Abstract
The pharmacokinetic and pharmacodynamic behaviors of 4-[3-[3-[Bis(4-isobutylphenyl) methylamino] benzoyl]-1H-indol-1-yl]-butyric acid (FK143), a new nonsteroidal steroid 5α-reductase inhibitor, in the ventral prostate were investigated after i.v. administration to rats. The relationship between blood concentrations at 24 hr and doses was linear in the range of 0.1 to 20 mg/kg. However, the levels of FK143 in the prostate were saturated over the dose of over 5 mg/kg. The dissociation constant (Kd) and maximum amount of binding substances (Bmax), calculated according to nonlinear kinetic analysis including a specific binding pool, was 0.0553 ± 0.0117 μg/ml (92 nM, estimated value ± S.D.) and 0.908 ± 0.092 μg/g tissue, respectively. A combined pharmacokinetic/pharmacodynamic (PK/PD) model was constructed using change in dihydrotestosterone (DHT) levels in the prostate after i.v. administration of FK143 as an index for its pharmacological effect and blood concentration as an input function. The apparent reaction rate constant of drug and enzyme (K) was 39.7 ± 25.1 g tissue/μg/hr (estimated value ± S.D.), the apparent turn-over rate constant of enzyme (k) was 0.140 ± 0.107 hr−1, the elimination rate constant of DHT (kel, DHT) was 1.13 ± 0.94 hr−1 and the fraction of FK143-insensitive DHT synthesis (F) was 0.461 ± 0.037. The PK/PD analysis suggested that the duration of the effect of FK143 was related to its accumulation in the binding pool of the prostate. After i.v. administration of FK143 in the range of 0.1 to 20 mg/kg, the DHT levels in the prostate decreased to about 40% of control value, after which despite the rapid decline of blood FK143 concentration, slowly recovered according to the elimination rate of FK143 in the prostate. Moreover, the PK/PD profiles of FK143 after repeated i.v. administration were predictable by using the PK/PD parameters obtained after single administration of FK143.
The active androgen in the prostate is hypothesized to be DHT, which is converted from testosterone by steroid 5α-reductase, a hormone secreted in the testis (Anderson and Liao, 1968; Bruchovsky and Wilson, 1968a, b; Hammond, 1978; Sandberg, 1980). In support of this concept, the level of DHT and steroid 5α-reductase activity in the prostate in patients with benign prostatic hyperplasia are higher than those in normal men (Gloyna et al., 1970a, b; Hudson et al., 1982a). Only finasteride N-tert-butyl-3-oxo-4-aza-5α-androst-1-ene-17β-carboxamide, Merck, Rahway, NJ), a 4-azasteroid, is presently in clinical use as a steroid 5α-reductase inhibitor. It reduces the prostate DHT level by approximately 85% and about 20% of the volume of the prostate, respectively, although more than 6 months are needed for the appearance of its effect (Peters and Sorkin, 1993; The Finasteride Study Group, 1994; Rittmaster, 1994; Steiner, 1996). Therefore, the prediction of its effect in the prostate from its blood concentration profiles would be useful for effective dosage planning in clinical studies.
FK143, a novel compound with a non-steroidal structure, which inhibits steroid 5α-reductase in a noncompetitive inhibition manner, decreases the level of DHT in the prostate (Hirosumi et al., 1995a) and reduces the size of the prostate in rats and dogs (Hirosumiet al., 1995b). We have previously investigated the disposition of FK143 in rats and reported that FK143 was transported from the blood to the prostate by a membrane-limited process and eliminated very slowly from the prostate as compared with from the blood (Katashima et al., 1997). In this study, we investigated the pharmacokinetic and pharmacodynamic behaviors of FK143 after a single i.v. administration to rats, using DHT levels in the prostate as an index for pharmacological effect due to the delay in change of size of the prostate. The effect of FK143 on steroid 5α-reductase activity after repeated administration was also quantitatively evaluated using the PK/PD model.
Materials and Methods
Chemicals
FK143 and FR130976 (4-[1-[3-[Bis(4-isobutylphenyl)methylamino]benzoyl]-1H-indol-3-yl]-butyric acid) were synthesized in Fujisawa Pharmaceutical Co. Ltd.(Osaka, Japan). Acetonitrile and n-hexane were purchased from Wako Pure Chemicals (Osaka, Japan) and were of HPLC grade. All other chemicals used were of analytical grade.
