QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: dmd.106.010793v1 34/9/1600    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Lu, C. Articles by Gan, L.-S. Search for Related Content PubMed PubMed Citation Articles by Lu, C. Articles by Gan, L.-S.

Comparison of Intrinsic Clearance in Liver Microsomes and Hepatocytes from Rats and Humans: Evaluation of Free Fraction and Uptake in Hepatocytes

Drug Metabolism and Pharmacokinetics, Drug Safety and Disposition, Millennium Pharmaceuticals, Inc., Cambridge, Massachusetts

(Received April 24, 2006; accepted June 19, 2006)

	Abstract

Top Abstract Materials and Methods Results Discussion References

 
Apparent intrinsic clearance (CL_int,app) of 7-ethoxycoumarin, phenacetin, propranolol, and midazolam was measured using rat and human liver microsomes and freshly isolated and cryopreserved hepatocytes to determine factors responsible for differences in rates of metabolism in these systems. The cryopreserved and freshly isolated hepatocytes generally provided similar results, although there was greater variability using the latter system. The CL_int,app values in hepatocytes are observed to be lower than that in microsomes, and this difference becomes greater for compounds with high CL_int,app. This could partly be attributed to the differences in the free fraction (f_u). The f_u in hepatocyte incubations (f_u,hep-inc) was influenced not only by the free fraction of compounds in the incubation buffer (f_u,buffer) but also by the rate constants of uptake (k_up) and metabolism (k_met). This report provides a new derivation for f_u,hep-inc, which can be expressed as f_u,hep-inc = [k_up/(k_met + k_up)]/[1 + (C_hep/C_buffer) x (V_hep/V_buffer)], where the C_hep, C_buffer, V_hep, and V_buffer represent the concentrations of a compound in hepatocytes and buffer and volumes of hepatocytes and buffer, respectively. For midazolam, the f_u,hep-inc was calculated, and the maximum metabolism rate in hepatocytes was shown to be limited by the uptake rate.

View larger version (12K): [in this window] [in a new window]   FIG. 1. Hepatocyte suspension model, a dynamic system where the rate of uptake equals the rate of metabolism plus diffusion from inside to outside at any given time. The rate constant of diffusion across either sides are assumed to be the same. The active uptake and efflux transporters were ignored for simplicity.

	Materials and Methods

Top Abstract Materials and Methods Results Discussion References

 
Reagents. Pooled human and rat liver microsomes and pooled cryopreserved rat hepatocytes were purchased from XenoTech LLC (Kansas City, KS). Cryopreserved human hepatocytes (pool of four in the experiment) were purchased from In Vitro Technologies, Inc. (Baltimore, MD). Fresh human hepatocytes were purchased from BD Gentest (Woburn, MA) and In Vitro Technologies, Inc. Fresh rat hepatocytes were prepared in-house. All hepatocytes used in this study had viability of >80%. Midazolam, 7-ethoxycoumarin, phenacetin, and propranolol were purchased from Sigma-Aldrich (St. Louis, MO).

CL_int,app Determination in Microsomes and Hepatocytes. Microsomes (0.5 mg/ml) were preincubated with 2 µM test compound for 5 min at 37°C in 0.1 M phosphate buffer, pH 7.4. The reactions were initiated by adding prewarmed cofactors (2 mM NADPH and 3 mM MgCl₂). After 0-, 3-, 7-, 12-, 20-, and 30-min incubations at 37°C, the reactions were stopped by adding an equal volume of acetonitrile containing 1 µM carbutamide (internal standard). The samples were kept in a refrigerator for 30 min and then centrifuged at 3000g for 10 min. The supernatants were analyzed with LC/MS/MS for the amount of parent compound remaining.

Calculation of apparent intrinsic clearance:

a: 20 and 45 g of liver/kg of body weight were used for human and rat, respectively (Lin et al., 1996).

Likewise, CL_int,app determinations in hepatocytes were performed in Krebs-Henseleit buffer, pH 7.4, with 1.0 x 10⁶ hepatocytes/ml (viability >80%) and 2 µM test compound. The incubations were carried out in a 37°C CO₂ (5%) incubator as described previously (Li et al., 1999a). The reactions were stopped at 0, 30, 60, 120, 180, and 240 min with the addition of an equal volume of acetonitrile containing 1 µM carbutamide. A value of 135 x 10⁶ hepatocyte/g of liver (Houston, 1994) was used in the CL_int,app calculation (equation above).

