Materials and Methods

Materials.

The 38 compounds for animal studies were discovery compounds from Schering Plough Research Institute (Kenilworth, NJ), and the 26 compounds for human studies were randomly selected from the literature and obtained from Sigma-Aldrich (St. Louis, MO) with the exception of sildenafil (obtained from Schering Plough Research Institute, Kenilworth, NJ). Cryopreserved hepatocytes were obtained from In Vitro Technologies (Baltimore, MD), and fresh and sandwich-cultured hepatocytes were obtained from Cedra (Austin, TX). Waymouth's 752/1 medium, with l-glutamine and without phenol red was obtained from Mediatech (Herndon, VA).

In Vivo Pharmacokinetic Data.

In vivo clearance data in rats, dogs, and cynomolgus monkeys were generated in-house after intravenous (0.6- to 10-mg/kg dose) administration to three or more animals per compound. Plasma samples were collected at various time points and analyzed. Total body clearances were calculated from the ratio of dose/AUC. Human in vivo clearance values were obtained from the literature.

Preparation of Hepatocytes.

Cryopreserved hepatocytes were thawed in a water bath at 37°C and transferred to a 50-ml tube containing 15 ml of medium at 4°C. The hepatocytes were centrifuged at 750 rpm at 4°C for 5 min. The supernatant was removed and the hepatocytes were washed two more times with medium at 4°C. The hepatocyte pellet was gently resuspended in medium to a final density of 2 million cells/ml. Fresh hepatocyte suspensions were shipped in 50-ml tubes containing a gel matrix. The hepatocyte suspension was thawed in a water bath at 37°C and transferred to a 50-ml tube containing 25 ml of medium at 37°C. The hepatocytes were washed and resuspended in the same way as cryopreserved hepatocyte. The sandwich-cultured hepatocyte plates shipped in a gel matrix were thawed in the incubator at 37°C and washed with medium twice before use.

Cryopreserved animal hepatocytes were received as pooled from five to seven individuals; therefore, no pooling was needed. For the cryopreserved human hepatocytes, each lot was from a single individual; therefore, five or more different lots were pooled before incubation. Because fresh animal hepatocytes were from a single individual, pooling was not possible; therefore, clearance studies were done with two separate lots. The viability of each hepatocyte preparation was determined by trypan blue staining method immediately before use and after 2 h of incubation.

In Vitro Hepatocyte Incubations.

Incubations were carried out at a final test compound concentration of 1 μM for cryopreserved and 2 μM for fresh hepatocytes. Stock solutions of the compounds were prepared in methanol and diluted to the desired concentrations before adding to the hepatocytes. Incubations were carried out with a hepatocyte concentration of 1 million cells/ml in Waymouth's medium. Samples (1 ml) were incubated in 24-well plates at 37°C for 2 h in a CO₂ incubator. At 0, 10, 20, 40, 60, and 120 min a 100-μl aliquot was removed and added to a 96-well plate containing 200 μl of acetonitrile with internal standard.

For sandwich-cultured hepatocytes, incubations were carried out at a final test compound concentration of 2 μM and with approximately 2 × 10⁵ viable cells/well (48 wells). A volume of 200 μl of each test compounds was added to each well and incubated at 37°C for 2 h in a CO₂incubator. At 0, 10, 20, 40, 60, and 120 min the contents of the wells were removed and added to a 96-well plate containing 200 μl of acetonitrile with internal standard. The wells were then washed with an additional 200 μl of acetonitrile and added to the same 96-well plate. The incubations were done with each type of hepatocyte using at least two different lots.

All samples were sonicated for 3 min and centrifuged at 4000 rpm for 20 min, and the supernatant was transferred into a 96-well plate. The plate was stored at −20°C until analyzed. Standard curves were also prepared in hepatocytes and analyzed with the samples.

HPLC/Atmospheric Pressure Chemical Ionization/MS.

