Advertisement
Research ArticleArticle

Sigmoidal Kinetics of CYP3A Substrates: An Approach for Scaling Dextromethorphan Metabolism in Hepatic Microsomes and Isolated Hepatocytes to Predict In Vivo Clearance in Rat

Lynn E. Witherow and J. Brian Houston
Journal of Pharmacology and Experimental Therapeutics July 1999, 290 (1) 58-65;

Abstract

The metabolism of a number of compounds by the cytochrome P-450 subfamily CYP3A does not exhibit classic Michaelis-Menten kinetics but displays a sigmoidal rate-substrate concentration relationship. Intrinsic clearance (CLint) cannot be calculated for these drugs due to the lack of a first order region in their kinetic profiles, and a suitable parameter has yet to be identified to allow such data to be scaled to predict in vivo clearance. As sigmoidal kinetics have only been observed with microsomal systems, we have investigated whether this behavior is demonstrable in freshly isolated hepatocytes. We have also evaluated the term maximum clearance (CLmax), which refers to the in vitro clearance when the enzyme is fully activated, to predict in vivo clearance. To these ends we have studied the metabolism of dextromethorphan to methoxymorphinan and dextrorphan; methoxymorphinan production is best described by sigmoidal kinetics in both hepatocytes and microsomes, dextrorphan production is best described by a two site Michaelis-Menten model in microsomes but is sigmoidal in hepatocytes. Total clearance, estimated from the CLmax and CLint terms, was scaled to give mean predictions of 127 to 319 ml/min/standard rat weight of 250 g. In vivo CLint, determined after infusion via the hepatic portal vein to steady state and correcting for plasma protein binding and blood-to-plasma concentration ratio, was 259 ± 59.2 ml/min/standard rat weight of 250 g. These investigations show that sigmoidal kinetics is not unique to microsomes and that CLmax is a useful parameter for scaling to the in vivo situation.

Intrinsic clearance (CLint) is a valuable parameter to describe the inherent metabolic stability of a drug in the absence of other rate-determining processes that may potentially influence its clearance in vivo (Wilkinson and Shand, 1976). For compounds that exhibit classic Michaelis-Menten kinetics this parameter can be readily estimated in vitro from the ratio ofVmax andKm values and scaled to the whole liver to allow in vivo clearance predictions (Houston, 1994). This approach has been successful for the prediction of in vivo CLint of a diverse range of cytochrome P-450 (CYP) substrates using in vitro data from both freshly isolated hepatocytes and hepatic microsomes (Houston and Carlile, 1997). The parameter CLint describes the rate of metabolism under first order conditions; this is believed to be of most relevance as therapeutic concentrations rarely approach theKm values commonly determined in vitro. However, for a drug displaying sigmoidal kinetics due to substrate activation, there is no first order region in the kinetic profile; this precludes the use of the standard approach for scaling the clearance of these compounds to the in vivo situation. The extent of activation can be substantial, thus the consequences of selecting an arbitrary substrate concentration and ignoring the phenomenon may result in a major underestimation of the clearance process. A number of CYP3A substrates have been reported to exhibit sigmoidal kinetics in microsomes (Ueng et al., 1997) but this distinctive activation phenomenon has yet to be demonstrated with other in vitro systems. CYP3A4 is the most abundant CYP present in the human liver (Shimada et al., 1994) and is responsible for the metabolism of at least 50% of the drugs currently in use (Benet et al., 1996). It is therefore important to devise and evaluate a procedure to allow the scaling of in vitro data for drugs showing sigmoidal kinetics and to provide a prediction of in vivo clearance.

Dextromethorphan (DEM) is an antitussive drug that undergoes two oxidative demethylations. The O-demethylation yielding dextrorphan (DOR) is mainly catalyzed by enzymes in the CYP2D subfamily and the N-demethylation to methoxymorphinan (MEM) is mainly catalyzed by members of the CYP3A subfamily in both humans (Kupfer et al., 1986; Coutts et al., 1994) and rats (Zysset et al., 1988; Kerry et al., 1993). Both primary metabolites can be further demethylated to yield 3-hydroxymorphinan (Fig. 1). In the rat in vivo, DOR is the main urinary metabolite (20%) and HOM and MEM represent 17% and 0.5% of dose, respectively (Bochner et al., 1994). Because Bochner et al. (1994) determined that 3-hydroxymorphinan was almost exclusively formed by the O-demethylation of MEM rather than the N-demethylation of DOR, the ratio of the two primary pathways is approximately one. However, this contrasts with reports using microsomal preparations (Kerry et al., 1993) in which DOR is the dominant pathway representing 95% of total clearance.

The initial aim of this study is to determine whether sigmoidal kinetics are unique to microsomes by studying DEM metabolism in both hepatic microsomes and freshly isolated hepatocytes in suspension from rat. Furthermore the term CLmax, defined as the clearance at maximal autoactivation of the enzyme, is evaluated as a suitable parameter for scaling data that show sigmoidal metabolite kinetics to predict in vivo CLint. For the purpose of scaling in vitro data, it would seem prudent to assume that full activation is the condition operating under in vivo conditions despite the lack of specific in vivo studies to confirm or refute this stance. We have evaluated our predictions from in vitro data using in vivo clearance values determined by i.v. infusion of DEM into the hepatic portal vein of rats with appropriate correction for plasma protein binding and red blood cell binding. These investigations also allow examination of the inconsistencies between the importance of the two demethylation pathways in vitro and in vivo.

Materials and Methods

Chemicals.