Hepatocyte Uptake. Cryopreserved hepatocytes were thawed out and suspended in Krebs-Henseleit buffer at 2.0 x 10⁶ hepatocytes/ml as described previously (Shitara et al., 2003). Centrifuge tubes were prepared by loading 150 µl of silicone/mineral oil on top of 50 µl of 5 N NaCl/0.2% saponin. In a 37°C water bath, 0.5-ml hepatocyte suspensions were preincubated in the presence or absence of 10 µM ketoconazole for 10 min. Uptake was initiated by adding 0.5 ml of 4 µM midazolam into the hepatocytes. At 0.5, 2, 4, 10, 20, and 30 min, 100 µl of the cell suspensions were transferred into the centrifuge tubes containing the silicone/mineral oil and the NaCl/0.2% saponin, and the uptake was terminated by separating the hepatocytes from the midazolam solution with a 10-s centrifugation at 10,000g. Midazolam concentrations in the hepatocytes, as well as the uptake solution, were analyzed using LC/MS/MS with appropriate standard curves.

Microsomal Protein Binding. Microsomal protein binding assay was adapted from a published procedure (Obach, 1997): microsomes (0.5 mg/ml) were mixed with 2 µM test compound in 0.1 M phosphate buffer, pH 7.4, containing 3 mM MgCl₂ (the dialysis buffer). The mixture was subjected to an overnight dialysis at 37°C against the dialysis buffer using the Spectrum apparatus (Spectrum, Los Angeles, CA). The retrieved microsomes were then diluted in 2 volumes of the dialysis buffer, and dialysate from the receiving side was diluted in half-volume of the control microsomes. After the protein in the samples from the donor and the receiver was precipitated in an equal volume of acetonitrile containing 1 µM carbutamide, the supernatants were analyzed using LC/MS/MS for the amount of parent compound remaining. The free fractions were calculated as:

LC/MS/MS Analyses. Peak area ratios of test compounds and carbutamide (internal standard) were determined by a LC/MS/MS system which consisted of an Agilent 1100 HPLC (Agilent, Palo Alto, CA), a Leap CTC PAL autosampler (LEAP Technologies Inc., Carrboro, NC), and a SCIEX API 4000 detector (Applied Biosystems, Concord, ON, Canada). Separation was performed on a Waters YMC Basic, 3 µM, 50 x 2.0-mm column (Waters, Milford, MA), eluted at a flow rate of 0.5 ml/min. Mobile phase A was 0.1% (v/v) formic acid in water, and mobile phase B was 0.1% (v/v) formic acid in acetonitrile. The gradient consisted of 30% of mobile phase B for 0.5 min after injection and increased linearly to 90% B from 0.5 to 1.5 min. Mobile phase B was held at 90% from 1.5 to 2.3 min, and the column was re-equilibrated to 30% B from 2.3 to 3.5 min. All compounds were detected by positive ion spray in the multiple-reaction monitoring mode using predetermined parent/product mass transition ion pairs.

	Results

Top Abstract Materials and Methods Results Discussion References

 
The apparent intrinsic clearance results are presented in Table 1. Phenacetin, 7-ethoxycoumarin (7-EC), propranolol, and midazolam are primarily metabolized by phase I enzymes. The CL_int,app values for all compounds in hepatocytes were much lower compared with those in microsomes, with midazolam showing the highest differential and, hence, has been studied in detail. All four compounds showed comparable CL_int,app in freshly isolated (three experiments) and cryopreserved (five experiments) rat hepatocytes with a similar interexperimental variation (Table 2). Furthermore, the mean CL_int,app values in freshly isolated and cryopreserved rat hepatocytes were comparable, suggesting that the enzyme activity in the cryopreserved rat hepatocytes was at levels comparable with those in the freshly isolated rat hepatocytes. In the cryopreserved human hepatocyte study, these four compounds also showed a similar interday variation in enzyme activity as that in cryopreserved rat hepatocytes. However, in freshly isolated human hepatocytes, the interexperimental variation was notably higher (Table 2). Unlike the cryopreserved hepatocyte study, which uses the same characterized and pooled hepatocytes, the fresh human hepatocyte studies were subject to interindividual variation in enzyme activities, limited characterization at the time of the experiment, and a possibility of damaged hepatocytes during shipment. On the other hand, cryopreservation is known to preserve most of the P450 activities of the original freshly isolated hepatocytes (Li et al., 1999b; Madan et al., 1999). Because the mean values of CL_int,app in cryopreserved and freshly isolated human hepatocytes in our study seemed to be similar, cryopreserved human hepatocytes could be a valid model for CL_int,app determinations.