The HPLC system consisted of an LC-10ADvp pump (Shimadzu, Koyoto, Japan) and Leap injector (Leap Technologies, Carrboro, NC). The column used was Genesis C₁₈2.0 × 50 mm (Jones Chromatography, Lakewood, CO). The mobile phase was a gradient with A (0.01 M ammonium acetate in 80:20 water/methanol) and B (0.01 M ammonium acetate in methanol). The flow rate was 0.35 ml/min. The initial liquid chromatography conditions were held at 40% B for 0.5 min, followed by a linear gradient from 40 to 95% B over 0.5 min, followed by 95% B for 2 min and back to 40% B over 0.5 min. The samples were analyzed in the positive ion mode using the heated nebulizer interface of a PE/Sciex 3000 (PerkinElmer Instruments, Norwalk, CT) triple quadrupole mass spectrometer with nebulizer probe temperature at 400°C.

Calculation of in Vitro Intrinsic Clearance.

Hepatocyte intrinsic clearances were calculated using the following formula:CLint=C0−C120 minAUC0−120 min·VN (μl/min/million hepatocyte) Equation 1where C₀ andC_120min are concentrations of compound at 0 and 120 min in μM, AUC_{0–120 min} is area under the concentration versus time curve from 0 to 120 min in minutes · micromolar, V is the volume of incubation (1000 μl), and N is number of hepatocytes (1 million hepatocytes/1000 μl). Assuming the in vitro clearance reaction follows a first order kinetics, thenCt=C0e−kt AUC0–120 min=∫0120 Ctdt=∫0120 C0e−kt dt =C0e−kt−k1200=C01−e−120kk=C0−C120 mink CL=C0−C120 minAUC0–120 min·VN=C0−C120 min(C0−C120 min)/k·1=k Traditional calculation of intrinsic clearance was as follows:CLint=C0AUC0–∞·VN (μl/min/million hepatocyte) Equation 2where C₀ is concentration of compound at 0 min in micromolar, and AUC_0-∞ is area under the concentration versus time curve from 0 to infinity in minutes · micromolar concentration.Ct=C0e−kt AUC0−∞=∫0∞Ctdt=∫0∞C0e−ktdt =C0 e−kt−k∞0=C0k CL=C0AUC0–∞·VN=C0C0/k·1=k Both the new equation and the traditional calculation of intrinsic clearance result in CL = kV/N =k (where V/N = 1).

Results

HPLC/MS-MS is a very important technique in drug discovery. It provides excellent sensitivity and selectivity, with a short analysis time. It also permits the high-throughput screening of large number of compounds for stability in the support of drug discovery. In the present study, all compounds were run with the same column, mobile phases, and gradient with a 3-min run time. No additional chromatographic method development was necessary for individual compounds. With the excellent selectivity of the mass spectrometer, the compounds could be separated by their characteristic parent and fragment ion masses and quantitated with minimal chromatographic separation.

The viability of each hepatocyte preparation was determined before and after incubation. Data suggested that the viability of the fresh and cryopreserved hepatocytes before incubation ranged from 85 to 95% and 50 to 70%, respectively. The viability remained the same after 2 h of incubation. Plated hepatocytes seem to be almost 100% viable before and after incubation.

The data provided by the supplier for both cryopreserved and fresh hepatocytes showed that phase I and II enzymes were still present and active. In addition, the same hepatocyte preparations have been used for in-house metabolite identification studies, both phase I and II metabolites were observed (data not shown). This indicates that both phase I and II enzymes are active in both fresh and cryopreserved hepatocytes.

The in vitro measurements of the rate of clearance were based on substrate disappearance over a 2-h incubation time. The CL_int was calculated using eq. 1 instead of the traditional way of extrapolating to infinity to determine the AUC_0-∞. The total in vivo plasma clearance values for rat, dog, and monkey (CL_tot) were determined experimentally from in-house i.v. data. In vivo human clearance data were obtained from literature references. Assuming that the drug is metabolized or excreted only by the liver and kidney, CL_tot is the sum of the hepatic (CL_hep) and renal (CL_renal) clearance. Therefore, hepatic clearance (CL_hep = CL_tot − CL_renal) was used in the correlation to CL_int. A linear regression curve fitting was used for all correlation analyses and the regression square (R²) was determined.

Rat, Dog, and Monkey Correlations.

