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

This Article Abstract Full Text (PDF) All Versions of this Article: dmd.105.005033v1 33/9/1319    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 Giuliano, C. Articles by Laufer, R. Search for Related Content PubMed PubMed Citation Articles by Giuliano, C. Articles by Laufer, R.

DIRECT DETERMINATION OF UNBOUND INTRINSIC DRUG CLEARANCE IN THE MICROSOMAL STABILITY ASSAY

Department of Pharmacology, Istituto di Ricerche di Biologia Molecolare (IRBM) P. Angeletti, Merck Research Laboratories, Rome, Italy

(Received April 6, 2005; accepted June 9, 2005)

	Abstract

Top Abstract Materials and Methods Results Discussion References

 
The microsomal stability assay is commonly used to rank compounds according to their metabolic stability. Determination of the unbound intrinsic clearance (CL_in,u) is essential for the accurate comparison of compounds, since nonspecific binding to microsomes can lead to an underestimation of the microsomal clearance. In this study, a new method (linear extrapolation in the stability assay, LESA) was established, which allows direct calculation of CL_in,u from microsomal stability data, without the need to independently determine the fraction of free (unbound) drug. The method was validated using nine drugs with different chemical structures and physicochemical properties. The CL_in,u of these compounds was extrapolated from the intrinsic clearance values obtained at different concentrations of human liver microsomes and compared with that calculated by the conventional method, using microsomal intrinsic clearance values and the free fraction of drug determined by equilibrium dialysis, ultracentrifugation, or ultrafiltration. A good agreement was observed between the data generated by the LESA method and those determined by conventional procedures. The method was further evaluated using a published dataset for 10 additional drugs and found to yield intrinsic clearance data comparable to the previously reported values. LESA provides a convenient and rapid method to determine the influence of microsome binding on intrinsic clearance in a single assay.

Many drugs are lipophilic organic compounds that can bind nonspecifically to the lipid-protein milieu of the microsomal membrane. The result of nonspecific binding is a reduction in the free concentration of drug that is available for interaction with microsomal drug-metabolizing enzymes. Depletion of unbound drug by extensive membrane partitioning leads to an underestimation of CL_in. The "true" CL_in, i.e., the value that would be observed in the absence of binding to microsomes, is termed unbound intrinsic clearance (CL_in,u). Unbound intrinsic clearance can be calculated by determining the free fraction of compound in microsomal incubations (f_u) according to the relationship:

(1)

Three different experimental methods are commonly used to determine f_u and, consequently, CL_in,u, namely, equilibrium dialysis, ultracentrifugation, and ultrafiltration. Of these, equilibrium dialysis is the most widely used method to determine f_u since it is experimentally easy and can be performed in a 96-well format (Kariv et al., 2001). In all three methodologies there is the possibility that nonspecific drug adsorption to equipment surfaces (dialysis membrane, ultrafiltration device, etc.) may distort the values obtained, leading to an underestimation of the CL_in,u (Lin et al., 1987). In addition, these methods are relatively laborious and time-consuming. The aim of the present work was to establish a methodology for the direct determination of CL_in,u, without the need for separate measurement of f_u. The new method is based on the assumption that compounds bind to or partition into microsomes in a nonspecific fashion, i.e., with low affinity, and that binding sites are not saturated at the concentrations used in microsomal stability assays. Under these conditions, the CL_in,u and f_u can be directly extrapolated from the microsomal stability data obtained at different microsome concentrations. The method was validated using a series of structurally diverse compounds that are subject to oxidative metabolism and are known to exhibit significant nonspecific binding to hepatic microsomes (Obach, 1999).

	Materials and Methods

Top Abstract Materials and Methods Results Discussion References

 
Materials. All chemicals were obtained from Sigma-Aldrich (Milan, Italy). Stock solutions of all compounds were prepared in dimethyl sulfoxide at a concentration of 10 mM. From these, working solutions containing 200 µM concentrations of compound were prepared in 50% methanol. The internal standard used in all LC-MS/MS analyses was a proprietary compound. Solvents and other reagents were from common sources and of HPLC grade or higher. Human liver microsomes (HLM pool, lot 24) were purchased from BD Gentest (Woburn, MA).