In the hepatocyte uptake study, after separating hepatocytes from the incubation buffer at different time points, the concentrations of midazolam in both hepatocytes and buffer were measured. In the human hepatocyte incubations, midazolam was depleted from the incubation buffer at the same rate as in the rat hepatocyte incubations. In contrast to hepatocytes, the clearance of midazolam in the liver microsomal incubation from rats was faster than that from humans (Table 1). When the selective CYP3A4 inhibitor ketoconazole was coincubated with midazolam (a selective CYP3A4 substrate), midazolam was no longer cleared from the incubation buffer (Fig. 2). These data suggested that 1) the removal of midazolam from the incubation buffer is also due to metabolism in hepatocytes, and 2) the uptake is the rate-limiting step. Taken together, these observations suggest that the midazolam uptake rates in rats and humans are similar to each other and that both rates are lower compared with the corresponding metabolism rates; i.e., as soon as midazolam gets into the hepatocytes, it is cleared rapidly by metabolism. It is reasonable to believe that midazolam uptake rates are similar in human and rat hepatocytes because they reflect simple diffusion across the cell membranes. Midazolam is not reported to be a substrate of any active uptake or efflux transporters; therefore, its uptake is not affected by ketoconazole. Ketoconazole is an inhibitor of several uptake and efflux transporters and CYP3A (Azer et al., 1995; Salphati and Benet, 1998).

View larger version (21K): [in this window] [in a new window]   FIG. 2. Midazolam uptake in human and rat hepatocytes. Measurement of midazolam disappearance in the incubation buffer after removing hepatocytes at each incubation time point.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
In our study, the CL_int,app values of 7-EC, phenacetin, propranolol, and midazolam, all cleared by phase I metabolism as the first step, were significantly lower in hepatocyte incubations compared with microsomal incubations. Similar observation was recently reported by Hallifax and colleagues (2005a), where they compared CYP1A2, 2C9, 2D6, 2E1, and 3A4 activities in cryopreserved hepatocytes from approximately 200 donors with that in microsomes from approximately 100 donors. The P450 activities in hepatocytes were found to be 2.5- to 20-fold lower than in microsomes. For example, using testosterone as the substrate, CYP3A4 activity was 11-fold lower in hepatocytes. Many factors could attribute to this difference, such as nonspecific binding or quality of cryopreserved hepatocytes. The present work provides an explanation for this difference from the perspective of free fraction in hepatocyte incubations for compounds that involve diffusional movement across cell membrane. Because hepatocytes are intact cells, compounds have to get into the cells before they can reach the metabolizing enzymes. The rate of compound clearance is, therefore, dependent on the uptake rate as well as the metabolism rate. Two important scenarios arise. If the uptake rate is much faster than the metabolism rate, then the overall clearance is metabolic rate limited. Conversely, if the metabolism rate is much faster than the uptake rate, the overall clearance is uptake rate limited.

Transcellular (diffusional) uptake is the primary route of entry into hepatocytes for most of compounds (Brayden, 1997). It is usually fast because it does not require an energy source or a salt gradient as most active uptake processes do. This discussion focuses on the transcellular uptake to quantitatively explore the relationship between hepatocyte clearance and hepatocyte uptake/metabolism. In hepatocytes, when the intracellular free concentration (C_hep,u) is « the Michaelis-Menten constant (K_m), the apparent metabolic rate is:

(1)

and the uptake rate in the hepatocytes is:

(2)

where k_up is the uptake rate constant, k_met is the metabolism rate constant, and C_buffer,u is the free concentration in buffer which is same as the extracellular free concentration (Fig. 1). Assuming that diffusion across membrane is identical in both directions, the removal rate from the hepatocytes is:

(3)

where the term k_met x C_hep,u represents metabolic rate, and k_up x C_hep,u represents the diffusion of compound from inside the hepatocytes to the outside buffer. After the compound is added to the incubation mixture, a dynamic equilibrium is established where the rate of compound being taken up by the hepatocytes equals the rate of compound being removed from the hepatocytes (by diffusional and metabolism) at any given time (Fig. 1), thus the uptake rate can be expressed as:

(4)

Dividing eq. 1 by eq. 4:

(5)

By multiplying both the numerator and denominator of the right side of eq. 5 by C_buffer,u and cancelling out the C_hep,u, eq. 5 becomes

(6)

Equation 6 can then be rewritten as:

(7)

where v_met,max = k_met x C_buffer is the maximum metabolic rate if C_buffer,u = C_hep,u. In other words, this would be the highest possible metabolic rate of the compound in hepatocytes as if the enzymes were exposed to the extracellular concentration. Equation 7 demonstrates the hyperbolic relationship between apparent metabolic rate and the maximum metabolic rate, where the uptake rate is the upper limit. For compounds that have a lower rate of metabolism, the effect of v_up on the clearance rate is minimal. However, for a rapidly metabolized compound, the apparent metabolic rate could plateau at v_up.

Intrinsic clearance can be calculated from the apparent intrinsic clearance by correcting with the free fraction of the compound to which the enzymes are exposed (Obach, 1999). The f_u in hepatocyte incubation (f_u,hep-inc) could be expressed as the ratio of intracellular free concentration (C_hep,u) to total incubation concentration (C_total), which is the amount of compound in hepatocytes (C_hep x V_hep) and buffer (C_buffer x V_buffer) divided by the incubation volume (V_buffer + V_hep):

(8)

where C_hep is the total concentration of compound in hepatocytes, i.e., free + bound to intracellular and membrane proteins, and C_buffer equals the C_buffer,u because no protein is present in the incubation buffer. The f_u,hep-inc could be simplified assuming that the volume of the hepatocytes compared with the volume of the buffer is minimal (V_hep « V_buffer):

(9)

Equation 9 can be rearranged to:

(10)

Equation 4 can be rearranged as:

(11)

By placing eq. 11 into eq. 10, we get:

(12)

At a true steady state where there is no metabolism to remove the compound (k_met = 0 or « k_up), the intracellular free drug concentration equilibriums with the extracellular free drug concentration. Equation 12 can be simplified to:

(13)

Thus, the intracellular free drug concentration approaches the extracellular free drug concentration. However, in eq. 12, when metabolism is very high compared with the uptake, the numerator becomes a small fraction, and the f_u,hep-inc becomes less than f_u,buffer. Therefore, the intracellular free concentration becomes much lower than the extracellular free concentration. This explains why the rapidly metabolized compound midazolam has a much lower CL_int,app in hepatocytes compared with that in microsomal incubations. As illustrated in eq. 12, the f_u in hepatocyte incubations can be calculated from the experimental data using C_hep, V_hep, C_buffer, V_buffer, k_met, and k_up.

The uptake rate in a hepatocyte incubation can be calculated from the amount of a compound appearing in hepatocytes at any given period of time. But this method cannot distinguish the fraction of compound nonspecifically bound to the outer membrane of the hepatocytes from the fraction uptaken into the hepatocytes. In an incubation where a compound's metabolism rate is greater than its uptake rate, monitoring the disappearance of the compound from the incubation buffer provides an easier way to determine the uptake rate. In our study, the metabolism rate constants of midazolam in rats and humans, converted from the microsomal data with correction of f_u, were 0.370 and 0.167/min/(million hepatocytes/ml), respectively. Putting values of rate constants of metabolism and uptake, as well the concentrations and volumes of the hepatocytes and incubation buffer in eq. 12, the f_u values of midazolam in rat and human hepatocyte incubations were calculated to be 0.075 and 0.129, respectively (Table 3). Noticeably, before being corrected for f_u, the apparent intrinsic clearance of midazolam in rat microsomal incubation was approximately 40-fold higher than that in hepatocytes. However, after a correction for f_u in both systems, the difference in CL_int is reduced to approximately 3.8-fold. Considering that the microsomal and hepatocyte incubations are two widely different experimental systems involving two different scale-up factors to calculate the f_u, this 3.8-fold difference may be considered acceptable. Likewise, a smaller difference (3.5-fold) was observed in intrinsic clearances between human microsomal and hepatocyte incubations after a correction of both values with f_u. The intrinsic clearance of midazolam using human hepatocytes was significantly lower than that in rat hepatocytes. This was consistent with the reported lower plasma clearance of 0.40 l/h/kg in humans (Hardman et al., 2001) compared with 4.75 l/h/kg in rats (Kotegawa et al., 2002). A recent study suggested that the metabolism of midazolam in human hepatocytes would not reach saturation until the total incubation concentration reaches 100 µM (Zhao et al., 2005). This is consistent with our study. The f_u,hep-inc of midazolam in human hepatocytes calculated from eq. 12 is 0.129 (Table 3). Therefore, a 100 µM concentration in hepatocyte incubation would have an intracellular free concentration around 12.9 µM. At such a concentration midazolam, which has K_m around 4 µM determined in human liver microsomes (Pelkonen et al., 1998), would have its metabolism rate at saturation.

