Lack of pharmacokinetic and pharmacodynamic interactions between ketoconazole and prednisolone The effects of ketoconazole on the pharmacokinetics and pharmacodynamics of intravenous prednisolone (14.8 mg) were assessed in six healthy volunteers. Subjects were studied with and without receiving ketoconazole, 200 mg orally for 6 days. The addition of ketoconazole did not significantly change the clearance (96 ± 11 versus 90 ± 11 mVhr/kg), mean residence time (4.29 ± 0.43 versus 4.45 ± 0.59 hours), volume of distribution (0.41 ± 0.02 versus 0.40 ± 0.02 L/kg), or plasma protein binding characteristics of prednisolone. The suppressive effects of prednisolone on serum cortisol, blood basophil, and helper T lymphocyte values, assessed by the ratio of the area under the curve (AUC) after prednisolone administration to the baseline AUC, was not altered significantly by ketoconazole. The 50% inhibitory concentration values derived from pharmacodynamic models developed to describe the direct suppressive effects of corticosteroids indicated no alteration in intrinsic sensitivity in the presence of ketoconazole. Ketoconazole does not appear to alter the pharmacokinetics or the pharmacodynamic response patterns of selected direct suppression effects of single low doses of prednisolone. (CLIN PHARMACOL THER

1991;49:558-70.)

Sharon K. Yamashita, PharmD,a Elizabeth A. Ludwig, PharmD, Elliott Middleton, Jr., MD, and William J Jusko, PhD Buffalo, N.Y.

Prednisolone, the active metabolite of prednisone, is eliminated predominantly in the liver by conjuga-

tion and hydroxylation by mixed-function oxidases,I thus rendering itself susceptible to enzyme induction and inhibition by other drugs.2.3 Ketoconazole is a known inhibitor of microsomal enzyme activity.4 Although a decrease in methylprednisolone clearance has been seen after the administration of ketoconazole,5 From the Departments of Pharmaceutics, Pharmacy, and Medicine, Schools of Pharmacy and Medicine, State University of New York at Buffalo, and Departments of Pharmacy and Medicine, Buffalo General Hospital. Supported in part by grant No. 24211 from the National Institute of General Medical Sciences, National Institutes of Health, Bethesda, Md. Presented at the Ninety-second Annual Meeting of the American Society for Clinical Pharmacology and Therapeutics, San Antonio, Texas, March 13-15, 1991. Received for publication Sept. 28, 1990; accepted Jan. 21, 1991. Reprint requests: William J. Jusko, PhD, Department of Pharmaceutics, State University of New York at Buffalo, 565 CookeHochstetter Complex, Buffalo, NY 14260. 1990 recipient of the Upjohn Excellence in Research Award at State University of New York at Buffalo for this study. 13/1/28205

558

the effects on prednisolone clearance have been contradictory.63 However, these differences are consistent with the steroid-specific interactions seen with other drugs such as troleandomycin, which inhibits the metabolism of methylprednisolone without an effect on prednisolone.8 Further elucidation of the interaction between ketoconazole and prednisolone is thus warranted. Of importance is an understanding of the pharmacodynamic interaction between ketoconazole and prednisolone. The direct inhibitory effects of ketoconazole on adrenal steroid synthesis as a result of its ability to inhibit the cytochrome P450-dependent enzymes involved in steroidogenesis have been well documented in the literature.9-i2 Ketoconazole may also alter the immune response as evidenced by in vitro inhibition of the lymphocyte response to mitogens and impairment of the phagocytic function of polymorphonuclear leukocytes. 13,14 The drug also acts as a competitive antagonist at the glucocorticoid receptor site, with the potential to displace steroids and reduce their effects. 15 Loose et al. 15 demonstrated that the 50% inhibitory concentration (IC50) of ketoconazole for the inhibition of dexa-