Microsomal Incubations. All incubations were conducted in quadruplicate. The incubation mixtures were prepared in 96-well cluster tubes (1.2 ml; Corning Life Sciences, Acton, MA) and contained 1 µM test compound, HLMs (0.1–2 mg of microsomal protein/ml), 3 mM MgCl₂, and 25 mM potassium phosphate buffer, pH 7.4, in a final volume of 1 ml. Reactions were initiated by the addition of NADPH (final concentration 1 mM) and kept in a shaking water bath at 37°C. Reactions were terminated by adding 100 µlofthe incubation mixture to 100 µl of acetonitrile/0.1% formic acid containing 1 µM internal standard. Immediately after the addition of NADPH, the sampling point for t = 0 min was taken, and further sampling points were taken at 5, 10, 30, 60, and 90 min. For incubations with the rapidly metabolized compounds diclofenac and midazolam, samples were taken at 0, 2, 4, 6, 8, 10, 15, 30, and 60 min. The samples were centrifuged for 10 min at 4000g to pellet precipitated microsomal protein, and the supernatant was subjected to LC-MS/MS analysis without further treatment. CL_in was calculated according to:

(2)

where Dose is the initial amount of drug in the incubation mixture (unit of mol/mg microsomal protein), and AUC is the area under the concentration versus time curve, extrapolated to infinity (unit of M · h). The unit for CL_in is l/h/mg protein. For all compounds tested, turnover was greater than 20%/h at the lowest concentration of microsomes. All CL_in values were also calculated using the half-life derived from fitting of the concentration time course data to a first-order kinetic model. No significant differences in CL_in were observed between the two calculation methods (data not shown).

Equilibrium Dialysis. Dialysis mixtures contained a 1 µM concentration of test compound, HLMs (0.2 and 1 mg/ml), 3 mM MgCl₂, and 25 mM potassium phosphate buffer, pH 7.4, in a final volume of 200 µl. Control mixtures did not contain microsomal proteins. Triplicate mixtures were subjected to equilibrium dialysis against 200 µl of phosphate-MgCl₂ buffer using a 96-well DispoDialyzer (Harvard Apparatus Inc., Holliston, MA). The dialyzing unit consists of two chambers separated by an ultrathin membrane with a molecular weight cut-off of 10 kDa. The plate was rotated for 12 h at 37°C in the perpendicular direction of the well orientation to ensure a constant contact between the two chambers, using a plate rotator (Harvard Apparatus Inc.). The solvent volumes in the two chambers did not change significantly during the course of the experiment. Upon completion of the dialysis, 100 µl of the samples from the microsome and buffer sides were processed as outlined for the metabolic stability samples and analyzed by LC-MS/MS. Recovery was found to be between 75% and 100% for all compounds, with the exception of chlorpromazine, where recovery was 61%. The free fraction was calculated according to eq. 3:

(3)

where C_b and C_m denote the concentrations of compound in the dialysis chambers containing buffer and microsomes, respectively.

Ultrafiltration. Mixtures were prepared as outlined for the equilibrium dialysis method. Aliquots of 200 µl were subjected to ultrafiltration using Centrifree filter devices (Millipore Corporation, Billerica, MA). The assembled filter unit was centrifuged for 1 h at 863g at 37°C. Upon completion of the filtration, 100 µl of ultrafiltrate were processed as outlined for the microsomal stability samples and analyzed by LC-MS/MS. Recovery was determined by analysis of filtered control samples prepared in the absence of microsomes and was found to be between 70% and 100% for all compounds with the exception of chlorpromazine, where recovery was 17%. Results were expressed as the concentration ratio of sample versus control samples:

(4)

Ultracentrifugation. Mixtures were prepared as outlined for the equilibrium dialysis method. Aliquots of 200 µl were placed in polycarbonate centrifuge tubes (8 x 34 mm; Beckman Coulter, Fullerton, CA) and centrifuged for 3 h at 356,000g at 37°C (Optima TL ultracentrifuge; Beckman Coulter). One hundred microliters of the resulting supernatant were processed as outlined for the metabolic stability samples and analyzed by LC-MS/MS. Recovery was determined by analysis of centrifuged control samples prepared in the absence of microsomes and was found to be between 75% and 100% for all compounds. Results were expressed as the concentration ratio of sample versus control samples:

(5)