DEM, MEM, DOR, and 3-hydroxymorphinan were supplied by Hoffmann-La Roche, Inc. (Basel, Switzerland). All other chemicals were purchased either from the Sigma Chemical Company (Poole, Dorset, UK) or BDH (Lutterworth, Leicester, UK).

Animals and Treatment.

Male Sprague-Dawley rats (240–260 g) were obtained from the University of Manchester Biological Services Unit. They were housed two to four per cage on a bedding of sawdust in rooms maintained at a temperature of 20 ± 2°C and a humidity of 45 to 55% with a 12-h light/dark cycle. They had free access to water and Chow Rat Mouse (Agro Foods, Peniston, UK) rat diet.

Hepatic Microsomal Studies.

Rats (n = 4) were sacrificed by cervical dislocation and washed microsomes were prepared as described previously (Hayes et al., 1995). Pretreatment of animals with dexamethasone and subsequent preparation of microsomes was carried out as described previously by Carlile et al. (1997).

Incubations were carried out under linear conditions with respect to time (10 min) and microsomal protein concentration (0.5 mg protein/ml). Microsomes and substrate (10 μl in phosphate buffer 0.1 M, pH 7.4) were preincubated in a shaking water bath at 37°C for 5 min and the reaction was started by mixing with a preincubated NADPH regenerating system (100 μl) consisting of 1 mM NADP, 7.5 mM isocitric acid, 1 U of isocitrate dehydrogenase/ml, 15 mM magnesium chloride, and 0.1 M phosphate buffer pH 7.4. The final incubation volume was 200 μl. The reaction was terminated by the addition of 30 μl of 60% perchloric acid. The final substrate concentration range studied was 0.5 to 500 μM and all incubations were carried out in duplicate.

Investigations into the extent of binding of DEM to microsomes were carried out using the protein concentration (0.5 mg/ml) and substrate concentration range (0.5–500 μM) used for kinetic studies. Samples (1 ml) containing microsomes, DEM, and 0.1 M phosphate buffer (pH 7.4) were preincubated in a shaking water bath at 37°C for 10 min. Aliquots (400 μl) were placed on 0.2 μm Anopore filters (VectaSpin 3, Whatman, Maidstone, Kent, UK) and spun in a microfuge at maximum speed for 5 min. An aliquot of the filtrate and the initial sample were analyzed and the amount of DEM bound determined from the ratio of the concentration in the filtrate to the initial concentration. There was no binding to the filters.

Hepatocyte Studies.

Untreated rats (n = 4) were administered heparin (500 U i.p.) 5 min before being sacrificed by cervical dislocation. The isolated hepatocytes were prepared by collagenase end of lobe perfusion as described by Hayes et al. (1995)with the hepatocytes being finally resuspended in albumin-free William’s media E.

Incubations were carried out at 37°C in a shaking water bath under linear conditions with respect to time (10 min) and cell density (0.75 × 106 cells/ml). DEM was preincubated with William’s media E for 5 min and the reaction was started by the addition of 0.5 ml of hepatocyte suspension; the final incubation volume was 3 ml. The DEM concentration range studied was 0.5 to 500 μM and the reaction was terminated by immersing incubates in liquid nitrogen. All incubations were carried out in duplicate.

The procedure used to determine the cell to media concentration ratio for DEM was based on that of Eaton and Klaassen (1978). Sodium hydroxide (50 μl of 0.5 M) was placed in an Eppendorf tube and silicone oil (510/50:550 3:2 v/v, density 1.002, 100 μl) placed on top to form a separate layer. William’s media E (250 μl) containing DEM (concentration range 0.5–500 μM), preincubated to 37°C, was added on top of the oil followed by 250 μl of cells to give a final cell density of 0.75 × 106cells/ml. The cells and substrate were allowed to equilibrate (2 min, based on preliminary studies) before spinning in a microfuge at maximum speed for 5 s. The Eppendorf tubes were then placed in a freezer and the tip containing the cells cut off into a test tube. Distilled water (0.5 ml) was added and the samples were left overnight before being analyzed. All incubations were carried out in duplicate.

Markers of intra- and extracellular volumes were used to determine the total cell volume in an incubation based on previous reports (Eaton and Klaassen, 1978; Iwamoto et al., 1984; McPhail et al., 1993) using [3H]water and [14C]sucrose, respectively (0.6 μCi/ml William’s media E for each marker). The cell volume was determined from the amount of [3H]water in the separated cells corrected for the adherent layer of water using the amount of [14C]sucrose in theseparated cells using eq. 1.Cell volume Equation 1=total 3H in separated cells 3H concentration in incubate−total 14C in separated cells 14C concentration in incubate The concentration of drug in the cells is calculated using eq.2:C=A−(M×Va)Vc Equation 2where C is the cell concentration, A is the amount of drug in the cells and adherent layer, M is the concentration in the media (calculated after correction for cell volume),Va is the volume of the adherent layer, and Vc is the volume of the cells in the incubation.

The unbound fraction in the cells (fucells) was calculated from the cell-media partition coefficient (Kp) using eq. 3.fucells=1Kp Equation 3The unbound concentration (Cu) and the fraction unbound of DEM (fumedia) in the hepatocyte incubation was calculated using eqs. (4) and (5), respectively.Cu=Ci1+Kp×VcVm Equation 4fumedia=CuCi Equation 5where Ci is the initial concentration and Vm is the volume of media.

In Vivo Studies.