VOLUME 49 NUMBERS

methasone binding to cytosolic steroid receptors in cultured cell preparations was 20 Rmol/L. Studies of chronic ketoconazole administration have yielded mean peak plasma concentrations (Croax) of 3 pAnol/L.16 Thus the percentage of bound ketoconazole to steroid receptors can be estimated as 13% from the ratio: Cmax/(Cmax + IC50). The affinity of prednisolone to the corticosteroid receptor, however, is only 15% of that of dexamethasone and hence may be displaced more easily.17 The possible overall effects of ketoconazole on the immunologic actions of prednisolone thus represent a complex interaction of several variables. The area under the curve of cortisol from 0 to 24 hours [AUC(0-24)] has been used as a measure of the adrenal suppressive actions of corticosteroids. By examining the ratio of AUC(0-24) after methylprednisolone administration to the AUC(0-24) under baseline conditions, Glynn et al.5 and Kandrotas et al.18 showed slightly enhanced cortisol suppression when ketoconazole was added to methylprednisolone. To date the pharmacodynamic interaction between ketoconazole and prednisolone (after administration of oral prednisone) has been studied only by Ludwig et al. ,7 who did not find a significant effect of ketoconazole on cortisol suppression in four healthy subjects. Confirmation of these findings is thus desired. In addition, pharmacodynamic models that integrated the pharmacokinetics of corticosteroids with several pharmacodynamic actions have recently been described. 19 The effects of ketoconazole on these pharmacodynamic parameters are of interest. Therefore this study was designed to reassess the pharmacokinetic interaction, as well as to examine the pharmacodynamic interactions, between ketoconazole and prednisolone in humans.

PHARMACODYNAMIC MODELS The pharmacodynamic activity of corticosteroids results from its reversible binding to cytosolic receptors, thus forming a steroid-receptor complex. Steroids can elicit both directly suppressive and gene-mediated effects. The latter effects are mediated through DNAdirected synthesis of secondary messengers and proteins and are characterized by a slow and delayed induction period. The direct suppressive effects, including suppression of cortisol, and redistribution of basophils (as represented by whole-blood histamine MBH1) and T lymphocytes probably occur too rapidly to be the result of DNA-mediated events. These immediate effects have been characterized with pharmacodynamic models (Fig. 1). Such models integrate both the pharmacokinetics of the administered cortico-

Ketoconazole and prednisolone

559

Cortisol Rcod

T-Helper Cells

Histamine (Basophils) kh

C pN , IC50

Fig. 1. Diagrammatic representation of the direct suppression models for cortisol, helper T lymphocytes, and whole blood basophils (histamine). Rc,, Circadian concentration; Rb, concentration amplitude; Rm, mean cortisol concentration; prednisolone concentration; IC50, 50% inhibitory concentration; C, cortisol: tz, peak time of circadian function; TH, helper T cell count; kr, helper T cell decline rate constant; H, histamine; kh, first-order rate constant for migration of basophils from blood to the extravascular compartment; kr", zero-order rate of return of the basophils from the extravascular compartment.

C,

steroid with the suppression and eventual return of the baseline physiologic rhythms of cortisol and basophils and has been extended to helper T lymphocytes. A cosine function describes the baseline circadian profile of cortisol:

kort = Rm + Rb cos (T

tz)

(1c)

(15/57.3)

(

1

)

(2)

in which Rcor, is the circadian concentration of cortisol, Rm is the mean cortisol concentration, Rb is the concentration amplitude, tc is the time in radians, T is the clock time within a 24-hour cycle, and tz is the ac-

rophase or peak time of the circadian function.

560

CLAN

Yamashita et al.

