Introduction

Top Abstract Introduction Materials & Methods Results Discussion References

In drug-drug interactions based on pharmacokinetics, inhibition of hepatic metabolism is one of the most important events and induces serious side effects. There have been many reports concerning the induction of various side effects and toxicities caused by azole antifungal agent-induced inhibition of drug metabolism (Baldwin et al., 1995; Neuvonen and Suhonen, 1995; Jurima-Romet et al., 1994). Furthermore, H₂ receptor antagonists (Pasanen et al., 1986; Knodell et al., 1991; Wright, 1991), new quinolone antibacterial agents (Thomsen et al., 1987; Wijnands et al., 1986; Beckmann et al., 1987), and macrolide antibiotic drugs (Warot et al., 1987; Gascon and Dayer, 1991) have also functioned as inhibitors of oxidative metabolism in the liver. Most of the enzymes related to drug-drug interactions of these drugs are CYPs.¹ The CYP superfamily is composed of families, subfamilies, and individual isoforms (Nelson et al., 1996). It is possible to qualitatively predict drug-drug interactions if the isoform related to the metabolism of each drug is identified. Moreover, we can quantitatively predict the extent of drug interactions if the metabolic K_i values of drugs are determined in liver microsomes and/or CYP-expressing cells. However, it is difficult to precisely predict the increasing ratio of the plasma concentrations of the interacting drug from this information alone. Therefore, we must solve the following problems (Sawada et al., 1996; Sawada and Iga, 1996; Sugiyama and Iwatsubo, 1996): 1) prediction of the hepatic disposition of inhibitors, 2) prediction of the concentrations of inhibitors in the hepatic vein after oral administration, and 3) prediction of drug-drug interactions in the gastrointestinal tract (Wacher et al., 1996). In this study, focusing on the first point, we attempted to predict the increasing ratio of the plasma concentrations based on in vivo and in vitro experiments in rats. To exclude the second and third points, the inhibitors were maintained at steady-state concentrations by combinations of iv administration and constant infusion, and intraportal administration of the interacting drugs was carried out. MDZ, an hypnotic drug, was used as a substrate and CIM and NZD, H₂ receptor antagonists, were used as inhibitors.

	Materials and Methods

Top Abstract Introduction Materials & Methods Results Discussion References

Materials. CIM was obtained from SmithKline Beacham Co. (Tokyo, Japan) or was purchased from Sigma Chemical Co. (St. Louis, MO). NZD and MDZ were obtained from Zeria Pharmaceutical Co. (Tokyo, Japan) and Yamanouchi Pharmaceutical Co. (Tokyo, Japan), respectively. Tetramethylammonium chloride was purchased from Nacalai Tesque (Kyoto, Japan). RAN and FAM were supplied from Glaxo Pharmaceutical Japan (Tokyo, Japan) and Yamanouchi Pharmaceutical Co. (Tokyo, Japan), respectively. All other chemicals used as reagents were of HPLC grade.

Animals. Wistar male rats (7-8 weeks of age) were purchased from Kuroda Experimental Animals Co. (Kumamoto, Japan) and Seac Yoshitomi (Fukuoka, Japan). Food and water were supplied ad libitum. The rats were maintained at a suitable temperature.

Preparation of Drug Solutions. The solution of CIM for bolus administration and constant infusion was prepared by dissolving CIM in 1 N HCl and was neutralized using 1 N NaOH. The total volume of CIM solution was adjusted to 3.5 ml by the addition of saline. The solution of MDZ used for intraportal administration was an MDZ injection solution (Dormicam Inj. Nippon Roche K. K., Tokyo, Japan) 10 mg/2 ml). The solution of MDZ used for the experiments on metabolism inhibition was the Dormicam Inj. solution diluted with purified water.