Animals
Male Sprague-Dawley rats aged 8 weeks (Nippon Bio-Supp, Center, Tokyo, Japan) were used in the experiments. Rats were maintained on a 12-hr light/dark cycle with food and water provided ad libitum.
Pharmacokinetic Study
FK143 was dissolved in PEG-400 and i.v. bolus administered from tail vein at a dose of 1 mg/kg to rats. At 0.05, 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, 10 and 24 hr after administration, blood samples were collected from the aorta using a heparinized syringe under light ether anesthesia (just before sample collection) and were immediately centrifuged at 2000 × g for 10 min. After collection of the blood samples, the ventral prostate was excised, weighed and homogenized on ice with 3 volumes of ice-cold 20 mM phosphate buffer (pH 7.0) using polytron homogenizer. In the same way, the blood and prostate samples were collected at 24 hr after i.v. administration of FK143 at doses of .1, .2, .5, 5, 10 and 20 mg/kg to rats. The samples were stored at -20°C until analysis using HPLC (Katashima et al., 1997). In the repeated dosing study, FK143 was administered at 1 mg/kg every 24 hr for 3 or 7 days, via a PE-10 polyethylene tube (Becton Dickinson & Co., sparks, MD) cannulated in the external jugular vein. The blood and prostate samples were collected at 24 hr after the first, third and seventh administration of FK143. Rats were killed at each point for blood or prostate sampling.
Blood concentrations and prostate levels of FK143 after single or repeated administration of 1 mg/kg have already been reported (Katashima et al., 1997).
Pharmacodynamic Study
FK143 was dissolved in PEG-400 and i.v. as a bolus administered to rats at 0.1, 0.2, 0.5, 1, 5, 10 and 20 mg/kg. The prostate samples were excised under light ether anesthesia (just before sample collection) at 1, 4, 10, 24, 30, 48, 72 and 240 hr after administration to rats (only 24-hr samples were collected in the case of 10 and 20 mg/kg dosing). In the repeated dosing study, FK143 was administered at 0.1, 1 and 20 mg/kg every 24 hr for 3 or 7 days and the prostate samples were excised at the same time points as in the “Pharmacokinetic study.” The vehicle was administered to rats in the control group, which were treated in the same way as FK143-administered rats. The prostate samples were weighed and homogenized on ice with 9 volumes of ice-cold distilled water. The prostate homogenate samples were stored at -20°C until assay of DHT levels with Radioimmunoassay (3H radioimmunoassay kit, Amersham, Buckinghamshire, UK).
Data Analysis
Pharmacokinetic analysis of the prostate levels of FK143.
The blood concentrations of FK143 (Cb = Cp · RB) were calculated from plasma concentrations (Cp) using blood to plasma concentration ratio (Rb = 0.68) (Katashimaet al., 1997) and fitted to the following equation by the nonlinear least squares method using a WinNonlin program (Version 1.1, Scientific Consulting Inc., Apex, NC)
Therefore, A2 is given by the the following equation (6).
Accordingly, the differential equation for At is as follows:
Furthermore, the following equations can be derived from equations (4) and (8).
The value of CL1 was previously estimated by integration plot (Katashima et al., 1997). The physical volume of the prostate, Vd, was assumed to be 1 ml/g. The prostate levels of FK143 after i.v. administration in the range of 0.1 to 20 mg/kg were fitted to the equations (4) and (9) by the nonlinear least squares method using a WinNonlin program and the kinetic parameters k2,Kd and Bmax were estimated. In the simulation study, the binding characteristics of FK143 in the prostate was assumed not to be changed during repeated administration.
PK/PD analysis of FK143.
In this PK/PD analysis (fig.1), the changes of DHT level in the prostate after i.v. administration of FK143 were used as an index of pharmacological effect. The inhibitory effects on steroid 5α-reductase were analyzed by the PK/PD model with inhibition parameters for the enzyme (Katashima et al., 1995; Sugiuraet al., 1992; Yamamoto et al., 1996), assuming that the FK143 in the precusor pool can react with steroid 5α-reductase. It was assumed that steroid 5α-reductase was synthesized at a constant rate, Ks, and eliminated with first order rate constant, k3, and DHT was synthesized at a constant rate, v0, by steroid 5α-reductase and eliminated with first order rate constant, kel, DHT (Ko and Jusko, 1995). The amount of active enzyme E and DHT levels in the prostate are given by the following equations.