LC-MS/MS Analysis. The LC-MS/MS system consisted of an Agilent 1100 series gradient HPLC pump (Agilent Technologies, Palo Alto, CA), a CTC HTS PAL Autosampler (CTC Analytics, Zwingen, Switzerland) and an Applied Biosystems/MDS Sciex API 2000 triple quadrupole mass spectrometer (Applied Biosystems, Foster City, CA) equipped with a turbo ionspray interface. Analytes in incubation mixtures were separated by reverse phase HPLC using an Ace Act RP C18 50 x 4.6 mm column (MAC-MOD Analytical, Inc., Chadds Ford, PA). A generic gradient elution program was used at a flow rate of 2 ml/min with a mobile phase of acetonitrile/0.1% formic acid (10% v/v) in water/0.1% formic acid for 0.2 min, after which time the acetonitrile concentration was increased to 90% over 1.7 min before restoring it back to 10% for the remaining 0.7 min. The injection volume was 20 µl. Approximately 10% of the eluent was introduced into the mass spectrometer source. The source temperature of the mass spectrometer was maintained at 450°C, and other source parameters (e.g., collision energy, declustering potential, curtain gas pressure etc.) were individually optimized for each compound. The most prominent fragment of the molecular ion (M + H⁺) was followed for each compound and the internal standard in the multiple reaction monitoring mode. Quantitation of each compound was achieved by comparison of the analyte/internal standard peak area ratios to those of a calibration curve ranging from 0.01 µM to 2 µM.

LESA Model. Two models for drug binding to microsomes have been proposed. The first model (McLure et al., 2000) assumes saturable association of drug to defined microsomal binding sites, according to the relationship:

(6)

where B and F are the concentrations of bound and free drug, respectively, B_max the concentration of binding sites, and K_D the equilibrium binding constant.

The second model (Austin et al., 2002) treats microsomal binding as a nonsaturable phase equilibrium process governed by a membrane partition coefficient, K_P:

(7)

Mathematically, this model is equivalent to the particular case of the defined binding site model where binding is nonsaturable, i.e., F << K_D. In this case, B = (B_max/K_D) x F, with K_P = B_max/K_D.

It should be noted that the membrane partition coefficient K_P defined in this way is directly proportional to the total number of membrane binding sites and subsequently to the total membrane protein concentration, M, i.e.,

(8)

with K' denoting the proportionality constant.

As pointed out by Austin et al. (1995), microsomal binding is normally independent of compound concentration, and saturation does not occur at the low micromolar concentrations used in microsomal stability assays. It is therefore appropriate to use eq. 7 to describe this process.

View larger version (17K): [in this window] [in a new window]   FIG. 1. Typical depletion profile for desipramine in pooled HLMs. The compound (1 µM) was incubated in the presence of NADPH with the indicated concentrations of HLMs. Each point represents the mean ± S.D. of triplicate determinations.

(9)

Substituting eqs. 8 and 9 into eq. 7 and rearranging, we obtain

(10)

If intrinsic clearance is determined in the presence of drug binding to microsomes, the relationship between the observed clearance, CL_in, and the "true" clearance of unbound drug, CL_in,u, is calculated according to eq. 1:

Substituting eq. 10 into eq. 1 and rearranging, we obtain

(11)

According to eq. 11, plotting the reciprocal of CL_in against the microsome concentration M should result in a straight line intersecting the y-axis at 1/CL_in,u. CL_in,u can thus be calculated without independently determining f_u. Values of CL_in,u obtained using this method were compared with those calculated using eq. 8, with f_u values determined by dialysis, ultrafiltration, or ultracentrifugation.

Statistical Analysis. Linear regression analysis and associated standard errors were determined using SigmaPlot 9.0 (Systat Software Inc., Richmond, CA).

	Results

Top Abstract Materials and Methods Results Discussion References

 
The LESA model (described under Materials and Methods) was applied to calculate the CL_in,u of nine drugs. The in vitro CL_in of chlorpromazine, desipramine, amitriptyline, imipramine, verapamil, diltiazem, propafenone, midazolam, and diclofenac was determined at five different concentrations of pooled HLMs, ranging from 0.1 to 2 mg/ml. A typical concentration-time curve is reported in Fig. 1 for NADPH-dependent desipramine consumption in HLMs. According to the LESA model, when 1/CL_in is plotted against the concentration of HLMs, a straight line intersecting the y-axis at 1/CL_in,u should be obtained (see eq. 11). As shown in Fig. 2 for desipramine, 1/CL_in was directly proportional to the concentration of HLMs. Similar linear plots were obtained for the other eight compounds investigated, with correlation coefficients (r²) ranging from 0.88 to 0.99. Table 1 summarizes the CL_in and the statistics of the linear correlations, as well as the values of CL_in,u extrapolated from the data.

View larger version (11K): [in this window] [in a new window]   FIG. 2. Linear correlation between 1/CLin and HLM concentration for desipramine. Data were fitted by linear regression (y = 0.0931x + 0.0244, r2 = 0.98). The y-axis intercept corresponds to 1/CLin,u (eq. 11). Each point represents the mean ± S.D. of quadruplicate determinations.