MAY 1991

Prednisolone concentrations (Cp) versus time (t) are biexponential: Cp = C,

e-x" +

C2

et

(3)

in which C, and C2 are intercept constants and X1 and X2 are the disposition slopes. After administration of prednisolone, the cortisol concentrations also fall in a biexponential fashion. Thus the time course of cortisol (C) after administration of a single dose of prednisolone can be described by the following equation: C

=

Ca

e

'

+

Ct,

et

into the study. All of the subjects were nonsmokers and were taking no medications before the study and throughout the duration of study. Exclusion criteria included hypersensitivity to corticosteroids or ketoconazole, chronic illness necessitating medications or contraindicating steroid or ketoconazole use, or history of alcohol, marijuana, or drug abuse. Written informed consent was obtained from all subjects before enrollment into the study. The study was approved by the Buffalo General Hospital Institutional Review Board. The study was divided into three phases with each phase separated by a washout period of week. On each study day an intravenous catheter was inserted into an arm vein and maintained patent with the instillation of small volumes of a heparinized saline solution (10 units/m1). The purpose of the first phase was to determine baseline cortisol, T lymphocyte, and WBH concentrations during a 32-hour period. Blood samples (5 to 10 ml) were drawn from the catheter starting at 8 AM, every 2 hours for 24 hours, and again at 28 and 32 hours. An additional 4 ml blood was drawn at 0, 1, 2, 4, 8, 12, 16, 20, and 24 hours for T lymphocyte analysis. During the second phase, subjects received a 20 mg intravenous bolus dose of prednisolone sodium phosphate (Hydeltrasol, Merck Sharp and Dohme, West Point Pa.; equivalent to 14.8 mg prednisolone) in the arm opposite to that in which the catheter was inserted. Blood samples collected at 0, 1/4, 1/2, 3/4, 1, 2, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 28, and 32 hours were used to determine the pharmacokinetics and pharmacodynamics of prednisolone. For the last phase of the study the subjects were given a 6-day supply of ketoconazole (Nizoral, Janssen Pharmaceuticals, Piscataway, N.J.) and instructed to take one 200 mg tablet at approximately 8 AM each day starting 5 days before the study day. On the morning of day 6 the same procedure as outlined for phase 2 of the study was repeated. The purpose of the final phase of the study was to evaluate the effect of ketoconazole on the pharmacokinetics and pharmacodynamics of prednisolone. After sample collection, 300 )1,1 whole blood was removed and stored for WBH analysis. All blood samples were then centrifuged immediately and the plasma was stored at -20° C until assay. Blood for T lymphocyte counts was collected in EDTA-containing collection tubes and analyzed within 12 hours of collection. Assays. Total concentrations of prednisolone and cortisol in plasma were determined by HPLC with the assay of Rose and Jusko.2° Plasma concentrations of 1

(4) Reort

(1

Cp +CPIC50)

in which Ca and Cb are the intercept constants and a and 13 are the biphasic slopes. Because helper T lymphocytes also exhibit a circadian variation (inverse to that of cortisol), a similar model can be used to integrate the pharmacokinetics of prednisolone with its effects of T cell suppression. Unlike cortisol, however, T lymphocytes appear to decline monoexponentially after the administration of steroids. The pharmacodynamic model describing the effects of corticosteroids on basophils measured as WBH is based on the premise that the rate of change in the basophil concentrations at any given time is a function

of the first-order rate constant for the migration of basophils from the blood to the extravascular compartment (kh) and a zero-order rate of return of the basophils from the extravascular compartment (kr°):

dHCpIC50 = kr" (1

PHARMACOI, THER

Cp +

kh

WBH

(5)

in which H = Ho at

t = 0 represents the baseline WBH concentration (Fig. 1). This relationship is slightly different from that of Kong et al. 19 and characterizes basophil trafficking more accurately.*

EXPERIMENTAL METHODS Subjects. Six healthy male volunteers between the ages of 22 and 37 years and within 20% of their ideal body weight were enrolled for each phase of the study. Although seven volunteers were initially recruited, one subject was removed from the study because of the development of an iron-deficiency anemia. Subjects were considered healthy on the basis of complete medical histories, physical examinations, and blood chemistry profiles (SMA-17) before entry *Wald JA, Jusko WJ. Abstract, Pharm Res 1990;7:5219.