Plasma and Liver Concentration Profiles for CIM and NZD. Rats anesthetized with ether were cannulated in the femoral artery and femoral vein. To determine the infusion rates for CIM and NZD, these drugs were administered through the femoral vein at a dose of 30 mg/kg. Blood samples were collected at 5, 10, 15, 20, 25, 30, 45, 60, 120, 180, and 240 min after administration. The blood was centrifuged at 12,000 rpm for 5 min to obtain the plasma. Plasma concentrations of the drugs were measured using HPLC. Pharmacokinetic parameters were calculated using the nonlinear least-squares method (MULTI program) (Yamaoka et al., 1981). Furthermore, both bolus injection and constant infusion were used to obtain steady-state concentrations of both drugs. Under ether anesthesia, the rats were cannulated in the femoral artery and femoral vein. After the rats had recovered from anesthesia, CIM was administered through the femoral vein at a dose of 3, 5, or 9 mg/rat and was then infused into the rats at one of three constant rates (K_o = 1.4, 2.8, or 5.7 mg/hr/rat), using a syringe infusion pump (model 11; Harvard Apparatus, S. Natick, MA), for 6 hr. NZD was administered through the femoral vein at a dose of 5, 7, or 10 mg/rat and was infused at one of three constant rates (K_o = 2.5, 5.1, or 11.4 mg/hr/rat) for 6 hr. Blood samples were collected at 0, 15, 30, 60, 120, 180, 240, and 360 min after the beginning of the infusion, and the liver was removed at the final sampling time. Blood was centrifuged at 12,000 rpm for 5 min to obtain the plasma. Plasma and liver samples were stored at 20°C, and the drug concentrations in the samples were measured using HPLC.

Effects of CIM or NZD on Plasma Concentration Profiles for MDZ. Under ether anesthesia, the rats were cannulated in the femoral artery, femoral vein, and portal vein. After the rats had recovered from anesthesia, CIM was administrated through the femoral vein at a dose of 9 mg/rat and was then infused into the rats at a constant rate of 5.7 mg/hr/rat, using a syringe infusion pump (model 11; Harvard Apparatus). NZD was administered through the femoral vein at a dose of 10 mg/rat and was infused at a constant rate of 11.4 mg/hr/rat. For treatment with both drugs, MDZ was administrated through the portal vein at a dose of 10 mg/kg at 100 min after the beginning of infusion. Blood samples were collected at 0, 2, 5, 10, 30, 60, 120, and 180 min after administration of MDZ. Blood was centrifuged at 12,000 rpm for 5 min to obtain the plasma. Plasma and liver samples were stored at 20°C, and the drug concentrations in the samples were measured using HPLC (Mandema et al., 1991).

Plasma Protein and Liver Tissue Binding Assays for CIM and NZD. The plasma and liver tissue protein binding of CIM and NZD was evaluated by using equilibrium dialysis. In the present study, we used 0.1 M phosphate buffer at pH 7.0 because the intracellular space is slightly acidic, compared with the pH value for blood (pH 7.4). In brief, 0.5 ml of plasma containing 6, 30, or 60 µM CIM or NZD and 0.1 M phosphate buffer (pH 7.0) containing no drugs were added to the dialysis board set on the dialysis membrane (Spectra/Por membrane; Spectrum Medical Industries, CA) and were incubated at 37°C for 6 hr. After the incubation, 0.1-ml samples were collected from both sides. Two milliliters of liver homogenate containing the drug and 0.1 M phosphate buffer (pH 7.0) containing no drugs were added to the dialysis board set on the dialysis membrane and were incubated at 37°C for 6 hr. After the incubation, a 0.5-ml sample of the homogenate and a 0.1-ml sample of the plasma were collected from the two sides. Plasma and liver homogenate concentrations of the drugs were measured using HPLC. The equations used for the calculation of f_p and f_H are as follows,

(1)

(2)

where C_f is the unbound drug concentration on the buffer side, C_t is the total drug concentration on the protein side, C_b is the bound drug concentration (C_b = C_t  C_f), and n is the percentage of homogenate. It was confirmed in preliminary studies that the protein binding was constant for 5 hr after incubation and the volumes in the buffer and protein cells were scarcely different. Moreover, tissue degradation might not have occurred, because enzymes were removed from the tissue homogenates by overnight equilibrium dialysis at 4°C before the binding study. Indeed, the sum of the unchanged drug concentrations in the buffer cell and the protein cell, as measured by HPLC, was equal to the drug concentration used in the protein binding study. Thus, it was confirmed that tissue degradation did not occur.