The CL_int,app values for midazolam in hepatocytes, measured at subK_m concentrations using the method of disappearance of parent compound, are consistent with those reported by Lave et al. (1997) and Lau et al. (2002). It is interesting to note that several reports (Obach, 1999; Kotegawa et al., 2002; Walsky and Obach, 2004; Hallifax et al., 2005) show different CL_int,app values measured using the alternate method of V_max/K_m. This discrepancy may be attributable to the V_max/K_m approach for estimating CL_int,app, where saturating concentrations of midazolam were used and enzymes in hepatocytes metabolized midazolam at full capacity. One has to be cautious when using the data from the V_max/K_m approach because midazolam, at high nonphysiological concentrations, is known to be a "substrate inhibitor" of CYP3A (Houston and Kenworthy, 2000; Galetin et al., 2003; Hallifax et al., 2005), thereby also showing reduced clearance in microsomes.

In conclusion, hepatocytes, being closer to the in vivo liver system, are ideally suited for the prediction of in vivo hepatic clearance. Microsomes are still valuable for clearance determination for compounds metabolized primarily by phase I enzymes. The determination of intrinsic clearance using hepatocytes is dependent on CL_int,app, which in turn, is dependent on the metabolism and cellular uptake rates. A derivation of an equation to calculate f_u,hep-inc is provided, and its use to account for the lower in vitro clearance in human and rat hepatocytes compared with microsomes for midazolam, a compound which shows high clearance using microsomal system, is demonstrated.

	Footnotes

 
Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.

doi:10.1124/dmd.106.010793.

ABBREVIATIONS: CL_int, intrinsic clearance; CL_int,app, apparent intrinsic clearance; P450, cytochrome P450; LC/MS/MS, liquid chromatography coupled to tandem mass spectrometry; 7-EC, 7-ethoxycoumarin; f_u,hep-inc, free fraction in hepatocyte incubations.

Address correspondence to: Chuang Lu, Millennium Pharmaceuticals, Inc., 40 Landsdowne Street, Cambridge, MA 02139. E-mail: chuang.lu{at}mpi.com

	References

Top Abstract Materials and Methods Results Discussion References

 


Austin RP, Barton P, Mohmed S, and Riley RJ (2005) The binding of drug to hepatocytes and its relationship to physicochemical properties. Drug Metab Dispos 33: 419–425.[Abstract/Free Full Text]

Azer SA, Kukongvirviyapan V, and Stacey NH (1995) Mechanism of ketoconazole-induced elevation of individual serum bile acids in the rat:relationship to the effect of ketoconazole on bile acid uptake by isolated hepatocytes. J Pharmacol Exp Ther 273: 1231–1237.

Bachmann K, Byers J, and Ghosh R (2003) Prediction of in vivo hepatic clearance from in vitro data using cryopreserved human hepatocytes. Xenobiotica 33: 475–483.[CrossRef][Medline]

Brayden D (1997) Human intestinal epithelial cell monolayers as prescreens for oral drug delivery. Pharm News 4: 11–15.

