showed minimal (<12%) binding during the 4-h experiment, implying that any change in the drug concentration observed in the cell experiments is essentially only due to the interaction between drug and hepatocytes. This assumption was supported by the ability to account for all added teicoplanin by measurement of compound in the cell suspension.

Cellular uptake of [14C]teicoplanin was studied in the presence of an extracellular marker ([3H]inulin). Apart from some initial variability, the cell suspension-to-WME ratio for [3H]inulin remained relatively constant throughout the experiment, whereas for teicoplanin this ratio increased slowly throughout the incubation period, eventually reaching a constant value 30 to 33% higher than the initial ratio (Fig.1). The different behavior of teicoplanin, compared with that of the extracellular marker, suggests that this antibiotic enters hepatocytes, albeit slowly.

Figure 1
Figure 1

Cell suspension-to-WME ratios for [14C]teicoplanin (A) and [3H]inulin (extracellular marker; B).

Each point represents the mean ± S.E. (n = 9).

Figure 2 shows the generally good agreement between cell and WME teicoplanin concentrations found experimentally and best fit model predictions. Teicoplanin uptake onto cells was virtually instantaneous followed by a slower uptake process, until steady state was reached. The estimates of the associated kinetic parameters are summarized in Table 1. The equilibrium distribution ratio, Kpuc(=1/fuc), of 42, far in excess of 1, indicates that hepatocytes have a reasonably high affinity for teicoplanin. The presence of additional nonradiolabeled teicoplanin did not influence the overall shape of the curves and showed no significant difference between the tracer alone and 1 μg/ml unlabeled teicoplanin, but some evidence for a concentration dependence was found at the highest concentration (100 μg/ml).

Figure 2
Figure 2

Comparison of predicted and observed time course of radiolabeled teicoplanin in rat hepatocytes (▪) and WME (×) in the absence (A) and presence (B and C) of teicoplanin (1 and 100 μg/ml, respectively).

Dashed and solid lines, the best fit predictions in WME and hepatocytes, respectively. The results from representative experiments are shown. Concentrations are normalized for the amount of radiolabeled teicoplanin added initially.

View this table:
Table 1

Kinetic parameters of [14C]teicoplanin uptake by hepatocytes in vitro (mean ± S.E.; n = 3) estimated in the presence and absence of additional teicoplanin (1 and 100 μg/ml)

In Vivo Data.

Figure 3 shows that a triexponential equation adequately fitted the plasma data obtained in vivo after bolus administration of teicoplanin to rats (Bernareggi et al., 1986; Zanolo et al., 1991). The fit of eqs. 5 and 7 to the hepatic concentration-time data is also shown. We observed generally good agreement between predicted and observed concentrations.

Figure 3
Figure 3

Fitted concentration-time curves (line) and observed concentrations (symbols) of teicoplanin in the plasma and liver after intravenous bolus administration of teicoplanin to rats.

The corresponding pharmacokinetic parameters are summarized in Table2. For the liver the estimated values were as follows: PSu, 4.4 ± 0.6 ml/h;Kvivo, 22 ± 4 ml; and fuc, 0.009 ± 0.001 (corresponding to a Kpuc of 106 ± 9).

View this table:
Table 2

Kinetic parameters in plasma and liver in vivo following intravenous administration of teicoplanin to rats (mean ± S.D.)

Discussion

Isolated hepatocytes, either in suspension or primary monolayer culture, have been used for various purposes in toxicological, biochemical, and pharmacological studies. They are relatively easy to prepare while retaining their functionality and many of the essential properties of the intact tissue, including similar permeability characteristics (Blaauboer et al., 1994).

Cell viability, essential to the meaningful interpretation of data, was assessed with a widely used method, which is based on the ability of only viable cells to exclude the dye trypan blue (Fariss et al., 1985). Although cell viability fell soon after incubation of hepatocytes with the medium (from 90–92% to 60–78%), it then stabilized for the remainder of the 4-h experiment needed to gain reasonable uptake data. Incubation times are usually only up to 2 h (Alpini et al., 1986;MacIntyre and Cutler, 1993) making direct comparison difficult. However, our viability figures are similar to those obtained by Kover et al. (1985) but somewhat lower than reported by Eaton and Klaassen (1978a), both estimated after 2 h of incubation. Such differences may be due to the different composition of the medium used as well as the procedure followed in the handling and isolation of cells (Blaauboer et al., 1994).