Measurement of the Concentrations of CIM, NZD, and MDZ. For determination of the plasma concentration of CIM, 0.1 ml of plasma, 0.1 ml of 75 µM NZD as an internal standard, 0.1 ml of 5 N NaOH, and 5 ml of CH₂Cl₂ were mixed, shaken for 10 min, and then centrifuged at 2000 rpm for 10 min. After the upper aqueous phase had been removed, 4 ml of the organic phase was transferred to another tube and completely evaporated under nitrogen gas. The residue was dissolved in 0.4 ml of the mobile phase, and 0.02 ml was injected into the HPLC system. For determination of the liver concentration of CIM, 0.5 g of liver was homogenized with saline for 1 min, on ice. Then 0.1 ml of 75 µM NZD, 0.1 ml of 0.5 N NaOH, and 5 ml of CH₂Cl₂ were mixed, shaken for 10 min, and then centrifuged at 3000 rpm for 10 min. After the upper aqueous phase had been removed, 3 ml of organic phase was transferred to another tube and completely evaporated under nitrogen gas. The residue was dissolved in 0.4 ml of the mobile phase and passed through a Ministar-RC 15 (Sartorius, Göttingen, Germany) cartridge, and then 0.02 ml was injected into the HPLC system. The extraction procedure for NZD was similar to that for CIM; after evaporation under nitrogen gas, the residue was dissolved in 0.1 ml of the mobile phase and 0.05 ml was injected into the HPLC system. As an internal standard, 75 µM CIM was used (Shimokawa et al., 1994).

In Vitro Metabolism of MDZ . The concentrations of MDZ for in vitro metabolism experiments were 1, 2, 5, 10, 20, and 50 µM. After 0.32 ml of a reaction mixture containing 0.04 ml of microsomes and an NADP-generating system (0.02 ml of 100 mM glucose-6-phosphate, 0.02 ml of 20 mM NADP, 0.02 ml of 20 units/ml glucose-6-phosphate dehydrogenase, 0.004 ml of 10 mM EDTA, 0.016 ml of 125 mM MgCl₂, and 0.2 ml of 0.2 M Na₂HPO₄/KH₂PO₄ buffer, with 0.4 mg/ml microsomal protein) had been preincubated for 2 min, the reaction was started by addition of 0.04 ml of a CIM (0.3 mM) or NZD, RAN, or FAM (1, 2, or 5 mM, respectively) solution and 0.04 ml of a MDZ solution. After incubation at 37°C for 5 min, the reactions were stopped by addition of 0.4 ml of acetonitrile and were centrifuged at 3000 rpm for 2 min. Then 0.4 ml of supernatant was removed to another tube, and the plasma concentration of MDZ was determined. The K_M and V_max values for MDZ and the K_i values for H₂ receptor blockers were calculated by using Lineweaver-Burk plots. For the in vitro kinetic study, we confirmed that there was a good linear relationship between incubation time and metabolite concentration until 5 min after incubation; thereafter, it showed nonlinearity.

Analysis of Data. As shown in Results, the K_M values for MDZ in the presence and absence of CIM and in the presence and absence of NZD were 30.3, 8.1, 9.6, and 5.9 µM, respectively. The average C_p,f values for MDZ were 0.9, 0.3, 0.1, and 0.1 µM, respectively. Thus, the increasing ratio of the plasma concentration of the inhibited drug (MDZ) can be estimated using the following equation (Sawada et al., 1996; Sawada and Iga, 1996; Sugiyama and Iwatsubo, 1996):

(3)

where C_p and AUC represent the blood concentration and the AUC in the absence of inhibitors (CIM or NZD), respectively. C_p' and AUC' represent values measured in the presence of inhibitors. C_H,f and K_i refer to H₂ blockers and H₂ blocker inhibition of MDZ metabolism, respectively. It is difficult to directly measure C_H,f, so that value was calculated using the total liver concentration (C_H) and f_H, as follows:

(4)

As another analytical method, the increasing ratio of the plasma concentration can be estimated using C_p,f instead of C_H,f, according to the following equation.