Galetin A, Clarke S, and Houston JB (2003) Multisite kinetic analysis of interactions between prototypical CYP3A4 subgroup substrates: midazolam, testosterone, and nifedipine. Drug Metab Dispos 31: 1108–1116.[Abstract/Free Full Text]

Griffin SJ and Houston JB (2004) Comparison of fresh and cryopreserved rat hepatocytes suspensions for the prediction of in vitro intrinsic clearance. Drug Metab Dispos 32: 552–558.[Abstract/Free Full Text]

Hallifax D, Griffin M, and Houston JB (2005a) Prediction of hepatic clearance from in vitro: Variability and bias among human microsomes and hepatocytes. Drug Metab Rev (Suppl 1) 37: 42.

Hallifax D, Rawden HC, Hakooz N, and Houston JB (2005) Prediction of metabolic clearance using cryopreserved human hepatocytes: kinetic characteristics for five benzodiazepines. Drug Metab Dispos 33: 1852–1858.[Abstract/Free Full Text]

Hardman JG, Limbird LE, and Gilman AG, eds (2001) Goodman & Gilman's The Pharmacological Basis of Therapeutics, pp 1983, McGraw-Hill, New York.

Houston JB (1994) Utility of in vitro drug metabolism data in predicting in vivo metabolic clearance. Biochem Pharmacol 47: 1469–1479.[CrossRef][Medline]

Houston JB and Carlile DJ (1997) Incorporation of in vitro drug metabolism data into physiologically-based pharmacokinetic model. Toxicol In Vitro 11: 473–478.

Houston JB and Kenworthy KE (2000) In vitro-in vivo scaling of CYP kinetic data not consistent with the classical Michaelis-Menten model. Drug Metab Dispos 28: 246–254.[Abstract/Free Full Text]

Ito K and Houston JB (2004) Comparison of the use of liver model for predicting drug clearance using in vitro kinetic data from hepatic microsomes and isolated hepatocytes. Pharm Res (NY) 21: 785–792.

Jones HM and Houston JB (2004) Substrate depletion approach for determining in vitro metabolic clearance: time dependencies in hepatocyte and microsomal incubations. Drug Metab Dispos 32: 973–982.[Abstract/Free Full Text]

Kotegawa T, Laurijssens BE, Von Moltke LL, Cotreau MM, Perloff MD, Venkatakrishnan K, Warrington JS, Granda BW, Harmatz JS, and Greenblatt DJ (2002) In vitro, pharmacokinetics, and pharmacodynamic interactions of ketoconazole and midazolam in the rat. J Pharmacol Exp Ther 302: 1228–1237.[Abstract/Free Full Text]

Lau YY, Sapidou E, Cui X, White RE, and Cheng KC (2002) Development of a novel in vitro model to predict hepatic clearance using fresh, cryopreserved, and sandwich-cultured hepatocytes. Drug Metab Dispos 30: 1446–1454.[Abstract/Free Full Text]

Lave T, Dupin S, Schmitt C, Chou RC, Jaeck D, and Coassolo P (1997) Integration of in vitro data into algometric scaling to predict hepatic metabolic clearance in man: application to 10 extensively metabolized drugs. J Pharm Sci 86: 584–590.[CrossRef][Medline]

Li AP, Hartman NR, Lu C, Collins JM, and Strong JM (1999a) Effects of cytochrome P450 inducers on 17a-ethynylestradiol (EE2) conjugation in primary human hepatocytes. Br J Clin Pharmacol 48: 733–742.[CrossRef][Medline]

Li AP, Lu C, Brent JA, Pham C, Fackett A, Ruegg CE, and Silber PM (1999b) Cryopreserved human hepatocytes: characterization of drug-metabolizing enzyme activities and applications in higher throughput screening assays for hepatotoxicity, metabolic stability, and drug-drug interaction potential. Chem-Biol Interact 121: 17–35.[CrossRef][Medline]

Lin JH, Chiba M, Balani SK, Chen IW, Kwei GY, Vastag KJ, and Nishime JA (1996) Species differences in the pharmacokinetics and metabolism of indinavir, a potent human immunodeficiency virus protease inhibitor. Drug Metab Dispos 24: 1111–1120.[Abstract]

Madan A, Dehaan R, Mudra D, Carrol K, Lecluyse E, and Parkinson A (1999) Effect of cryopreservation on cytochrome P-450 enzyme induction in cultured hepatocytes. Drug Metab Dispos 27: 327–335.[Abstract/Free Full Text]