Untreated rats (n = 9) with previously inserted cannulae in the lineal vein and carotid artery (Harms and Ojeda, 1974) received a loading dose of DEM (9.9, 14.8, and 29.7 mg/kg in saline) into the hepatic portal vein via the lineal vein followed immediately by an infusion of DEM (in polyethylene glycol/ethanol/saline, 2:1:1, v/v) via the same route at a rate of either 8.8, 11.7, or 23.4 mg/h/kg (0.15 ml/h for 3 h). Loading doses and infusion rates were calculated from clearance and volume of distribution values (Ashforth et al., 1995) determined from preliminary experiments (260 ± 32 ml/min/kg and 19.8 ± 2.5 l/kg, respectively). Blood samples were taken at 30-min intervals from the carotid artery up to 3 h to determine steady-state plasma concentration (Css). After centrifugation to obtain plasma, the samples were stored at −20°C until analysis. In vivo clearance was calculated by dividing the infusion rate (mg/h/kg) by Css(mg/liter).

Assay Procedure for Analysis of DEM, DOR, and MEM.

After termination of the reaction of microsomal incubations, the incubate was centrifuged and the supernatant (100 μl) injected onto the HPLC. Hepatocyte incubations (1 ml for 0.5–2.5 μM samples and 0.5 ml for 5–500 μM samples) were incubated with 0.5 ml β-glucuronidase with sulfatase activity (200 U/ml in sodium acetate, pH 4.5) for 1 h in a shaking water bath at 37°C to hydrolyze any conjugates. After deconjugation, 10 M sodium hydroxide (10 μl) was added along with codeine (10 μl of 1 mg/ml solution in methanol) as internal standard. The sample was extracted withtert-butyl-methyl ether (5 ml) by rotary mixing for 20 min. The samples were then centrifuged (2500 rpm, 10 min) and the organic layer was removed and evaporated to dryness under a stream of oxygen-free nitrogen at 50°C. The extraction efficiencies were 95.2 ± 5.3% (n = 10), 97.6 ± 2.9%, and 92.1 ± 6.3% for DOR, MEM, and DEM, respectively. The sample was reconstituted in mobile phase (300 μl). Plasma samples were extracted as for hepatocytes with the exception of deconjugation. Standard curves were constructed over the range of 0.01 to 10 nmol for the two metabolites and 0.02 to 10 nmol for DEM; the amount of compound was quantified by interpolation from the standard curve for microsomes or by a peak area ratio method with respect to the internal standard codeine for hepatocyte and plasma samples.

The HPLC method used is based on that of Chen et al. (1990). The sample (100 μl) was injected (Gilson 231 autoinjector, Anachem, Luton, Bedfordshire, UK) onto a 25 cm × 4.6 mm Spherisorb C8 column with a mobile phase of 59.9% water, 40% acetonitrile, 0.1% triethylamine, pH 3, at 0.6 ml/min (Gilson 302 pump). Fluorescence detection (Hewlett Packard 1046A) was used with excitation and emission wavelengths of 280 and 310 nm, respectively. The retention times were 4, 6.5, 10.5, and 12 min for codeine, DOR, MEM, and DEM, respectively. The relationship between peak area and the amount of DOR, MEM, and DEM was linear over the range of the standard curve (r2 = 0.99). The variabilities of the slope of the standard curve over a 4-month period were 11.8%, 10%, and 5.9% for DOR, MEM, and DEM, respectively. The “on column” limit of detection was 5 pmol for DOR and MEM and 25 pmol for DEM, respectively. The minimal rate determinable for the production of DOR and MEM in microsomes was 0.01 nmol/min/mg and in hepatocytes was 0.001 nmol/min/106cells.

Determination of Plasma Protein Binding and Blood-to-Plasma Ratio.

Rat plasma (2.5 ml) was preincubated with DEM (final concentration 0.5–500 μM) in a shaking water bath at 37°C for 10 min. An aliquot (1 ml) was placed in an equilibrium dialysis cell (Dianorm, Munich, Germany) separated by semipermeable membrane (molecular weight cut-off 5000, Dianorm) from a cell containing 1 ml of 0.1 M phosphate buffer, pH 7.4. All incubations were carried out in duplicate. Preliminary experiments had determined that equilibrium was reached after 2 h and after 4 h the buffer and plasma were removed from the cells into clean test tubes and analyzed as described previously. The fraction unbound of DEM in rat plasma was calculated using eq. 6.fu=1−[plasma]−[buffer][plasma] Equation 6Blood (2 ml) was spiked with DEM (final concentration range 1–10 μM) and preincubated in a shaking water bath at 37°C for 10 min. An aliquot (200 μl) was analyzed as described previously in duplicate. The sample was then spun (2500 rpm, 10 min) and an aliquot of the plasma (200 μl) was analyzed in duplicate. The blood-plasma ratio was calculated from the amount of DEM per milliliter of blood and plasma respectively.

Data Analysis.

In vitro data were analyzed using least-squares nonlinear regression with a weighting of 1/y(Siphar, version 3.3, Simed, Creteil, France). Log average substrate concentrations were used for calculation of kinetic parameters thereby correcting for loss of substrate from the incubation due to metabolism. MEM production in both microsomes and hepatocytes and DOR production in hepatocytes was best described by the Hill eq. 7.v=Vmax×SnS50n+Sn Equation 7where v and Vmax are the observed and maximal rates of metabolism, S50is the concentration at half Vmax, n is the Hill coefficient, and S the substrate concentration.