VOLUME 49 NUMBERS

Ketoconazole and prednisolone

steroids as low as 5 ng/ml can be detected with this method. Both the intraday and interday coefficients of variation for 40 and 600 ng/ml samples were less than 5%. No interference was seen with ketoconazole added at a concentration of 10 p,g/m1 or from plasma samples taken from subjects receiving ketoconazole alone. The concentrations of unbound prednisolone were determinied at 37° C by an ultrafiltration technique (Centrifree Micropartition System; Amicon Corp., Danvers, Mass.). WBH was measured by a commercial radioimmunoassay method (NMS Pharmaceuticals Inc., Newport Beach, Calif.). A total leukocyte count was performed by an automated hemocytometer (Coulter Counter S-Plus IV; Coulter Electronics Inc., Hialeah, Fla.). The proportion of lymphocytes and monocytes was determined microscopically, and the number of total circulating lymphocytes per milliliter was calculated. Mononuclear cells were separated on Ficoll Hypaque and reacted with monoclonal antibody (anti-CD4 and CD8; Becton Dickinson and Co., Cockeysville, Md.) and analyzed on an automated flow cytometer (FACS 440; Becton Dickinson and Co.). The total number of circulating T helper cells was determined by multiplying the proportion of fluorescent cells by the number of circulating lymphocytes. The area under the prednisolone concentration time curve to infinity (AUC) and the area under the first moment curve to infinity (AUMC) were determined with the slopes and intercepts of equation 3 generated by least-squares regression analysis of the plasma concentration data with the PCNONLIN computer program,21 where:

+

AUC =

AUMC =

C ATII2

(6) C2

± x22

(7)

The following noncompartmental pharmacokinetic parameters for unbound and total prednisolone were then obtained: CL = Intravenous dose/AUC MRT = AUMC/AUC VSS

CL x MRT

in which CL is clearance; MRT is mean residence time, and Vss is the steady-state volume of distribution. These parameters are apparent values because the effects of reversible metabolism of prednisolone

561

and prednisone are not accounted for. This also assumes rapid and complete hydrolysis of the sodium phosphate ester to prednisolone.22 For the cortisol, T lymphocyte, and histamine data, AUC(0-24) was calculated according to the trapezoidal rule. The binding of prednisolone to albumin and transcortin was described by the following equation23: DB

NTKTPTDF ± KTDF

NAKAPADF

(11)

1

in which DB and DF are bound and free drug, NT and NA are the number of binding sites on the transcortin (T) and albumin (A) molecules, KT and KA are the affinity constants, and PT and PA are the molar concentrations of the proteins in plasma. Three proteinbinding parameters (KT, NTKTPT, and NAKAPA) were

fitted with PCNONLIN. 21 The net pharmacodynamic effects of prednisolone on cortisol, T lymphocytes, and histamine were determined by calculating the ratio of cortisol, T lymphocyte, and histamine AUC(0-24) values after prednisolone administration (with and without ketoconazole) to the respective baseline AUC(0-24) values. This ratio was used as a measure of the net suppressive effects of prednisolone on these physiologic parameters. The direct suppressive effects of prednisolone on cortisol, T lymphocytes, and histamine were analyzed further by extensions of the pharmacodynamic models of Kong et al.19 The cortisol, helper T lymphocyte, and WBH data were fitted to the pharmacodynamic models with PCNONLIN.21 For the prednisolone phase a simultaneous fitting of the baseline and prednisolone phase was done. The data from the prednisolone plus ketoconazole phase was fitted simultaneously with the baseline phase. Goodness of fit of the data to the models was assessed by visual inspection of the fittings, as well as examination of the residual plots, correlation coefficients, and sums of squared deviations. The prednisolone pharmacokinetic parameters and the cortisol, T lymphocyte, and histamine AUC(0-24) ratios for two treatment phases were compared with the paired t test. One-way analysis of variance for repeated measures was used to compare the cortisol, T lymphocyte, and histamine AUC(0-24) values, as well as the 24-hour serum concentrations during the three phases. Tukey's multiple comparison test was used to determine which of the mean values was different. The different parameters derived from the pharmacodynamic and protein binding models were compared by either the paired t test or one-way analysis of variance for repeated measures where appropriate. The

CLAN

562

PHARMAC01, THER

Yamashita et al.