The integrity of the hepatocyte membrane can also be assessed by comparing the extracellular volume at the different incubation times (Alpini et al., 1986). No significant changes with incubation time were observed, suggesting that despite the low viability obtained by the trypan blue exclusion test, the duration of the incubation does not affect membrane integrity.

Regarding the uptake profiles, we interpret the positive intercept on the amount of teicoplanin associated with the hepatocytes obtained immediately after adding teicoplanin to the hepatocyte suspension (15–30 s) as representing drug bound to the exterior cell membrane, which has also been demonstrated for other compounds (Eaton and Klaassen, 1978b; Miyauchi et al., 1988). Such binding can occur even with nonviable hepatocytes (MacIntyre and Cutler, 1993). However, this last artifact was avoided by centrifuging the cells through dibutyl phthalate, a procedure that ensures that viable and nonviable cells are separated, since only the heavier viable cells can penetrate the organic oil (Fariss et al., 1985). Also, our study shows that teicoplanin enters hepatocytes, albeit slowly (Fig. 2), and binds there (as evidenced by the high Kpu value of 42), which contrasts with findings with erythrocytes, where exterior surface binding but no internal permeation occurs (Reinoso et al., 1998). Reasonably high affinity of teicoplanin for liver components was also shown in vitro using liver homogenate (Reinoso et al., 1998).

In the model used to describe the data, we assume that the species permeating across the hepatocytes is unbound drug, with the driving force being the concentration gradient of this species across the cell membrane. This is in keeping with the permeation of most drug molecules across cell membranes, although we recognize that the relationship between bound and unbound drug within the cell may be complex and cannot be unambiguously resolved based on the present data.

A linear closed two-compartment model adequately described the hepatocyte distribution data (Fig. 2). Concentration-independent distribution was observed in vitro with various tissue homogenates (Reinoso et al., 1998) and in vivo in humans (based on events in plasma), after the infusion of single increasing doses of teicoplanin of 15 to 25 mg/kg, yielding total plasma concentrations not in excess of 150 μg/ml, corresponding to an unbound concentration of 15 μg/ml (Del Favero et al., 1991). However, this study suggests that hepatocyte cell surface binding is saturable at an unbound medium concentration of 100 μg/ml. This may be true for other cells, although saturation of binding to erythrocytes was not evident when evaluated at least over the unbound medium concentration range, 1 to 10 μg/ml (Reinoso et al., 1998).

The intracellular aqueous volume (3.9 ± 2.1 μl per 106 hepatocytes), determined in this study by the difference between the total and extracellular aqueous spaces, is similar to that reported (3.4 μl per 106hepatocytes; Petzinger and Fückel, 1992) and is comparable with the cellular volume estimated by the model for teicoplanin (Vc, 8.1 ± 1.3 μl per 106 hepatocytes).

Because of the discontinuous nature of the capillary membrane of the liver (sinusoids), the hepatocyte membrane is the first barrier that most drugs encounter on entering the liver (Wisse et al., 1985). Hepatic distribution kinetics depends upon the unbound fraction of drug in blood (fu), drug permeation through tissue membranes (PSu), and tissue blood flow (Q) (Rowland and Tozer, 1995). Chou et al. (1995) concluded that tissue distribution is determined by both membrane permeability and tissue perfusion when the (fu·PSu)-to-Q ratio lies between 0.07 and 5.7; values higher than 5.7 indicate perfusion rate-limited distribution, and values lower than 0.07 indicate permeability rate-limited distribution. With a (fu·PSu)/Q of only 5.1 × 10−4 this latter condition certainly holds in the case of teicoplanin. This ratio was calculated using the PSu determined in vivo (0.07 ± 0.01 ml/min), 0.1 as the unbound fraction in plasma (Wittendorf et al., 1987) and 7.5 ml/min as the hepatic plasma flow (based on a hepatic blood flow of 13.6 ml/min) (Bernareggi and Rowland, 1991), recognizing that teicoplanin does not enter erythrocytes.