DOR production in control and dexamethasone-induced microsomes required a two-site Michaelis-Menten eq. 8.v=Vmax1×SKm1+S+Vmax2×SKm2+S Equation 8where Vmax1 is the maximal rate for the high-affinity, low-capacity site,Km1 is the Michaelis constant for the high-affinity, low-capacity site, andVmax2 andKm1 are the corresponding parameters for the second low-affinity, high-capacity site.

MEM production in dexamethasone-induced animals was best described by the classical Michaelis-Menten eq. 9.v=Vmax×SKm+S Equation 9To obtain the total clearance for DEM metabolism in vitro, the maximum clearance for the two parallel pathways were added. The maximal clearance corresponds to CLint for MEM production in dexamethasone-induced microsomes (Vmax/Kmratio) and for DOR production in control and in dexamethasone-induced microsomes (the sum of theVmax/Kmratio for the two sites). For MEM production in control microsomes and hepatocytes and for DOR production in hepatocytes, clearance was calculated for each substrate concentration from rate/S and the CLmax was obtained graphically (see Fig. 5B).

Figure 5
Figure 5

Typical sets of data showing clearance (v/S) of DEM by the DOR (▪) and MEM (●) pathways and as total metabolism (×) in hepatic microsomes (A) and isolated hepatocytes (B). The solid lines show the best lines of fit from usingv/S = (Vmax1/Km1 + S) + (Vmax2/Km2 + S) for DOR in microsomes and v/S =Vmax. Sn −1/S50n + Sn for MEM in microsomes and both metabolites in hepatocytes.

Both observed and predicted clearance terms are expressed per standard rat weight of 250 g (SRW). In vitro clearance andVmax values were scaled using the scaling factors of 660 mg microsomal protein/SRW and 1200 × 106 cells/SRW for microsomes and hepatocytes, respectively (Carlile et al., 1997). In vivo blood Clint values were obtained from the plasma clearance, the blood-plasma ratio, and the fraction unbound in plasma. The use of the hepatic portal vein avoids the hepatic blood flow constraints (20 ml/min/SRW) but assumes that the venous equilibration liver model adequately describes hepatic events (Wilkinson and Shand, 1976).

Results

Kinetics of Metabolite Formation in Hepatocytes and Hepatic Microsomes.

MEM production, catalyzed by CYP3A isoforms, displays sigmoidal kinetics in both in vitro systems; this can be clearly seen from the characteristic convex Eadie-Hofstee plots (Fig.2). Although the production of DOR is best described by a two-site Michaelis-Menten model consisting of a high-affinity, low-capacity site and a low-affinity, high-capacity site in microsomes, it displays sigmoidal kinetics in hepatocytes (Fig.3). The kinetic parameters obtained by nonlinear regression of the rate data for both pathways in the two in vitro systems are given in Table 1. The S50 value for MEM production is lower in hepatocytes than that in microsomes and there is an approximately 2-fold higher n value in the former system. Very similar S50 values are obtained for both pathways in hepatocytes but the n value is greater for MEM than for DOR. For both in vitro systems, Vmax is larger for MEM than DOR production. The secondary metabolite, 3-hydroxymorphinan, was not detected in either the microsomal or hepatocyte incubations.

Figure 2
Figure 2

Typical sets of data describing MEM production in hepatic microsomes (A; 0.5 mg protein/ml, 10-min incubation; ●) and isolated hepatocytes (B; 0.75 × 106 cells/ml, 10-min incubation) as a function of total substrate concentration (B; ▪) or unbound substrate concentration (■) shown as Eadie-Hofstee plots.

Figure 3
Figure 3

Typical sets of data describing DOR production in hepatic microsomes (A; 0.5 mg protein/ml, 10-min incubation; ●) and isolated hepatocytes (B; 0.75 × 106 cells/ml, 10-min incubation) as a function of total substrate concentration (▪) or unbound substrate concentration (■) shown as Eadie-Hofstee plots.

View this table:
Table 1

Kinetic parameters for DEM metabolism in rat hepatic microsomes and isolated hepatocytes (mean ± S.D., n = 4)

There is an increase in the total CLint for DOR production in dexamethasone-induced microsomes as compared with control microsomes due mainly to a significant decrease in theKm value (p < .005) of the high-affinity, low-capacity site in these induced microsomes (Table 1). As CYP2D isoforms are not inducible by dexamethasone, additional isoforms (e.g., CYP3A members) must contribute to this pathway. A significant increase inVmax for MEM production (p < .005) in dexamethasone-induced microsomes as compared with control is also evident (Table 1).

Substrate Binding within In Vitro Matrices.

The extent of binding to hepatic microsomes was measured over the substrate concentration range used for the kinetic studies. As can be seen from Table 2 there is minimal binding of DEM to the microsomes and no concentration dependence is apparent over the 1000-fold range studied.

View this table:
Table 2

Effect of DEM concentration on hepatocyte concentrations and fractions unbound in media, cells and microsomes

Cellular uptake of DEM was also determined over the substrate concentration range (0.5–500 μM) used for kinetic studies with hepatocyte suspensions. The time course of DEM uptake was found to be rapid with plateau concentrations reached within 30 s and maintained over the incubation time of 2 min. DEM accumulates within the hepatocyte resulting in cell-to-media partition coefficients as high as 50 at low concentrations and saturation evident at concentrations greater than 10 μM (Fig.4). On the assumption that these high values result from cellular binding rather than active uptake into the cells, unbound fractions in the cell and in the total incubation were calculated (Table 2). Whereas most of the DEM within the cell is bound (>90%), the unbound media (and consequently the unbound cellular) concentration is only slightly lower than the initial media concentration. The unbound fraction in the total incubation exceeds 0.8 at all concentrations. Using the latter data rather than the initial concentrations to calculate kinetic parameters made very little difference, as can be seen from the Eadie-Hofstee plot in Figs. 2B and3B.