(5)

C_p,f can be calculated as follows.

(6)

where C_p represents the total plasma concentration.

Statistical Analysis. The statistical analysis was performed by using the Student t test, and p = 0.05 was taken as the minimum level of significance. Data were expressed as mean ± SD.

	Results

Top Abstract Introduction Materials & Methods Results Discussion References

Plasma and Liver Concentration Profiles for CIM and NZD. Plasma concentration profiles for CIM (fig. 1A) and NZD (fig. 1B) at three infusion rates are shown in fig. 1. The plasma concentrations reached steady state 2 hr after injection of the two drugs at any infusion rate, and the plasma concentrations at steady state were almost proportional to the infusion rates for both drugs. Fig. 2 shows the plasma concentration dependence of the K_p,H values for CIM (fig. 2A) and NZD (fig. 2B). The K_p,H values were within the range of 2-5 for CIM and 2-4 for NZD, regardless of the plasma concentration of the drugs.

View larger version (15K): [in this window] [in a new window]   Fig. 1.   Plasma concentration profiles for CIM (A) and NZD (B) at three infusion rates. A, CIM was administered through the femoral vein at a dose of 3 (), 5 (), or 9 () mg/rat and was then infused at one of three constant rates [Ko = 1.4 (), 2.8 (), or 5.7 () mg/hr/rat], using a syringe infusion pump, for 6 hr. Plasma concentrations were determined by HPLC, as described in Materials and Methods. Each point represents the mean ± SD (n = 3 or 4). B, NZD was administered through the femoral vein at a dose of 5 (), 7 (), or 10 () mg/rat and was then infused at one of three constant rates [Ko = 2.5 (), 5.1 (), or 11.4 () mg/hr/rat], using a syringe infusion pump, for 6 hr. Plasma concentrations were determined by HPLC, as described in Materials and Methods. Each point represents the mean ± SD (n = 3).

View larger version (9K): [in this window] [in a new window]   Fig. 2.   Plasma concentration dependence of the Kp,H values for CIM (A) and NZD (B). Kp,H values are the ratio of the liver concentration at 360 min to the plasma concentration at 60 min. Each point represents the mean ± SD (n = 3 or 4).

Effects of CIM and NZD on Plasma Concentrations of MDZ. Fig. 3 shows the plasma concentration-time profile for MDZ after administration through the portal vein, in the presence or absence of CIM (fig. 3A) or NZD (fig. 3B). Table 1 summarizes the kinetic parameters for the metabolism of MDZ in vivo

View larger version (10K): [in this window] [in a new window]   Fig. 3.   Effects of CIM and NZD on plasma concentrations of MDZ. A, CIM was administered through the femoral vein at a dose of 9 mg/rat and was then infused at a constant rate of 5.7 mg/hr/rat, using a syringe infusion pump. MDZ was administered through the portal vein, at a dose of 10 mg/kg, at 100 min after the beginning of the infusion. Plasma concentrations of MDZ were determined by HPLC, as described in Materials and Methods. Each point represents the mean ± SD (n = 5). Significant differences were determined by Student t test (*, p < 0.05). , Plasma concentrations of MDZ in the absence of CIM; , plasma concentrations of MDZ in the presence of CIM. B, NZD was administered through the femoral vein at a dose of 10 mg/rat and was then infused at a constant rate of 11.4 mg/hr/rat. MDZ was administered through the portal vein, at a dose of 10 mg/kg, at 100 min after the beginning of the infusion. Plasma concentrations of MDZ were determined by HPLC as described in Materials and Methods. Each point represents the mean ± SD (n = 4 or 5). , Plasma concentrations of MDZ in the absence of NZD; , plasma concentrations of MDZ in the presence of NZD.