In the steady state, the amounts of steroid 5α-reductase and DHT are maintained at constant values of E0 and DHT0, respectively.
After administration of FK143, drug in the precusor pool is assumed to react with steroid 5α-reductase with second-order rate constant, K, and active form of enzyme E is assumed to be transformed to inactive form Ec. If the elimination rate of E and Ec are the same, the total amount of enzyme is kept constant, E0 (= E + Ec). Assuming that Ecrecovered to E with first order rate constant, k4, the amount of E is expressed by the following equation:
Assuming steroid 5α-reductase activity (ε) is linearly related to the ratio of active enzyme to total enzyme (E/E0), the following equation is derived from equations (13) and (15).
The synthesis rate of DHT is assumed to be related to ε, therefore DHT levels after administration of FK143 are given by the following equation (17):
FK143 levels in the precursor pool (A1) were calculated from blood concentrations and FK143 levels in the prostate by equation (9). Then, equations (16) and (18) were fitted to the changes of DHT levels after administration of FK143 in the range of 0.1-20 mg/kg by nonlinear least squares method using a WinNonlin program to estimate the PK/PD parameters K, k, kel, DHT and F.
Results
Pharmacokinetics of FK143.
The blood concentrations and prostate levels of FK143 after i.v. administration of 1 mg/kg to rats are shown in figure 2. The pharmacokinetic parameters from equation 1 were 7.62 ± 4.49 μg/ml (estimated value ± S.D.) for A, 0.692 ± 0.370 μg/ml, 0.105 ± 0.021 μg/ml for C, 17.5 ± 10.0 hr−1 for α, 1.77 ± 0.78 hr−1 for β and 0.0672 ± 0.0157 hr−1 for γ. FK143 was very slowly eliminated from the prostate (t1/2 = 113 hr) compared with that from the blood (t1/2 = 10.3 hr). The concentrations of FK143 in the blood and prostate at 24 hr after i.v. administration are shown in figure3. The blood concentrations were linear up to 20 mg/kg, although FK143 level profile in the prostate was saturable with the doses of 5 mg/kg or more. FK143 levels in the blood and prostate at 0.1 mg/kg were under determination limit (blood, 10 ng/ml; prostate, 40 ng/g).
We carried out our pharmacokinetic analysis using the model with binding pool in the prostate (fig. 1). The estimated parameters of binding were 0.0553 ± 0.0117 μg/ml (92.0 nM, estimated value ± S.D.) for Kd , 0.908 ± 0.092 μg/g for Bmax and 0.214 ± 0.022 hr−1 for k2, assuming Vd equals 1 ml/g. A good agreement was observed between the fitted curves and the observed values (fig.4). Furthermore, the simulation curve based on the above kinetic parameters showed good agreement with the observed data after repeated administration of FK143 at 1 mg/kg once a day (fig. 5).
Pharmacokinetic/pharmacodynamic analysis of FK143.
DHT levels in the prostate at 24 hr after administration of FK143 decreased with a dose-dependent manner in the range of 0.1 to 20 mg/kg and appeared to be nonlinear at more than 5 mg/kg. The estimated dynamic parameters were 39.7 ± 25.1 g tissue/μg/hr (estimated value ± S.D.) for K, 0.140 ± 0.107 hr−1, for k 1.13 ± 0.94 hr−1 and for kel, DHT 0.461 ± 0.037 for F. A good agreement was noted between the fitted curves and observed values for prostate DHT level in the range of 0.1 to 20 mg/kg (fig. 6). Also, the simulation curves, for the change of DHT level after repeated administration of FK143 at 0.1, 1 and 20 mg/kg, based on dynamic parameters after single administration of FK143 were in good agreement with the observed values (fig. 7).