Figure 4
Figure 4

Hepatocyte-to-media partition coefficient for DEM. Cellular concentrations of DEM calculated using eq. 2.

In Vivo Studies.

The binding of DEM to rat plasma proteins was independent of substrate concentration (0.5–500 μM) and the fraction unbound was determined as 0.45 ± 0.06 (mean ± S.D., four concentrations in duplicate). The blood-to-plasma ratio was calculated to be 1.76 ± 0.37 (mean ± S.D., four concentrations in duplicate) over the concentration range studied (1–10 μM).

Administration of a bolus loading dose followed by a constant rate infusion into the hepatic portal vein achieved a steady-state plasma concentration within 30 min for all infusion rates investigated. Table3 shows the targetedCss, the corresponding infusion rate and loading dose, and the actual Cssobserved. There is good agreement between the targeted and observedCss for the three regimens used. Plasma clearance did not differ for the three infusion conditions studied. It was not possible to extend the range ofCss studied (0.53–2 μM) due to the limits of assay sensitivity at lower concentrations and toxicity of DEM at higher concentrations. The clearance after hepatic portal vein administration is blood flow-independent and when corrected for plasma protein binding and blood-to-plasma ratio provides an in vivo CLint of 259 ± 59 ml/min/SRW.

View this table:
Table 3

Comparison of target steady-state concentration, with corresponding loading dose and infusion rate, and steady-state concentrations achieved

Scaled In Vitro Parameters.

Scaled clearances based on the rate of metabolite formation divided by the substrate concentration for microsomes and hepatocytes are shown as a function of incubation concentration in Fig. 5. This graphical representation shows the progressive increase in the magnitude of the clearance until maximal activation is achieved and, subsequently, saturation of the enzyme system is evident. The value for CLmax was selected to provide a prediction of in vivo CLint for the pathways displaying sigmoidal kinetics. For DOR production in microsomes CL decreases monotonically from the CLint, which is the sum of theVmax/Kmvalues for the two sites. Table 4 shows the scaled CLint andVmax values for DOR and MEM pathways for both in vitro systems. For both pathways the scaledVmax values are similar in the two in vitro systems, whereas the Clint values differ significantly. CLint (calculated from CLmax) is 5-fold higher in hepatocytes than microsomes for MEM. Conversely, DOR CLint is 8-fold higher in microsomes (calculated from the sum of theVmax/Kmratios) than in cells (a CLmax estimate).

View this table:
Table 4

Comparison of scaled CLint and Vmax for both pathways of DEM metabolism in microsomes and hepatocytes

Discussion

Despite the many known complexities of the CYP-450 enzyme system (Lewis, 1996) the kinetics of metabolite formation can frequently be described by simple Michaelis-Menten type models. An exception to this general trend is the kinetic behavior of the most abundant CYP isoform in human liver, CYP3A4. Sigmoidal kinetics, indicative of substrate activation, have been reported in both human liver microsomal and cDNA-expressed systems for substrates of CYP3A4 (Andersons et al., 1994; Kerr et al., 1994; Schmider et al., 1995; Ueng et al., 1997). Activation of this isoform by α-naphoflavone can result in the loss of sigmoidal kinetics for several of these substrates (Ueng et al., 1997). It would appear that this particular subfamily of the CYP system has multiple binding sites. Recent investigations (Korzekwa et al., 1998) have indicated that other isoforms also display sigmoidal kinetics. To date sigmoidal kinetics have been observed only in microsomal preparations.

DEM was selected for study due to the simplicity of the metabolite pathway (see Fig. 1) and the known isoform involvement in these pathways. We have demonstrated that MEM production exhibits sigmoidal kinetics in both hepatic microsomes and isolated hepatocytes and the degree of sigmoidicity, as measured by the Hill coefficient, is greater in hepatocytes than in microsomes. Other investigations of MEM formation in rat hepatic microsomes have not observed sigmoidal kinetics, however, two of these studies were carried out in female rats (Zysset et al., 1988; Kerry et al., 1993) and the low CYP3A content in this gender is well known (Cooper et al., 1993). Roos et al. (1991)carried out microsomal incubations using the same strain and gender as our study but the substrate concentration range investigated was not reported. Activation is a phenomenon that occurs at low substrate concentrations and if the substrate concentration range does not extend to low concentrations (<10 μM) then the sigmoidicity may not be detected.