To assess the influence of binding protein in the media (fum) on the shape of the cell uptake profiles, using the mean estimated in vitro values for PSu,Vc, K, and fuc (Table 1) we performed simulations that show a progressive but modest increase in t1/2and a pronounced decrease in Kpc with decreasing fum (Fig. 4). These findings support the notion that comparison with in vivo data should be made after correcting Kpc and PS for the fraction unbound in plasma (i.e., with Kpuc, PSu). When made, Kpuc estimated from isolated hepatocytes (42 ± 10) is similar although somewhat smaller than the in vivo value (106 ± 9). The latter estimate is likely to be reasonable because the same value for Kpuc (112) is obtained using a nonmodeling approach. Namely, estimating Kpc from the same in vivo total plasma and hepatic concentration-time data (8.3) (Reinoso et al., 1998), using the area ratio method (Gallo et al., 1985), and then correcting for the plasma fraction unbound and the vascular and interstitial space volumes (Khor and Mayersohn, 1991). Moreover, compared with the Kpc determined in vitro using liver homogenate (4 ± 1) (Reinoso et al., 1998), after applying the same corrections as in in vivo, excellent agreement is observed between the in vitro liver homogenate and hepatocyte Kpucvalues (54 ± 11 versus 42 ± 10 for liver homogenate and hepatocyte, respectively).

Figure 4
Figure 4

Simulations of the influence of changes in the drug unbound fraction in the media outside the cells (fum) on the time course of teicoplanin in hepatocytes (A) and on the values of the distribution half-life (■, t1/2) and equilibrium distribution ratio (□, Kpc) (B).

Hepatocyte concentrations are normalized for the amount of radiolabeled teicoplanin added initially.

To compare in vitro with in vivo data, PSu values have to be normalized to a common surface area, taken to be that associated with the total number of hepatocytes in the liver (1.3 × 108 cells/g) (Seglen, 1973). The corresponding PSu value for hepatocytes is 5.5 ml/min based upon a PSu of 0.003 ± 2 × 10−4 ml/min per 0.83 × 106 cells and a liver weight of 10.3 g (250-g rat) (Bernareggi and Rowland, 1991). The resultant hepatocyte PSu is considerably greater than that obtained from in vivo data upon correction for the teicoplanin unbound fraction in plasma (PSu; 0.74 ± 0.10 ml/min).

The most likely explanation for the difference between in vitro and in vivo findings is the available surface area for permeation. Whereas in isolated hepatocytes all the external surface is exposed to the outer media, in vivo hepatocytes adjoin other hepatocytes and are in contact with both bile canaliculi and the perisinusoidal space of Disse (occupying 13 and 15 to 37% of the membrane area, respectively; Weibel et al., 1969), thereby effectively reducing the surface area available for permeation. Indeed, comparison of in vitro and in vivo PSu values after correcting the former for the percentage of cell surface in contact with the perisinusoidal space supports this statement (0.82–2.03 versus 0.74 ml/min).

Another explanation could be related to liver cell heterogeneity (Gumucio and Miller, 1981), which may result in different contributions of the hepatocytes to global drug uptake process, depending on their localization in the liver. During isolation, selection of subpopulations of hepatocytes could explain the in vitro-in vivo discrepancies found for teicoplanin, as has been proposed for other compounds (Blom et al., 1982; Sandker et al., 1994).

In conclusion, the results of the present study indicate that teicoplanin can enter hepatocytes in keeping with the results obtained from in vitro liver homogenate binding studies (Reinoso et al., 1998). The cellular uptake is characterized by being slow and independent of the drug concentration certainly for unbound concentrations less than 1 μg/ml, corresponding to plasma concentrations of 10 μg/ml, and probably even higher. The former is in agreement with the slow hepatic distribution of teicoplanin in vivo (Bernareggi et al., 1986; Zanolo et al., 1991).

Acknowledgments

We are grateful to Glynis Nicholls and Ivan Nestorov for their helpful advice.

Footnotes

  • Send reprint requests to: Prof. Malcolm Rowland, School of Pharmacy and Pharmaceutical Sciences, University of Manchester, Manchester M13 9PL, UK. E-mail: MRowland{at}fs1.pa.man.ac.uk

  • This research was supported by a grant from the Wellcome Trust.