To avoid the extensive use of expensive and time-consuming animal studies, it is more strategic to establish an in vitro system that can screen out compounds with poor animal pharmacokinetics. A total of 27, 18, and 26 Schering compounds for rat, dog, and monkey, respectively, were dosed intravenously to determine their in vivo clearance values. The in vitro clearances for these compounds were determined using cryopreserved, fresh, and sandwich-cultured hepatocytes. The in vitro intrinsic clearances and the in vivo clearances for rats, dogs, and monkeys are summarized in Tables 1,2, and 3. The in vitro intrinsic clearance values are plotted versus actual clearance values in Figs. 1,2, and 3. Good correlations were obtained between in vitro clearance and in vivo clearance values. For rats, the R²value for fresh, sandwich-cultured, and cryopreserved hepatocytes was 0.72, 0.72, and 0.65, respectively. For dogs, theR² value for fresh, sandwich-cultured, and cryopreserved hepatocytes was 0.85, 0.68, and 0.71, respectively. However, for monkeys, much poorer correlations were seen, with theR² value for fresh, sandwich-cultured, and cryopreserved hepatocytes only 0.45, 0.35, and 0.67, respectively.

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Figure 1

Correlation of predicted (in vitro) and observed in vivo clearance using fresh, sandwich-cultured, and cryopreserved rat hepatocytes.

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Figure 2

Correlation of predicted (in vitro) and observed in vivo clearance using fresh, sandwich-cultured, and cryopreserved dog hepatocytes.

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Figure 3

Correlation of predicted (in vitro) and observed in vivo clearance using fresh, sandwich-cultured, and cryopreserved monkey hepatocytes.

Cryopreserved hepatocytes are more readily available than fresh hepatocytes. Therefore, it is necessary to determine whether cryopreserved hepatocytes can be used instead of fresh hepatocytes to provide data that could predict in vivo clearances. The in vitro values obtained from all three animal species using fresh and cryopreserved hepatocytes were compared. The results are shown in Table4. Very good correlations were observed between intrinsic clearance data from fresh and cryopreserved hepatocytes with R² value equal to 0.77, 0.82, and 0.70, for rat, dog, and monkey, respectively. A similar correlation was also obtained using sandwich-cultured and cryopreserved hepatocytes. Figure 5 contains plots of accuracy of in vitro clearance values using rat, dog, and monkey hepatocytes versus the respective in vivo clearance values. All data points in rats and dogs were within 2-fold error. In monkeys, a few low-clearance compounds fall outside the 2-fold error boundaries, indicating less correlation between the in vivo and in vitro data.

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Figure 5

Accuracy of clearance values predicted from rat, dog, monkey, and human hepatocytes versus actual in vivo clearance values.

a, rat hepatocyte studies; b, dog hepatocyte studies; c, monkey hepatocyte studies; d, human hepatocyte studies. Fresh hepatocyte (⋄), sandwich-gel hepatocyte (■), and cryopreserved hepatocyte (○). n is the number of compounds used in the study. Dotted lines signify 2-fold errors between in vitro and in vivo clearance values using in vivo data as reference point. Errors for tenoxicam and warfarin in d are well off scale (2000–6500% error) and are not depicted.

Human Correlation.

An in vitro-in vivo correlation to human clearance values is particularly important in the screening of new chemical entities in drug discovery because in vitro data are the only human information available before clinical trials. Twenty-six compounds with published in vivo clearance values were randomly selected from the literature and obtained from Sigma-Aldrich. In vitro intrinsic clearance values were determined from the incubation of pooled cryopreserved hepatocytes. A very good correlation was obtained between in vitro and in vivo clearances, with R² = 0.87. A summary of the human intrinsic clearances obtained in vitro is presented in Table 5. The in vitro intrinsic clearance values are plotted versus actual clearance values in Fig.4. The accuracy of in vitro clearance values using human hepatocytes versus the respective in vivo human clearance values was plotted in Fig. 5d. Only five data points with very low in vivo clearances fall outside the 2-fold error boundary.

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Figure 4

Correlation of predicted (in vitro) and observed in vivo clearance in human using cryopreserved hepatocytes.