McGinnity DF, Soars MG, Urbanowicz RA, and Riley RL (2004) Evaluation of fresh and cryopreserved hepatocytes as in vitro drug metabolism tools for the prediction of metabolic clearance. Drug Metab Dispos 32: 1247–1253.[Abstract/Free Full Text]

Naritomi Y, Terashita S, Kimura S, Suzuki A, Kagayama A, and Sugiyama Y (2001) Prediction of human hepatic clearance from in vivo animal experiments and in vitro metabolic studies with liver microsomes from animals and humans. Drug Metab Dispos 29: 1316–1324.[Abstract/Free Full Text]

Obach RS (1997) Nonspecific binding to microsomes: impact on scale-up of in vitro intrinsic clearance to hepatic clearance as assessed through examination of warfarin, imipramine, and propranolol. Drug Metab Dispos 25: 1359–1369.[Abstract/Free Full Text]

Obach RS (1999) Prediction of human clearance of twenty-nine drugs from hepatic microsomal intrinsic clearance data: an examination of in vitro half-life approach and nonspecific binding to microsomes. Drug Metab Dispos 27: 1350–1359.[Abstract/Free Full Text]

Obach RS, Baxter JG, Liston TE, Silber BM, Jones BC, MacIntyre F, Rance DJ, and Wastall P (1997) The prediction of human pharmacokinetic parameters from preclinical and in vivo metabolism. J Pharmacol Exp Ther 283: 46–58.[Abstract/Free Full Text]

Pelkonen O, Maenpaa J, Taavitsainen P, Rautio A, and Raunio H (1998) Inhibition and induction of human cytochrome P450 (CYP) enzymes. Xenobioteca 28: 1203–1253.

Salphati L and Benet LZ (1998) Effects of ketoconazole on digoxin absorption and disposition in rat. Pharmacology 56: 308–313.[CrossRef][Medline]

Shitara Y, Li AP, Kato Y, Lu C, Ito K, Itoh T, and Sugiyama Y (2003) Function of uptake transporters for taurocholate and estradiol 17ß-D-glucuronide in cryopreserved human hepatocytes. Drug Metab Pharmacokinet 18: 33–41.[CrossRef][Medline]

Tan E and Pang S (2001) Sulfation is the rate limiting in the futile cycling between estrone and estrone sulfate in enriched periportal and perivenous rat hepatocytes. Drug Metab Dispos 29: 335–346.[Abstract/Free Full Text]

Walsky RL and Obach RS (2004) Validated assay for human cytochrome P450 activities. Drug Metab Dispos 32: 647–660.[Abstract/Free Full Text]

Witherow LE and Houston JB (1999) Sigmoidal kinetics of CYP3A substrates: an approach for scaling dextromethorphan metabolism in hepatic microsomes and isolated hepatocytes to predict in vivo clearance in rats. J Pharmacol Exp Ther 290: 58–65.[Abstract/Free Full Text]

Zhao P, Kunze KL, and Lee CA (2005) Evaluation of time-dependent inactivation of CYP3A in cryopreserved human hepatocytes. Drug Metab Dispos 32: 335–346.

This article has been cited by other articles:

				J. B. Dennison, M. A. Mohutsky, R. J. Barbuch, S. A. Wrighton, and S. D. Hall Apparent High CYP3A5 Expression Is Required for Significant Metabolism of Vincristine by Human Cryopreserved Hepatocytes J. Pharmacol. Exp. Ther., October 1, 2008; 327(1): 248 - 257. [Abstract] [Full Text] [PDF]

				M. G. Soars, K. Grime, J. L. Sproston, P. J. H. Webborn, and R. J. Riley Use of Hepatocytes to Assess the Contribution of Hepatic Uptake to Clearance in Vivo Drug Metab. Dispos., June 1, 2007; 35(6): 859 - 865. [Abstract] [Full Text] [PDF]

				P. Zhao, C. A. Lee, and K. L. Kunze Sequential Metabolism Is Responsible for Diltiazem-Induced Time-Dependent Loss of CYP3A Drug Metab. Dispos., May 1, 2007; 35(5): 704 - 712. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: dmd.106.010793v1 34/9/1600    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Lu, C. Articles by Gan, L.-S. Search for Related Content PubMed PubMed Citation Articles by Lu, C. Articles by Gan, L.-S.