DOR production is best described in microsomes by a two-site Michaelis-Menten model consisting of a high-affinity, low-capacity site and a low-affinity, high-capacity site, as reported earlier by Kerry et al. (1993) and Zysset et al. (1988). Although DOR formation is regarded as a CYP2D pathway, our incubations with dexamethasone-induced microsomes demonstrated induction of DOR, as well as MEM, production providing good evidence for other isoform involvement (e.g., CYP3A) in the former pathway. We also report that the formation of DOR in hepatocytes shows sigmoidal kinetics albeit to a lesser degree than MEM, which may be a consequence of the contribution of the two isoform subfamilies to DOR production. With dexamethasone-induced microsomes, DOR production was best described by a two-site Michaelis-Menten model as was seen for untreated microsomes. Surprisingly, sigmoidicity was not evident for MEM production in dexamethasone-induced microsomes, in contrast to untreated microsomes, and the former data were best described by classical Michaelis-Menten kinetics. This may be due to a difference in the main CYP3A isoforms present in untreated and dexamethasone-induced animals. In the former, CYP3A2 represents 4.9% of total CYP (40 pmol/mg protein) and CYP3A1 is not detectable, whereas in the latter CYP3A1 (660 pmol/mg protein) and CYP3A2 (360 pmol/mg protein) are 30% and 16% of the total CYP content of the liver, respectively (Cooper et al., 1993). It may be speculated that CYP3A2 is the isoform responsible for the sigmoidal kinetic profile for MEM. Interestingly, CYP3A2-mediated acetaminophen oxidation has also been identified as being particularly sensitive to activation by caffeine and the mechanism implicated involves enhancement of electron transfer (Lee et al., 1991, 1997).

As sigmoidal kinetics have not been reported previously for isolated hepatocyte preparations, it is important to establish that this phenomenon is enzyme-related. For example, binding within the cell could result in lower concentrations available to the enzyme than would be anticipated from the initial media concentration, and if this effect were saturable then sigmoidal kinetics could artifactually arise. To determine the importance of saturable binding of DEM within the cell, the uptake of DEM into hepatocytes was investigated and extensive accumulation was shown. However, if it is assumed that this accumulation results solely from cellular binding, and the substrate concentration is expressed as an unbound concentration within the incubation, the rates of production of MEM and DOR in hepatocytes maintain their sigmoidal characteristics (Figs. 2B and 3B). Furthermore, although there is saturation of cellular uptake, this occurs at a relatively high concentration (100 μM; see Fig. 4). Conversely, if a carrier-mediated transport system were responsible for the extensive accumulation of DEM, then the substrate concentrations available for metabolism in the cell would be higher than the initial media concentration. When cell concentrations are used to calculate clearance, a low value is obtained, which is independent of substrate concentration. Because these clearance characteristics were inconsistent with all other data, the possible role of hepatic transporters was not pursued.

For simple Michaelis-Menten models, the use of theVmax/Km ratio, as a CLint parameter has proved to be valuable for scaling in vitro data to a whole liver basis to allow both predictions of in vivo kinetics and comparison between different in vitro systems (Houston and Carlile, 1997). When sigmoidal kinetics is evident, an alternative approach to the scaling of in vitro data is required. Sigmoidal kinetics can be identified by a number of classic graphical plots including the Eadie-Hofstee. Figure 5 shows an alternativev/S (i.e., clearance) versus S plot that has a characteristic maximum. We propose the use of this maximum, CLmax, for scaling in vitro data in a similar manner to CLint for drugs whose kinetics is characterized by Michaelis-Menten models.

Predicted CLint were calculated by summing the clearance terms for both pathways. For the two in vitro systems the mean predictions are quite similar, 304 ml/min/SRW and 126 ml/min/SRW for microsomes and hepatocytes, respectively. The range of predictions from both in vitro systems and the range of in vivo values observed are compared in Fig. 6. Although microsomal predictions tend to provide higher predictions than hepatocytes, there is good overlap between the predicted CLintvalues from both in vitro systems with the observed in vivo CLint values.

Figure 6
Figure 6

Comparison of predicted CLint from hepatic microsomes and isolated hepatocytes with the observed in vivo CLint. Data shown for individual animals in vivo (n = 9) and individual microsomal (n = 4) and isolated hepatocyte (n = 4) preparations.

Consideration of the kinetic parameters describing the individual pathways in the two in vitro systems (Table 4) shows that the scaledVmax values are very similar for both pathways yet there is a large difference in the scaled CLint values (9-fold and 5-fold for DOR and MEM, respectively). This observation emphasizes that although both in vitro systems are similar in their capacities to produce the two metabolites, the concentration-response relationships (for activation and/or metabolism) differ between the in vitro systems resulting in different clearances. This difference between the systems is particularly evident in Fig. 5, which illustrates the relative clearance for both pathways.Bochner et al. (1994) demonstrated that both pathways are of equal importance in vivo (see introduction) using a dose of 20 mg/kg DEM to ensure plasma concentrations less than 4 μM. It can be seen from Fig.5A that DOR is the predominant pathway in microsomes and at a substrate concentration of 4 μM the clearance ratio of DOR to MEM is 7.1 ± 2.6. In contrast, the clearance ratio of DOR to MEM in hepatocytes is 0.96 ± 0.17 at 4 μM (Fig. 5B). Also if the relative importance of the pathways is assessed assuming full autoactivation (by the use of Clmax) then the ratio of DOR to MEM is markedly overestimated (18 ± 5) for microsomes whereas for hepatocytes there is reasonable concordance (0.4 ± 0.1) in vivo. Thus, overall hepatocytes are a better reflector of the relative importance of the metabolic pathways of DEM in vivo than microsomes, although both in vitro systems provide accurate prediction of the total metabolic clearance of DEM.

In conclusion, we report that sigmoidal kinetics is not unique to microsomes and can be demonstrated in isolated hepatocytes. This implies that the activator present in vivo is washed out from both in vitro systems during their preparation. We were unable to extend the range of our in vivo studies to establish whether activation can be observed in vivo. To investigate this would require detailed and judiciously planned studies. However, the currently available information would support the assumption of full activation in vivo is not unreasonable. The maximal clearance in vitro would appear to be a useful term for scaling sigmoidal data to the in vivo situation but its general applicability and the validity of the underlying assumption of full activation of the enzymes under in vivo conditions will require further investigation.