f_p and f_H Values for CIM and NZD. We measured the f_H values for CIM (36 µM) and NZD (36 µM) using liver homogenates. The f_H values for CIM and NZD were obtained by extrapolation of data from 5, 10, 20, and 25% homogenates to 100% homogenates, according to eq. 2, and examined the effect of the dilution ratio of the homogenate on the unbound fraction. The f_H values obtained for CIM and NZD were within the ranges of 0.4-0.5 and 0.7-0.8, respectively, suggesting weak binding of both drugs. Thus, a linear relationship between f_H values and protein concentrations was not observed. Furthermore, the f_p values for CIM and NZD were 0.80 and 0.96, respectively, regardless of the drug concentration (data not shown).

C_H,f/C_p,f Ratios. Table 2 shows the liver concentration, plasma concentration, K_p,H, f_H, and f_p for CIM and NZD, determined from the experiments described above, and the C_H,f/C_p,f ratios calculated using those values. The C_H,f/C_p,f ratios for CIM and NZD were 1.9 and 2.4, respectively, and were greater than 1. 

Inhibitory Effects of Histamine H₂ Receptor Antagonists on the Metabolism of MDZ. Fig. 4 shows the inhibitory effects of CIM, NZD, FAM, and RAN on the metabolism of MDZ by rat liver microsomes, as analyzed using Lineweaver-Burk plots. In the presence or absence of CIM, NZD, FAM, or RAN, the metabolism of MDZ showed only one pattern; metabolism showed competitive inhibition by all of the histamine H₂ receptor antagonists. The calculated K_M and V_max values were 12.75 ± 11.79 µM and 3.15 ± 1.0 nmol/min/mg protein, respectively. The K_i values for CIM, NZD, FAM, and RAN were 108, 2562, 9380, and 6814 µM, respectively.

View larger version (20K): [in this window] [in a new window]   Fig. 4.   Inhibitory effects of histamine H2 receptor antagonists on the metabolism of MDZ. The inhibitory effects of CIM (A), NZD (B), RAN (C), and FAM (D) on the metabolism of MDZ by rat liver microsomes were assessed with Lineweaver-Burk plots. Drug concentrations were determined by HPLC, as described in Materials and Methods. The initial concentrations of MDZ for inhibition of metabolism were 1, 2, 5, 10, 20, and 50 µM, and the concentrations of inhibitors were as follows: CIM, 0.3 mM; NZD, 1, 2, and 5 mM; RAN, 1, 2, and 5 mM; FAM, 1, 2, and 5 mM. A, , MDZ only; , with 0.3 mM CIM; B, C, and D, , MDZ only; , with 1 mM NZD, RAN, or FAM; , with 2 mM NZD, RAN, or FAM; , with 5 mM NZD, RAN, or FAM.

Comparison Between Predicted and Observed Values for the Increasing Ratio of the Plasma Concentration of MDZ. Table 3 shows the predicted increasing ratio of the plasma concentration of MDZ (R_p or R_H) in the presence of CIM or NZD, calculated according to eqs. 3 and 5, and the observed R value (AUC'/AUC). The increasing ratio (R_p) calculated from C_p,f using eq. 5 was underestimated, compared with the observed increasing ratio (R). However, the increasing ratio (R_H) calculated from C_H,f using eq. 3 was very close to the observed value.

	Discussion

Top Abstract Introduction Materials & Methods Results Discussion References

There has been little attempt to precisely predict the increasing ratios of blood concentrations of interacting drugs. For complete prediction, the following problems must be solved (Sawada et al., 1996; Sawada and Iga, 1996; Sugiyama and Iwatsubo, 1996): 1) prediction of the disposition of inhibitors in the liver, 2) prediction of the concentrations of inhibitors in the hepatic vein after oral administration, and 3) prediction of drug-drug interactions in the gastrointestinal tract. The first point is especially important because many drugs are concentratively taken up by liver cells (Yamazaki et al., 1996). To evaluate this point, von Moltke et al. (1996) estimated K_p,H values using a mixture of plasma and liver homogenate in an in vitro study, and they attempted to predict the increasing ratios of the blood concentrations of the interacting drugs. However, they did not take into consideration the concentrative hepatic uptake of drugs through active transport. Nakamura et al. (1994) reported the concentrative hepatic uptake of H₂ receptor antagonists such as CIM and NZD, using isolated rat hepatocytes. From those findings, it has been concluded that, when histamine H₂ receptor antagonists are used as inhibitors of drug metabolism, C_H,f but not C_p,f is an important determinant (Sawada et al., 1996; Sawada and Iga, 1996; Sugiyama and Iwatsubo, 1996).