  • Abbreviations used are::
    WME
    Williams' medium E
    PSu
    permeability-surface area product of cell membrane
    Kpuc
    cell-to-unbound media concentration ratio
    • Received November 2, 2000.
    • Accepted December 8, 2000.

References

  1. ↵
    1. Alpini G,
    2. Garrick RA,
    3. Jones MJT,
    4. Nunes R,
    5. Tavoloni N
    (1986) Water and nonelectrolyte permeability of isolated rat hepatocytes. Am J Physiol 251:C872–C882.
    OpenUrlAbstract/FREE Full Text
  2. ↵
    1. Antony KK,
    2. Lewis EW,
    3. Kenny MT,
    4. Dulworth JK,
    5. Brackman MB,
    6. Kuzma R,
    7. Yuh L,
    8. Eller MG,
    9. Thompson GA
    (1991) Pharmacokinetics and bioavailability of a new formulation of teicoplanin following intravenous and intramuscular administration to humans. J Pharm Sci 80:605–607.
    OpenUrlCrossRefPubMed
  3. ↵
    1. Assandri A,
    2. Bernareggi A
    (1987) Binding of teicoplanin to human serum albumin. Eur J Clin Pharmacol 33:191–195.
    OpenUrlCrossRefPubMed
  4. ↵
    1. Baur H,
    2. Kasperex S,
    3. Pfaff E
    (1975) Criteria of viability of isolated liver cells. Hoppe-Seyler's Z Physiol Chem 356:827–838.
    OpenUrlPubMed
  5. ↵
    1. Bernareggi A,
    2. Borghi A,
    3. Borgonovi M,
    4. Cavenaghi L,
    5. Ferrari P,
    6. Vékey K,
    7. Zanolo M,
    8. Zerilli LF
    (1992) Teicoplanin metabolism in humans. Antimicrob Agents Chemother 36:1744–1749.
    OpenUrlAbstract/FREE Full Text
  6. ↵
    1. Bernareggi A,
    2. Cavenaghi L,
    3. Assandri A
    (1986) Pharmacokinetics of [14C]-teicoplanin in male rats after single intravenous dose. Antimicrob Agents Chemother 30:733–738.
    OpenUrlAbstract/FREE Full Text
  7. ↵
    1. Bernareggi A,
    2. Danese A,
    3. Cometti A,
    4. Buniva G,
    5. Rowland M
    (1990) Pharmacokinetics of individual components of teicoplanin in man. J Pharmacokinet Biopharm 18:525–543.
    OpenUrlCrossRefPubMed
  8. ↵
    1. Bernareggi A,
    2. Rowland M
    (1991) Physiologic modeling of cyclosporin kinetics in rat and man. J Pharmacokinet Biopharm 19:21–50.
    OpenUrlCrossRefPubMed
  9. ↵
    1. Berry MN,
    2. Friend DS
    (1969) High yield preparation of isolated rat liver parenchymal cells. J Cell Biol 43:506–519.
    OpenUrlAbstract/FREE Full Text
  10. ↵
    1. Blaauboer BJ,
    2. Boobis AR,
    3. Castell JV,
    4. Coecke S,
    5. Groothuis GMM,
    6. Guillouzo A,
    7. Hall TJ,
    8. Hawksworth FM,
    9. Lorenzon F,
    10. Miltenburger HG,
    11. Rtogiers V,
    12. Skett P,
    13. Villa P,
    14. Wiebel FJ
    (1994) The practical applicability of hepatocyte cultures in routine testing. The report and recommendations of ECVAM workshop 1. Alternatives Lab Anim 22:231–241.
    OpenUrl
  11. ↵
    1. Blom A,
    2. Scaf AHJ,
    3. Meier DKF
    (1982) Hepatic drug transport in the rat. A comparison between isolated hepatocytes, the isolated perfused liver and the liver in vivo. Biochem Pharmacol 31:1553–1565.
    OpenUrlCrossRefPubMed
  12. ↵
    1. Borghi A,
    2. Coronelli C,
    3. Faniuolo L,
    4. Allievi G,
    5. Pallanza R,
    6. Gallo GG
    (1984) Teichomycins, new antibiotics from Actinoplanes teichomyceticus Nov. Sp. IV. Separation and characterization of the components of teichomycin (teicoplanin). J Antibiot 37:615–620.
    OpenUrlPubMed
  13. ↵
    1. Chou C-H,
    2. McLachlan AJ,