Discussion

With recent advances in combinatorial chemistry, thousands of compounds are going through drug discovery. To decrease the amount of expensive and time-consuming animal studies and be able to exclude compounds with undesirable human pharmacokinetics, a validated in vitro system is needed. The most common in vitro systems used include incubations of compounds in microsomes, hepatocytes, and liver slices. Microsomal studies are simple, cheap, and easy to conduct; however, microsomes only carry out phase I metabolism when supplemented with NADPH and therefore, this system is only predictive when oxidative metabolism predominates over other phase II metabolic routes such as conjugation, reduction, and hydrolysis. Incubations in liver slices are poor predictors of the in vivo state (Houston and Carlile, 1997) due to delayed accessibility of substrate to the cells within the core of the slices. In addition, human liver slices are difficult to obtain and the results are not reproducible. According to Houston and Carlile (1997), freshly isolated hepatocytes show high fidelity and provide accurate predictions of in vivo clearance. In the present study, satisfactory correlations were obtained not only from fresh hepatocyte suspensions but also with sandwich-cultured and cryopreserved hepatocytes, indicating that cryopreserved hepatocytes have predictive power similar to that of fresh hepatocytes. The good correlation to human in vivo clearance values provides the basis for wider use of the more available and accessible human cryopreserved hepatocytes. This allows pooling of hepatocytes from different individuals to eliminate interindividual variability.

The basic assumptions of the in vitro-in vivo clearance correlation model are 1) in vitro enzymatic kinetics is applicable to in vivo kinetic properties, 2) intrinsic clearance follows first order kinetics, and 3) metabolic clearance in the liver is the major route of clearance. These assumptions have been adopted in most studies in the search for better in vitro-in vivo correlation (Houston and Carlile, 1997; Obach, 1999). The general approach (eq. 2) for the calculation of intrinsic clearance was from the ratio of the initial amount of the test compound added to the incubation medium to the corresponding AUC_0-∞. AUC_0-∞ was calculated using the linear trapezoidal rule and extrapolation to time infinity by using the last few data points in the terminal phase of the log concentration-time curve. This approach requires longer incubation times (up to 72 h) to reach the terminal phase followed by linear regression to extrapolate to infinity time. Obviously over prolonged incubation times, cell viability and consequent nonconstant clearance are important concerns.

In vitro intrinsic clearance can also be obtained from the Michaelis-Menten constant (K_m) and maximum velocity of the metabolic reaction (V_max) parameters for the initial rate of metabolite formation or parent drug loss. Under the first order condition, CL_int can be defined asV_max/(K_m+ S). Only when S ≪K_m does the in vitro intrinsic clearance correspond toV_max/K_m. A separate method to derive in vitro clearance values from in vitro half-lives has also been reported (Obach et al., 1997b; Lavé et al., 1999). The in vitro half-life (t_1/2) is determined from the rate of the disappearance of the parent compound. The half-life value is then converted to the corresponding intrinsic clearance using the Michaelis-Menten equation (k = 0.693/t_1/2, CL = kV). The above-mentioned two methods require linear regression analysis of the raw data for the estimation of CL_int. In addition, half-lives are difficult to estimate when compounds have very low intrinsic clearance. In the present study, neither approach was used because both methods involved some extrapolation that may alter the outcome of the correlation.

In the present study, we have implemented a short incubation procedure of just 2 h and developed a mathematical formula (eq. 1) for obtaining intrinsic clearance directly from experimental data with no extrapolations. The potential validity of this new approach has been tested by comparing the intrinsic clearance and in vivo clearance using the hepatocyte system. The good correlations obtained in rats, dogs, and humans suggest that this model represents an accurate approach for predicting in vivo clearance. In a side-by-side comparison, the use of the general approach (eq. 2) yielded anR² = 0.72 (Schneider et al., 1999) versus R² = 0.87 obtained in this study using human hepatocytes.

Several recent studies incorporated protein binding to improve the in vitro-in vivo correlation. However, protein binding is significant only for highly protein-bound (>90%) and low-clearance drugs (Oravcová et al., 1996). Because the purpose of this study was to screen for high-clearance drugs, this factor may not be important to early discovery phase. Obach (1997a) also indicated that excluding plasma protein binding resulted in better predictions of clearance from in vitro microsomal data. Data from our own laboratory (data not shown) indicated that compounds highly bound to plasma protein are usually highly bound to the liver microsomes. Therefore, in this study the effect of protein binding was not taken into account.