To investigate whether the results obtained with the LESA method reflected the true CL_in,u, the unbound fractions (f_u) of the nine drugs investigated were determined by equilibrium dialysis, ultracentrifugation, and ultrafiltration at two different microsome concentrations, 0.2 mg/ml and 1 mg/ml (Table 2). For chlorpromazine, f_u could not be determined by ultrafiltration, since the compound displayed very low mass balance in this system (see Materials and Methods). CL_in,u was then calculated by the conventional method using eq. 1 (CL_in,u = CL_in/f_u; Table 3). For all nine drugs, the results for CL_in,u obtained by direct measurement with the LESA method were in good agreement with those obtained with the other three (or two in the case of chlorpromazine) methodologies (Table 3).

In Fig. 3, CL_in,u obtained by the LESA method is compared for the nine compounds investigated with that determined by direct determination of f_u, using the average value from equilibrium dialysis, ultracentrifugation, and ultrafiltration. It should be noted that the nine compounds differ in their structures, physicochemical properties, and CL_in,u. Furthermore they have greatly differing degrees of nonspecific binding to microsomes with f_u ranging from 25 to near 100%. The correlation obtained was excellent at both microsome concentrations, with r² = 0.92 and r² = 0.96 for 1 mg/ml and 0.2 mg/ml microsomal protein, respectively.

View larger version (14K): [in this window] [in a new window]   FIG. 3. Correlation between CLin,u values for nine drugs obtained by LESA versus that calculated using experimentally determined values of fu. The value of CLin,u used was the average of the values determined by equilibrium dialysis, ultrafiltration, and ultracentrifugation at two different concentrations of HLMs, 1 mg/ml (plot A; r2 = 0.92) and 0.2 mg/ml (plot B; r2 = 0.96). Standard errors of CLin,u determined by LESA were derived from those of 1/CLin,u as calculated by the curve fitting software.

	Discussion

Top Abstract Materials and Methods Results Discussion References

The methodologies most frequently used (equilibrium dialysis, ultrafiltration, and ultracentrifugation) determine CL_in,u indirectly via measurement of f_u (eq. 1). Equilibrium dialysis (Lin et al., 1987) is technically simple, a variety of apparatus are commercially available, and, using 96-well plates, it is possible to determine the f_u of several compounds in a single experiment. However, equilibrium time can be long, and unstable drugs or proteins may degrade during long equilibration times. Drug adsorption to the dialysis membrane or dialysis device tends to be greater than drug adsorption to ultracentrifugation tubes, and recovery of the parent compound is not always quantitative. Another problem that can increase the error in the measurement of f_u by equilibrium dialysis is the potential for volume shift due to the Donnan effect (Lin et al., 1987). However, the methodology is widely applied and yields satisfactory results if appropriate controls are included.

Ultrafiltration is faster than equilibrium dialysis, but an increased protein concentration during filtration, as well as a potential decrease in the filter pore size due to protein accumulation, may cause errors in the measurement of f_u. Ultracentrifugation is not affected by membrane or Donnan effects. However, the technique is of low throughput and potentially subject to artifacts due to surface adsorption and variation of the protein concentration during centrifugation.

All the drugs selected for the present study are mainly subject to hepatic oxidative metabolism (Obach, 1999). Desipramine, amitriptyline, imipramine, verapamil, diltiazem, propafenone, and chlorpromazine are basic compounds; midazolam is neutral; and diclofenac is acidic. Seven basic compounds were selected because compounds with a pK_a > 7.4 generally show greater nonspecific binding than do neutral and acidic compounds (Austin et al., 1995). This is expected because basic compounds exhibit enhanced affinity for membrane phospholipids, as demonstrated by liposome binding studies (Austin et al., 1995; Kramer et al., 1998). Furthermore, all of the drugs used were reported to display appreciable binding to hepatic microsomes (Obach, 1999). The substrate depletion approach was used because formal kinetic characterization and metabolite quantification are not required. The CL_in was calculated as Dose/AUC rather than with the more rigorous approach that uses enzyme kinetic data (i.e., maximum enzyme velocity, V_max, and Michaelis-Menten constant, K_M). This simplified approach is appropriate, since the substrate concentration used (1 µM) is below the apparent K_M for substrate turnover, and no significant product inhibition or mechanism-based inactivation of the enzyme is present (Obach, 1999). All the drugs selected were metabolized in HLMs with CL_in,u ranging between 38 µl/min/mg for diltiazem and 344 µl/min/mg for diclofenac (Table 1).