Footnotes

  • Send reprint requests to: Dr. J.B. Houston, School of Pharmacy and Pharmaceutical Sciences, University of Manchester, Manchester M13 9PL, UK. E-mail: bhouston{at}man.ac.uk

  • ↵1 This work was supported by the European Center for the Validation of Alternative Methods (ECVAM) Contract 11277–95-10. A portion of this study was presented at the meeting of the British Pharmacological Society, December 10–12 1997, Harrogate, UK and appeared in abstract form in Br J Clin Pharmacol45:P519–P520 (1998).

  • Abbreviations:
    CLint
    intrinsic clearance
    CYP
    cytochrome P-450
    DEM
    dextromethorphan
    DOR
    dextrorphan
    MEM
    methoxymorphinan
    CLmax
    clearance at maximal activation
    Css
    steady-state plasma concentration
    SRW
    standard rat weight of 250 g
    • Received October 28, 1998.
    • Accepted February 15, 1999.

References

  1. ↵
    1. Andersson T,
    2. Miners JO,
    3. Veronese ME,
    4. Birkett DJ
    (1994) Diazepam metabolism by human liver microsomes is mediated by both S-mephenytoin hydroxylase and CYP3A isoforms. Br J Clin Pharmacol 38:131–137.
    OpenUrlPubMed
  2. ↵
    1. Ashforth EIL,
    2. Carlile DJ,
    3. Chenery R,
    4. Houston JB
    (1995) Prediction of in vivo disposition from in vitro systems: Clearance of phenytoin and tolbutamide using rat hepatic microsomal and hepatocyte data. J Pharmacol Exp Ther 274:761–766.
    OpenUrlAbstract/FREE Full Text
  3. ↵
    1. Hardman JG,
    2. Limbird LE,
    3. Molinoff PB,
    4. Ruddon RW,
    5. Gilman AG
    1. Benet LZ,
    2. Kroetz DL,
    3. Sheiner LB
    (1996) Pharmacokinetics: The dynamics of drug absorption, distribution and elimination. in Goodman and Gilmans The Pharmacological Basis of Therapeutics, eds Hardman JG, Limbird LE, Molinoff PB, Ruddon RW, Gilman AG (McGraw-Hill, New York), 9th ed. p 14.
  4. ↵
    1. Bochner F,
    2. Somogyi AA,
    3. Chen ZR
    (1994) Dextromethorphan metabolism in the rat: Interstrain differences and the fate of individually administered oxidative metabolites. Xenobiotica 24:543–552.
    OpenUrlPubMed
  5. ↵
    1. Carlile DJ,
    2. Zomorodi K,
    3. Houston JB
    (1997) Scaling factors to relate drug metabolic clearance in hepatic microsomes, isolated hepatocytes and the intact liver: Studies with induced livers involving diazepam. Drug Metab Dispos 25:903–911.
    OpenUrlAbstract/FREE Full Text
  6. ↵
    1. Chen ZR,
    2. Somogyi AA,
    3. Bochner F
    (1990) Simultaneous determination of dextromethorphan and three metabolites in plasma and urine using high-performance liquid chromatography with application to their disposition in man. Ther Drug Monit 12:97–104.
    OpenUrlPubMed
  7. ↵
    1. Cooper KO,
    2. Reik LM,
    3. Jayyosi Z,
    4. Bandiera S,
    5. Kelley M,
    6. Ryan DE,
    7. Daniel R,
    8. McCluskey SA,
    9. Levin W,
    10. Thomas PE
    (1993) Regulation of two members of the steroid-inducible cytochrome P450 subfamily (3A) in rats. Arch Biochem Biophys 301:345–354.
    OpenUrlCrossRefPubMed
  8. ↵
    1. Coutts RT,
    2. Su P,
    3. Baker GB
    (1994) Involvement of CYP2D6, CYP3A4, and other cytochrome P-450 isozymes in N-dealkylation reactions. J Pharmacol Toxicol Methods 31:177–186.
    OpenUrlCrossRefPubMed
  9. ↵
    1. Eaton DL,
    2. Klaassen CD
    (1978) Carrier-mediated transport of ouabain in isolated hepatocytes. J Pharmacol Exp Ther 205:480–488.
    OpenUrlFREE Full Text
  10. ↵
    1. Harms PG,
    2. Ojeda SR
    (1974) A rapid and simple procedure for chronic cannulation of the rat jugular vein. J Appl Physiol 36:391–392.
    OpenUrlFREE Full Text
  11. ↵
    1. Hayes KA,
    2. Brennan B,
    3. Chenery R,
    4. Houston JB
    (1995) In vivo disposition of caffeine predicted from hepatic microsomal and hepatocyte data. Drug Metab Dispos 23:349–353.
    OpenUrlAbstract
  12. ↵
    1. Houston JB
    (1994) Utility of in vitro drug metabolism data in predicting in vivo metabolic clearance. Biochem Pharmacol 47:1469–1479.
    OpenUrlCrossRefPubMed
  13. ↵
    1. Houston KB,
    2. Carlile DJ
    (1997) Prediction of hepatic clearance from microsomes, hepatocytes and liver slices. Drug Met Rev 29:891–922.
    OpenUrlPubMed
  14. ↵
    1. Iwamoto K,
    2. Furune Y,