Discussion
We have already suggested the existence of a binding pool in the prostate, a part of which might be related to steroid 5α-reductase or its associated substances (Katashima et al., 1997). In this study, blood concentrations of FK143 at 24 hr after administration were linear with dose escalation (0.1-20 mg/kg).However, the prostate levels of FK143 were nonlinearly increased (fig. 3) withKd value of 92.0 nM. TheKd values of 4-methyl-aza-steroid, which is another steroid 5α-reductase inhibitor, were 7 nM for the prostate microsome and 6.5 nM for the liver microsome, respectively, and were comparable with Ki value (5 nM) for steroid 5α-reductase (Liang et al., 1983). In addition, NADPH-dependent binding capacities of 4-methyl-aza-steroid correlated with the steroid 5α-reductase activity in several tissues (Lianget al., 1983). On the other hand, the affinity of FK143 for binding pool in the rat prostate (Kd , 92.0 nM) in vivo was more than 10 times larger than IC50 (=Ki , 4.2 nM) obtained from the inhibitory effect on steroid 5α-reductase in vitro. Further study will be required to relate binding parametersin vivo to those in vitro.
In the developed PK/PD model (fig. 1), FK143 in the precursor pool (A1) directly reacts with steroid 5α-reductase to transform the enzyme to inactive form. The apparent turn-over rate constant of enzyme, k, defined as k3 + k4, was estimated to be 0.140 hr−1 and the apparent half life of the enzyme, calculated from k, was 5.0 hr. It is reported that the turn-over half lives of NADPH P-450 reductase, cytochrome b5 and drug metabolic enzymes, P-450 (PB) and P-450 (MC), in rat liver are 35, 50, 25 and 15 hr, respectively (Sadano and Omura, 1982). Assuming that the turn-over of steroid 5α-reductase also takes as long as scores of hours, k4 should be much larger than k3, resulting in k ≈ k4. Accordingly, the half life of recovery from inactive form to active form of enzyme was estimated to be 5.0 hr. Then, the apparent Ki value in vivo (k/K) was estimated to be 6 nM, which is similar to the IC50 (4.2 nM) (Hirosumi et al., 1995a)in vitro.
The recovery half-life of enzyme (5.0 hr) was shorter than the terminal phase elimination half life of FK143 from the blood (10.3 hr, fig. 2). However, the levels of DHT in the prostate after administration of FK143 decreased gradually, reaching minimum value at about 10 to 24 hr after administration, and then recovered very slowly (fig. 6). The elimination of FK143 from the prostate was slow with a half-life of 113 hr (fig. 2). This finding suggested that the duration of the pharmacological effect of FK143 is influenced by the existence of the binding pool, which characterized the tissue distribution profile of the drug in the prostate.
It is reported that the time course of the recovery of vitamin K epoxide reductase activity is parallel with that of the level of microsome-free warfarin binding sites in the liver after administration of warfarin to rats and that the half life of recovery of enzyme activity is about 7 days (Thijssen and Janssen, 1994). Similarly to warfarin, the tissue binding of FK143 was closely related to its inhibitory effect on the enzyme. We have previously analyzed the inhibitory effect of proton pump inhibitors, omeprazole and lansoprazole, on acid secretion (Sugiura et al., 1992;Katashima et al., 1995) and the antiplatelet effect of aspirin (Yamamoto et al., 1996), using a PK/PD model similar to that used in this study. The long duration of inhibitory effect of these drugs, compared with elimination from the plasma was proved to be controlled by the irreversible binding of drugs and enzymes. It seems that our PK/PD model is useful for evaluating the quantitative relationship between plasma drug concentration and enzyme inhibition with various mechanisms.