View larger version (12K): [in this window] [in a new window]   FIG. 4. Correlation between published CLin,u values with those extrapolated by LESA. Data are from Table 4. Each point represents the mean ± S.E. of triplicate determinations.

Austin et al. (2002) measured the CL_in and f_u for 13 drugs at three different concentrations of rat liver microsomal protein, 0.25, 1, and 4 mg/ml, and determined the CL_in,u from these data. This set of 13 compounds includes five neutral, four acidic, and four basic drugs covering a wide range of lipophilicity. We applied the LESA method to calculate CL_in,u from the reported values of CL_in (Austin et al., 2002). The LESA method could not be applied to isradipine, since only two experimental CL_in values were reported (Austin et al., 2002). As shown in Table 4, there was a generally good agreement between CL_in,u extrapolated by LESA and that calculated using the experimentally measured f_u values (Austin et al., 2002). For 10 of the 12 compounds analyzed, the difference between the results obtained with the two methods was less than 2-fold. The two outliers were amiodarone and astemizole. Both compounds were reported to bind extensively to microsomes even at the lowest concentration tested (0.25 mg/ml), with f_u of 0.006 and 0.076, respectively (Austin et al., 2002), which may introduce a significant error in the calculation of CL_in,u by either method. Since CL_in,u is the ratio between CL_in and f_u, compounds with high CL_in and very low f_u will yield very high estimates of CL_in,u (16,000 and 10,000 µl/min/mg for amiodarone and astemizole, respectively), associated with an amplified statistical error. Obviously, the reciprocal value 1/CL_in,u will be close to zero, posing a practical limit to the applicability of the LESA method. Thus, for amiodarone, the extrapolation yielded a negative intercept, which has no physical meaning. On the other hand, extrapolation of the data for astemizole yielded a significantly lower CL_in,u than that calculated by the conventional method (Austin et al., 2002), raising the possibility that the latter was biased by an underestimation of f_u for that compound. Further studies will be needed to clarify this point. Excluding amiodarone, astemizole, and isradipine from the comparison, a good correlation was obtained between CL_in,u values calculated with LESA versus the conventional method, with a linear regression coefficient (r²) of 0.96, as shown in Fig. 4.

The main limitation of LESA is due to the fact that it is utilizing the substrate depletion approach. For this reason, the CL_in,u can only be calculated with sufficient accuracy in the case of appreciable turnover of the substrate (at least 20%) (Jones and Houston, 2004). On the other hand, the CL_in,u in LESA is extrapolated linearly from a range of CL_in values obtained at different microsome concentrations. This increases the confidence in the experimental data. Notably, in the other three methodologies, CL_in,u is usually obtained from the f_u at a single microsome concentration. Another potential limitation of the LESA method is that it is based on the assumption that drug binding to microsomes is truly nonspecific, i.e., of low affinity. The method would not be valid for compounds whose binding is saturated at the concentrations used in the microsomal stability assay. However, as discussed by Austin et al. (2002), this is unlikely to occur at the low micromolar concentrations used in modern metabolic assays. Notwithstanding these potential limitations, the LESA method provides a convenient and rapid method to determine the influence of microsome binding on intrinsic clearance, without the need for separate determination of the unbound fraction. The method should be particularly useful in cases where the unbound fraction cannot be determined by conventional methods due to technical limitations such as nonspecific adsorption to dialysis apparatus or compound solubility (Walsky et al., 2005). It may also be applicable to studies of kinetic parameters of drug interaction with microsomal enzymes (e.g., cytochrome P450 inhibition) (Margolis and Obach, 2003; Walsky et al., 2005) and to other in vitro systems, such as hepatocytes, where clearance can be influenced by cellular accumulation (Jones and Houston, 2004).

In summary, LESA was shown to accurately determine the CL_in,u in the microsomal stability assay by comparison with three traditionally used methods. Furthermore, LESA could be applicable to investigate the influence of nonspecific binding of drugs to protein or lipids in enzyme inhibition/induction studies (Tran et al., 2002).

	Acknowledgments

 
We thank Edith Monteagudo and Elena Fraschini for helpful discussion and suggestions.

	Footnotes

 
This work was supported in part by a grant from the Ministero dell'Istruzione, dell'Università e della Ricerca.

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

doi:10.1124/dmd.105.005033.