    3. Rowland M
    (1995) Membrane permeability and lipophilicity in the isolated perfused rat liver: 5-Ethyl barbituric acid and other compounds. J Pharmacol Exp Ther 275:933–940.
    OpenUrlAbstract/FREE Full Text
  14. ↵
    1. Del Favero A,
    2. Patoia L,
    3. Rosina R,
    4. Buniva G,
    5. Danese A,
    6. Bernareggi A,
    7. Molini E,
    8. Cavenaghi L
    (1991) Pharmacokinetics and tolerability of teicoplanin in healthy volunteers after single increasing doses. Antimicrob Agents Chemother 35:2551–2557.
    OpenUrlAbstract/FREE Full Text
  15. ↵
    1. Dykhuizen RS,
    2. Harvey G,
    3. Stephenson N,
    4. Nathwani D,
    5. Gould IM
    (1995) Protein binding and serum bactericidal activities of vancomycin and teicoplanin. Antimicrob Agents Chemother 39:1842–1847.
    OpenUrlAbstract/FREE Full Text
  16. ↵
    1. Eaton DL,
    2. Klaassen CD
    (1978a) Carrier-mediated transport of the organic cation procaine amide ethobromide by isolated rat liver parenchymal cells. J Pharmacol Exp Ther 206:595–606.
    OpenUrlAbstract/FREE Full Text
  17. ↵
    1. Eaton DL,
    2. Klaassen CD
    (1978b) Carrier-mediated transport of ouabain in isolated hepatocytes. J Pharmacol Exp Ther 205:480–488.
    OpenUrlFREE Full Text
  18. ↵
    1. Fariss MW,
    2. Brown MK,
    3. Schmithz JA,
    4. Reed DJ
    (1985) Mechanism of chemical-induced toxicity. I. Use of a rapid centrifugation technique for the separation of viable and nonviable hepatocytes. Toxicol Appl Pharmacol 79:283–295.
    OpenUrlCrossRefPubMed
  19. ↵
    1. Gallo M,
    2. Lam FC,
    3. Perrier DG
    (1985) Area methods for the estimation of partition coefficients for physiological pharmacokinetic models. J Pharmacokinet Biopharm 15:271–281.
  20. ↵
    1. Gumucio JJ,
    2. Miller D
    (1981) Functional implications of liver cell heterogeneity. Gastroenterology 80:393–403.
    OpenUrlPubMed
  21. ↵
    1. Hayes KA,
    2. Brennan B,
    3. Chenery R,
    4. Houston BJ
    (1993) In vivo disposition of caffeine predicted from hepatic microsomal and hepatocyte data. Drug Metab Dispos 23:349–353.
    OpenUrlAbstract
  22. ↵
    1. Khor SP,
    2. Mayersohn M
    (1991) Potential errors in the measurement of tissue to blood distribution coefficients in physiological pharmacokinetic modeling. Drug Metab Dispos 19:478–485.
    OpenUrlAbstract
  23. ↵
    1. Kover K,
    2. Hung CH,
    3. Moore WV
    (1985) Enhancement of rat growth hormone binding by membrane disulphide reduction. Endocrinology 116:1017–1023.
    OpenUrlCrossRefPubMed
  24. ↵
    1. MacIntyre AC,
    2. Cutler DJ
    (1993) Kinetics of chloroquine uptake into isolated rat hepatocytes. J Pharm Sci 82:592–600.
    OpenUrlCrossRefPubMed
  25. ↵
    1. McNamara PJ,
    2. Gibaldi M,
    3. Stoeckel F
    (1983) Fraction unbound in interstitial fluid. J Pharm Sci 72:834–836.
    OpenUrlCrossRefPubMed
  26. ↵
    1. Miyauchi S,
    2. Sugiyama Y,
    3. Iga T,
    4. Hanano M
    (1988) Membrane-limited hepatic transport of the conjugative metabolites of 4-methylumbelliferone in rats. J Pharm Sci 8:688–692.
    OpenUrlCrossRef
  27. ↵
    1. Petzinger E,
    2. Fückel D
    (1992) Evidence for a saturable, energy-dependent and carrier-mediated uptake of oral antidiabetics into rat hepatocytes. Eur J Pharmacol 213:381–391.
    OpenUrlCrossRefPubMed
  28. ↵
    1. Raghoebar M,
    2. Huisman JAM,