Although the results demonstrated the predictive potential of intrinsic clearance for rats, dogs, and humans, the approach showed some limitation for predicting clearance in monkey. The correlation for monkey was better for cryopreserved (R² = 0.668) than for fresh (R² = 0.450) or sandwich-cultured (R² = 0.349) hepatocytes. These observations might result from the following reasons: 1) higher interindividual variability occurs in monkeys than other animal species; and 2) more variation in age and size of animal. Fresh and sandwich-cultured hepatocytes are only available from one individual in each incubation. It is possible that high interindividual variability resulted in the lack of correlation when fresh and sandwich-cultured hepatocytes are used. Therefore, it is important to be able to use hepatocytes that are pooled from different individuals.

The compounds for rat, dog, and monkey studies were randomly chosen from nine different programs at Schering-Plough with multiple chemotypes and vast structural differences. Yet good correlations were obtained across these multiple chemotypes, suggesting that the method presented could be applied to other chemotypes and structures. Because the in-house compounds were in early discovery phase, only the general pharmacokinetic properties were determined. In some special cases, the metabolic fate of the lead compounds was determined. Based on these limited metabolic studies, it seems that most of the in-house compounds are eliminated by phase I reaction followed by phase II reaction. In addition, the compounds chosen have reasonable Caco-2 permeability, suggesting that they are not transport limited.

The accuracy of in vitro clearance values from rat, dog, monkey, and human hepatocytes versus actual in vivo clearance values were shown in Fig. 5. Although the correlations for the low-clearance compounds are not as good as for the high-clearance compounds, all predicted clearance values are within 2-fold error for rat and dog. For the monkey, there are a few compounds that the predicted values are more than 2-fold error due to the poor correlation. Likewise, there are also a few compounds in the human study that showed high percentage error. Although the in vitro data overestimated the in vivo clearance of those few compounds, the predicted data still suggested that those compounds were low-clearance compounds.

The commercial compounds used in the human hepatocyte studies were divided into two groups. Group I includes the 16 compounds that are eliminated by phase I reaction and group 2 included 10 compounds by phase II or phase I and II. Separate linear regression analysis showed that group I and group II are very similar in the slopes of the correlation curves, and the R² values were 0.96 and 0.82, respectively. This analysis suggests that the predictive ability apply to both phase I and phase II compounds. The cytochromes P450 involved in the elimination of those compounds include the following major isoforms: 1A2, 2C9, 2C19, 2D6, 2E2, and 3A4. After carefully examined each data point in Fig. 4, no particular outlier that is eliminated by any particular isoform of cytochrome P-450 was observed. Indicating that none of the major cytochromes P450 is deficient in the human hepatocyte. Hence, the knowledge of metabolic pathways or the cytochrome P450 isoforms involved is not crucial to the predictability of the modeling approaches put forth in this article.

In general, cryopreserved hepatocytes from rat or dog have similar predictive power as the fresh and sandwich gel hepatocytes. Cryopreserved monkey hepatocytes exhibit greater predictive power than fresh or sandwich hepatocytes. Cryopreserved hepatocytes are more readily available particularly in the case of human hepatocytes. Making experimental planning using fresh human hepatocytes ahead of time is not possible. In addition, pooling of fresh hepatocytes was not possible due to the limited availability of fresh liver from more than one individual at one time. Therefore, cryopreserved hepatocytes were our top choice among the three different types. In vitro clearance data obtained using the hepatocyte models presented in this study can only predict in vivo hepatic clearance. It will not be predictive on compounds that are cleared mainly through renal and other extrahepatic clearance.

In conclusion, the equation used in this study for calculating in vitro intrinsic clearance provides a more direct estimation than previously published methods that relied on extrapolation and linear regression analysis. In addition, the extended incubation time that was required for the previously established method is no longer needed in the method presented herein. Therefore, our approach eliminates the necessity for keeping the hepatocytes functionally active for long periods of time. This is especially important because commercially available cryopreserved human hepatocytes do not retain viability for very long under routine tissue culture conditions. The data presented herein indicate that hepatocytes processed and preserved by different methods would generate equally satisfactory correlations with in vivo data. This study provides a basis for wider use of human cryopreserved hepatocyte preparations. Pooled hepatocytes derived from several donors can help eliminate individual variation. In addition, this study presents a method that can rapidly evaluate in vitro intrinsic clearance. Satisfactory correlation between in vitro and in vivo clearance values validates the usefulness of the method in predicting in vivo human clearance. This method presents a way to substantially increase the drug-screening efficiency and provide quantitative information on drug metabolism before pharmacokinetic studies in vivo.