As shown in table 2, the predicted C_H,f/C_p,f values for CIM and NZD were approximately 1.9 and 2.4, respectively, suggesting the existence of concentrative hepatic uptake of both drugs. In contrast, Nakamura et al. (1994) reported that the intracellular/medium concentration ratios for CIM and NZD were 7-10 and 1-3, respectively, as determined using isolated hepatocytes. Those values were higher than our results. One of the reasons for this discrepancy could be that the concentrative hepatic uptake was saturated because of the high concentrations of CIM and NZD in our experiments. Furthermore, heterogeneous distribution of inhibitors in the liver can be hypothesized. In this study, the C_p,f for the cytosol may be underestimated if the inhibitors are not distributed to certain organelles in liver cells. However, we could not avoid this problem, because liver homogenates were used to determine the nonspecific binding of drugs to liver tissue in this study. This problem may be resolved in future studies by performing in vitro uptake experiments using ATP-depleted hepatocytes without an energy source for active transport of drugs (Sugiyama and Iwatsubo, 1996). As shown in table 3, the increasing ratio of the plasma concentration of MDZ estimated from the C_H,f data was very close to the observed value, suggesting the suitability of the hypothesis in this analysis. It may possible to predict the extent of drug-drug interactions after iv administration to humans based on the pharmacokinetic parameters (K_M, K_i, and V_max) for inhibitors obtained using human liver microsomes and/or human CYP-expressing systems, the f_H and K_p,H values obtained using experimental animals such as rats, and the time profiles of the total plasma concentration and the unbound concentrations of inhibitors.

Many clinical cases and clinical tests of the interaction of MDZ and H₂ receptor antagonists (CIM, RAN, and FAM) have been reported (Ellwood et al., 1983; Dundee et al., 1984; Fee et al., 1987; Ochs et al., 1986). To predict this interaction, the second and third points mentioned above are important because both drugs are almost always orally administered in clinical settings. Recently, it was reported that MDZ was mainly metabolized by CYP3A4 in the intestine (Wacher et al., 1996). However, at the present time, the extent of the inhibitory effects of CIM and NZD on metabolism in the intestine is unclear, and further studies are necessary. Moreover, it should be considered that drug concentrations in the portal vein are higher than those in the systemic circulation after oral administration of inhibitors (Hoffman et al., 1995; Fujieda et al., 1996; Paine et al., 1996). Therefore, we must take into consideration the point mentioned above, to estimate C_p,f and C_H,f as the concentrations of drugs near metabolic enzymes. The extent of the inhibitory effect is underestimated if the concentrations of drugs in the systemic circulation are used as the concentrations of inhibitors. Therefore, it is necessary to estimate the drug concentrations in the portal vein, considering the absorption ratio of the drugs, and to combine those values with the blood concentrations of the drugs in the systemic circulation to predict the concentrations of inhibitors near metabolic enzymes.

In conclusion, the increasing ratio of the blood concentration of MDZ in the presence of H₂ blockers could be quantitatively estimated from the C_H,f values for the inhibitors and the K_i values obtained using liver microsomes. Moreover, when the inhibitors were actively transported into the liver, the inhibition ratio was underestimated using the C_p,f values for the inhibitors. To predict the increasing ratio of blood concentrations in the case of oral administration of both an interacting drug and an inhibitor, in future studies, the development of models considering metabolism and absorption in the gastrointestinal tract would be necessary.