    3. Watanabe J
    (1984) Difference in hepatic uptake kinetics of aspirin and salicylamide in rats. Biochem Pharmacol 33:3089–3095.
    OpenUrlCrossRefPubMed
  15. ↵
    1. Kerr BM,
    2. Thummel KE,
    3. Wurden CJ,
    4. Klein SM,
    5. Kroetz DL,
    6. Gonzalez FJ,
    7. Levy RH
    (1994) Human liver carbamazepine metabolism: Role of CYP3A4 and CYP2C8 in 10,11-epoxide formation. Biochem Pharmacol 47:1969–1979.
    OpenUrlCrossRefPubMed
  16. ↵
    1. Kerry NL,
    2. Somogyi AA,
    3. Mikus G,
    4. Bochner F
    (1993) Primary and secondary oxidative metabolism of dextromethorphan: In vitro studies with female Sprague-Dawley and Dark Agouti rat liver microsomes. Biochem Pharmacol 45:833–839.
    OpenUrlCrossRefPubMed
  17. ↵
    1. Korzekwa KR,
    2. Krishnamachary N,
    3. Shou M,
    4. Ogai A,
    5. Parise RA,
    6. Rettie AE,
    7. Gonzalez FJ,
    8. Tracy TS
    (1998) Evaluation of atypical cytochrome P450 kinetics with two-substrate models: Evidence that multiple sites can simultaneously bind to cytochrome P450 active sites. Biochemistry 37:4137–4147.
    OpenUrlCrossRefPubMed
  18. ↵
    1. Kupfer A,
    2. Schmid B,
    3. Pfaff G
    (1986) Pharmacogenetics of dextromethorphan O-demethylation in man. Xenobiotica 16:421–433.
    OpenUrlPubMed
  19. ↵
    1. Lee CL,
    2. Thummel KE,
    3. Kalhorn TF,
    4. Nelson SD,
    5. Slattery JT
    (1991) Inhibition and activation of acetaminophen reactive metabolite formation by caffeine. Drug Metab Dispos 19:348–353.
    OpenUrlAbstract
  20. ↵
    1. Lee CL,
    2. Manyike PT,
    3. Thummel KE,
    4. Nelson SD,
    5. Slattery JT
    (1997) Mechanism of cytochrome P450 activation by caffeine and 7,8-benzoflavone in rat liver microsomes. Drug Metab Dispos 25:1150–1156.
    OpenUrlAbstract/FREE Full Text
  21. ↵
    1. Lewis DFV
    (1996) Cytochromes P450. Structure, Function and Mechanism. (Taylor & Francis, London).
  22. ↵
    1. McPhail ME,
    2. Knowles RG,
    3. Salter M,
    4. Dawson J,
    5. Burchell B,
    6. Pogson CI
    (1993) Uptake of acetaminophen (paracetamol) by isolated rat liver cells. Biochem Pharmacol 45:1599–1604.
    OpenUrlCrossRefPubMed
  23. ↵
    1. Roos FJ,
    2. Zysset T,
    3. Reichen J
    (1991) Differential effect of biliary and micronodular cirrhosis on oxidative drug metabolism. In vivo-in vitro correlations of dextromethorphan metabolism in rat models. Biochem Pharmacol 41:1513–1519.
    OpenUrlCrossRefPubMed
  24. ↵
    1. Schmider J,
    2. Greenblatt DJ,
    3. Von Moltke LL,
    4. Harmatz JS,
    5. Shader RI
    (1995) N-demethylation of amitriptyline in vitro: Role of cytochrome P450 3A (CYP3A) isoforms and effect of metabolic inhibitors. J Pharmacol Exp Ther 275:592–597.
    OpenUrlAbstract/FREE Full Text
  25. ↵
    1. Shimada T,
    2. Yamazaki H,
    3. Minura M,
    4. Inui Y,
    5. Guengerich FP
    (1994) Interindividual variations in human liver cytochrome P450 enzymes involved in the oxidations of drugs, carcinogens and toxic chemicals: Studies with liver microsomes of 30 Japanese and 30 Caucasians. J Pharmacol Exp Ther 270:414–423.
    OpenUrlAbstract/FREE Full Text
  26. ↵
    1. Ueng Y-F,
    2. Kuwabara T,
    3. Chun Y-J,
    4. Guengerich FP
    (1997) Cooperativity in oxidations catalyzed by cytochrome P450 3A4. Biochemistry 36:370–381.
    OpenUrlCrossRefPubMed
  27. ↵
    1. Wilkinson GR,
    2. Shand DG
    (1976) A physiological approach to hepatic drug clearance. Clin Pharmacol Ther 18:377–390.
    OpenUrl
  28. ↵
    1. Zysset T,
    2. Zeugin T,
    3. Kupfer A
    (1988) In vivo and in vitro dextromethorphan metabolism in SD and DA rat. An animal model of the debrisoquine-type polymorphic oxidation in man. Biochem Pharmacol 37:3155–3160.
    OpenUrlCrossRefPubMed
View Abstract
Back to top

In this issue

Download PDF
Article Alerts
Email Article
Citation Tools

Research ArticleArticle

Sigmoidal Kinetics of CYP3A Substrates: An Approach for Scaling Dextromethorphan Metabolism in Hepatic Microsomes and Isolated Hepatocytes to Predict In Vivo Clearance in Rat

Lynn E. Witherow and J. Brian Houston
Journal of Pharmacology and Experimental Therapeutics July 1, 1999, 290 (1) 58-65;
Twitter logo Facebook logo Mendeley logo

Jump to section

Advertisement