    3. Van Den Berg WB,
    4. Van Ginneken CAM
    (1987) An in vitro approach to study cellular kinetics of drugs. J Pharmacol Methods 18:239–251.
    OpenUrlCrossRefPubMed
  29. ↵
    1. Reinoso RF,
    2. Telfer BA,
    3. Rowland M
    (1998) In vitro studies of teicoplanin binding to rat tissues and erythrocytes Eur J Pharm Sci 6:145–152.
  30. ↵
    1. Rowland M,
    2. Tozer TN
    (1995) Clinical Pharmacokinetics. Concepts and Applications (Williams & Wilkins, Baltimore), 3rd ed..
  31. ↵
    1. SAAM Institute
    (1994) SAAM II Users Guide Version 1.0. (University of Washington, Seattle, WA).
  32. ↵
    1. Sandker GW,
    2. Weert B,
    3. Olinga P,
    4. Wolters H,
    5. Slooff MJH,
    6. Meijer DKF,
    7. Groothuis GMM
    (1994) Characterization of transport in isolated human hepatocytes. A study with the bile acid taurocholic acid, the uncharged ouabain and the organic cations vecuronium and rocuronium. Biochem Pharmacol 47:2193–2200.
    OpenUrlCrossRefPubMed
  33. ↵
    1. Seglen PO
    (1973) Preparation of rat liver cells. III. Enzymatic requirement for tissue dispersion. Exp Cell Res 82:391–398.
    OpenUrlCrossRefPubMed
  34. ↵
    1. Strom SC,
    2. Jirtle RL,
    3. Jones RS,
    4. Novicki DL,
    5. Rosenberg MR,
    6. Novotny A,
    7. Irons G,
    8. McLain JR,
    9. Michalopoulos G
    (1982) Isolation, culture and transplantation of human hepatocytes. J Natl Cancer Inst 68:771–778.
  35. ↵
    1. Weibel ER,
    2. Staubli W,
    3. Gnagi HR,
    4. Hess FA
    (1969) Correlated morphometric and biochemical studies on the liver cell. I. Morphometric model, stereologic methods, and normal morphometric data for rat liver. J Cell Biol 42:68–91.
    OpenUrlAbstract/FREE Full Text
  36. ↵
    1. White CO,
    2. Reeves DS
    (1991) Problems with the assay of teicoplanin caused by differential binding of the A2 components to plastics and to glass. Program and Abstracts of the 31st Interscience Conference on Antimicrobial Agents and Chemotherapy, (Abstracts 1312 and 317), American Society for Microbiology, Washington..
  37. ↵
    1. Wisse E,
    2. De Zanger RB,
    3. Charels K,
    4. Van Der Smissen R,
    5. McCuskey RS
    (1985) The liver sieve: Considerations concerning the structure and function of endothelial fenestrae, the sinusoidal wall and the space of Disse. Hepatology 5:683–692.
    OpenUrlCrossRefPubMed
  38. ↵
    1. Wittendorf RW,
    2. Swagzdis JE,
    3. Gifford R,
    4. Mico BA
    (1987) Protein binding of glycopeptide antibiotics with diverse physical-chemical properties in mouse, rat and human serum. J Pharmacokinet Biopharm 15:5–13.
    OpenUrlCrossRefPubMed
  39. ↵
    1. Zanolo G,
    2. Bernareggi A,
    3. Cavenaghi L,
    4. Giachetti C,
    5. Tognolo C
    (1991) Distribution and excretion of teicoplanin in rats after single and repeated intravenous administration. Eur J Drug Metab Pharmacokinet (Suppl 3) 85–93.
View Abstract
Back to top

In this issue

Download PDF
Article Alerts
Email Article
Citation Tools
Research ArticleArticle

Uptake of Teicoplanin by Isolated Rat Hepatocytes: Comparison with in Vivo Hepatic Distribution

Raquel F. Reinoso, Brian A. Telfer, Beverley S. Brennan and Malcolm Rowland
Drug Metabolism and Disposition April 1, 2001, 29 (4) 453-459;
del.icio.us logo Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo

Jump to section