ABBREVIATIONS: CL_in, in vitro intrinsic clearance; CL_in,u, intrinsic clearance of unbound drug; f_u, unbound fraction; HLM, human liver microsomes; LESA, linear extrapolation in the stability assay; LC-MS/MS, liquid chromatography-tandem mass spectrometry.

Address correspondence to: Ralph Laufer, Department of Pharmacology, IRBM, MRL Rome, Via Pontina Km 30.600, 00040 Pomezia (RM), Italy. E-mail: Ralph_Laufer{at}Merck.com

	References

Top Abstract Materials and Methods Results Discussion References

 


Austin RP, Barton P, Cockroft SL, Wenlock MC, and Riley RJ (2002) The influence of nonspecific microsomal binding on apparent intrinsic clearance and its prediction from physicochemical properties. Drug Metab Dispos 30: 1497–1503.[Abstract/Free Full Text]

Austin RP, Davis AM, and Manners CN (1995) Partitioning of ionizing molecules between aqueous buffers and phospholipid vesicles. J Pharm Sci 84: 1180–1183.[CrossRef][Medline]

Houston JBKK and Galetin A (2003) Typical and atypical enzyme kinetics, in Drug Metabolizing Enzymes. Cytochrome P450 and Other Enzymes in Drug Discovery and Development (Lee JS, Obach RS, and Fisher MB eds), pp 211–254, Marcel Dekker, New York.

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]

Kariv I, Cao H, and Oldenburg KR (2001) Development of a high throughput equilibrium dialysis method. J Pharm Sci 90: 580–587.[CrossRef][Medline]

Kramer SD, Braun A, Jakits-Deiser C, and Wunderli-Allenspach H (1998) Towards the predictability of drug-lipid membrane interactions: the pH-dependent affinity of propanolol to phosphatidylinositol containing liposomes. Pharm Res (NY) 15: 739–744.

Lin JH, Cocchetto DM, and Duggan DE (1987) Protein binding as a primary determinant of the clinical pharmacokinetic properties of non-steroidal anti-inflammatory drugs. Clin Pharmacokinet 12: 402–432.[Medline]

Madan AUE, Burton LA, Ogilvie BW, and Parkinson A (2002) In vitro approaches for studying the inhibition of drug-metabolising enzymes and identifying the drug-metabolising enzymes responsible for the metabolism of drugs, in Drug-Drug Interactions (Rodriques D ed), pp 217–293, Marcel Dekker, New York.

Margolis JM and Obach RS (2003) Impact of nonspecific binding to microsomes and phospholipid on the inhibition of cytochrome P4502D6: implications for relating in vitro inhibition data to in vivo drug interactions. Drug Metab Dispos 31: 606–611.[Abstract/Free Full Text]

McGinnity DF and Riley RJ (2001) Predicting drug pharmacokinetics in humans from in vitro metabolism studies. Biochem Soc Trans 29: 135–139.[CrossRef][Medline]

McLure JA, Miners JO, and Birkett DJ (2000) Nonspecific binding of drugs to human liver microsomes. Br J Clin Pharmacol 49: 453–461.[CrossRef][Medline]

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 vitro metabolism data. J Pharmacol Exp Ther 283: 46–58.[Abstract/Free Full Text]

Rodrigues AD (1997) Preclinical drug metabolism in the age of high-throughput screening: an industrial perspective. Pharm Res (NY) 14: 1504–1510.

Tran TH, Von Moltke LL, Venkatakrishnan K, Granda BW, Gibbs MA, Obach RS, Harmatz JS, and Greenblatt DJ (2002) Microsomal protein concentration modifies the apparent inhibitory potency of CYP3A inhibitors. Drug Metab Dispos 30: 1441–1445.[Abstract/Free Full Text]

Walsky RL, Obach RS, Gaman EA, Gleeson JP, and Proctor WR (2005) Selective inhibition of human cytochrome P4502C8 by montelukast. Drug Metab Dispos 33: 413–418.[Abstract/Free Full Text]

This article has been cited by other articles:

				S. S. De Buck, V. K. Sinha, L. A. Fenu, M. J. Nijsen, C. E. Mackie, and R. A. H. J. Gilissen Prediction of Human Pharmacokinetics Using Physiologically Based Modeling: A Retrospective Analysis of 26 Clinically Tested Drugs Drug Metab. Dispos., October 1, 2007; 35(10): 1766 - 1780. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: dmd.105.005033v1 33/9/1319    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 Giuliano, C. Articles by Laufer, R. Search for Related Content PubMed PubMed Citation Articles by Giuliano, C. Articles by Laufer, R.