In our PK/PD model, DHT is assumed to be synthesized at a constant rate and eliminated with first-order process. DHT in the prostate is mainly metabolized by 3α-HSOR, 3β-HSOR and 17β-HSOR. Among these enzymes, 3α-HSOR plays an important role in controlling DHT levels in the prostate (Isaacs and Coffey, 1981; Sandberg, 1980; Hudson, 1982b). The elimination rate constant of DHT (kel, DHT) was estimated to be 1.13 hr−1 in the present PK/PD analysis. Intrinsic clearance of DHT metabolism (CLint = kel, DHT · Vd) in the prostate can be calculated to be 1.13 ml/hr/g tissue. CLint of DHT for the metabolism by 3α-HSOR in rat prostate microsome in vitro is estimated to be 0.0509 ml/hr/mg protein based on the report by Fukabori et al. (1992), which is further calculated to be 3.33 ml/hr/g tissue based on the report by Haaparanta et al. (1983). CLint estimated from the in vitrostudy was only about three times larger than that estimated from kel, DHT obtained by PK/PD analysis in vivo. The difference between in vivo and in vitro may be related to the location of 3α-HSOR in the prostatic cells, because 3α-HSOR exists not only in microsomes but also in cytosol and nuclear fraction (Fukabori et al., 1992; Abalainet al., 1989). Furthermore, the CLintin vivo may be underestimated because 3α-androstanediol produced from DHT by 3α-HSOR is partly returned to DHT in vivo(Isaacs and Coffey, 1981; Sandberg, 1980; Hudson, 1982b).
Although FK143 almost completely inhibited steroid 5α-reductasein vitro (Hirosumi, 1995a), DHT levels in the rat prostates were not decreased to less than 40% of control by FK143 (figs. 6 and7), which is similar to the results reported by Hirosumi et al., 1995b. In this PK/PD model, a route of DHT synthesis was assumed which is not inhibited by testosterone, but other possibilities cannot be excluded. For example, testosterone levels in the prostate may be increased by the inhibition of steroid 5α-reductase by FK143, resulting in the increase in DHT synthesis rate. But, it is reported that the levels of testosterone do not influence the inhibitory effect of FK143 on the enzyme (Hirosumi et al., 1995a). Another possibility is that the concentrations of FK143 may not be sufficient for substrate of steroid 5α-reductase in the prostate in vivo. The reason for incomplete reduction of DHT level remains unclear.
In conclusion, the PK/PD behavior of FK143 in the prostate was clarified by nonlinear distribution data of FK143 in the prostate and the change in DHT levels in the prostate as index for pharmacological effect. The PK/PD profiles of FK143 after repeated administration could be successfully predicted from the profiles after single administration.
Acknowledgments
The authors thank Dr. Toshitaka Manda for his critical comments on this paper, and Dr. Susumu Tsujimoto and Ms. Sanae Matsumoto for technical assistance.
Footnotes
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Send reprint requests to: Masataka Katashima, Pharmaceutical and Pharmacokinetic Research Laboratories, Fujisawa Pharmaceutical Co. LTD., 1-6, Kashima 2-chome, Yodogawa-ku, Osaka 532, Japan.
- Abbreviations:
- FK143
- 4-[3-[3-[Bis(4-isobutylphenyl) methylamino] benzoyl]-1H-indol-1-yl]-butyric acid
- PK/PD
- pharmacokinetic/pharmacodynamic
- DHT
- dihydrotestosterone
- Cb
- blood concentration of FK143
- A1
- amount of drug in the precursor pool
- A2
- amount of drug in the binding pool
- CL1
- tissue uptake clearance from blood compartment to precursor pool
- k2
- efflux rate constant from precursor pool to blood compartment
- Kd
- dissociation constant of drug-binding substances complex
- Bmax
- maximum amount of specific binding substances of drug
- E
- amount of active steroid 5α-reductase
- Ec
- amount of inactive steroid 5α-reductase
- K
- apparent reaction rate constant of FK143 and steroid 5α-reductase
- Ks
- biosynthesis rate of steroid 5α-reductase
- k3
- elimination rate constant of steroid 5α-reductase
- k4
- recovery rate constant of inactive steroid 5α-reductase
- k
- apparent turn-over rate constant of enzyme (k3+k4)
- v0
- biosynthesis rate of DHT
- kel, DHT
- elimination rate constant of DHT
- ε
- ratio of active and total steroid 5α-reductase
- F
- fraction of DHT synthesis, which is not inhibited by the drug
- 3α-HSOR
- 3α-hydroxysteroid oxidoreductase
- 3-β-HSOR
- 3β-hydroxysteroid oxidoreductase
- 17β-HSOR
- 17β-hydroxysteroid oxidoreductase
- Received February 18, 1997.
- Accepted November 26, 1997.
- The American Society for Pharmacology and Experimental Therapeutics