MAY 1991

1000.0

M

100.0

LLI CD

0 0

p-

10.0

cc

cc

a.

z

1.0

0.1 0

8

12

16

20

TIME (HOURS)

Fig. 2. Plasma concentrationtime profile of total (circles) and unbound (squares) prednisolone (PN) in a representative subject with (open symbols) and without (solid symbols) ketoconazole (K) treatment. Inset, Clearances of total prednisolone in individual subjects. P, prednisolone.

Kolmogorov-Smirnov test24 was used to assess deviations in the prednisolone binding curves when ketoconazole was added. Significance for all tests was considered to be at the p < 0.05 level.

RESULTS Pharmacokinetics. The plasma concentrationtime profile of prednisolone from a typical subject in the presence and absence of ketoconazole is depicted in Fig. 2. The treatment with ketoconazole did not appear to have an effect on the elimination of prednisolone, as evidenced by the disposition curves, which are nearly superimposable. The mean pharmacokinetic parameters for the six subjects are listed in Table I. The clearance of total prednisolone with and without ketoconazole (96 ± 11 versus 90 ± 11 ml/hr/kg; p > 0.05) remained unchanged. Although the absolute clearance decreased slightly in most subjects, the magnitude of these changes was clinically insignificant as demonstrated in the inset in Fig. 2. Furthermore, no significant differences in the Vss and MRT were observed. Although the change in elimination rate constant achieved statistical significance, the difference between 0.21 -± 0.02 and 0.22 ± 0.02 hr-1 is not meaningful. A small (5%) but consistent change resulted in a small difference in the SEM. Similar differences were found with

the pharmacokinetic parameters for unbound pred-

nisolone. The relationship between the total prednisolone concentrations and the fraction of prednisolone bound to plasma proteins is depicted in Fig. 3. Over the linear binding range, ketoconazole did not appear to alter the percent of prednisolone bound to plasma proteins (92.4% ± 2.7% versus 92.6% ± 1.7%). In addition there were no differences in the protein-binding parameters obtained from the regression analysis when ketoconazole was administered with prednisolone. However, when the Kolmogorov-Smirnov test was applied to the residuals from the least squares regression analysis, the binding curve during the ketoconazole phase was significantly different from the phase in which prednisolone was administered alone in four of six subjects. In three of these subjects the protein binding was slightly lower with ketoconazole. Cortisol dynamics. Under baseline conditions the plasma cortisol concentrations exhibited a normal circadian variation, with peaks in the morning (8 AM) and nadirs in the early evening (4 to 6 PM; Fig. 4). When prednisolone was administered with and without ketoconazole, the normal rhythm of cortisol was suppressed, as evidenced by the rapid biexponential decline in the cortisol concentrations followed by its prolonged suppression. After dissipation of pred-

VOLUME 49 NUMBER

Ketoconazole and prednisolone

1.2

563

-

0.8

0.4 PN PN + K

0.0 10

1000

100 PREDNISOLONE CONCENTRATION (NG/ML)

Fig. 3. Relationship between total prednisolone (PN) concentration and fraction of prednisolone bound to plasma proteins during prednisolone phase (open circles) and combined prednisoloneketoconazole (PN + K) phase (solid circles).

Table I. Pharmacokinetic parameters for prednisolone in the presence and absence of ketoconazole Prednisolone Total prednisolone CL (ml/hr/kg) (L/kg) Terminal slope (hr AUC (ng hr/ml) MRT (hr)

1)

Unbound prednisolone CL (ml/hr/kg) Vs, (L/kg) Terminal slope (hr- t) AUC (ng hr/m1) MRT (hr)

Prednisolone plus ketoconazole

96 ± 11 0.41 ± 0.02 0.22 ± 0.02 1727 ± 297 4.29 ± 0.43

90 ± 0.40 ± 0.21 ± 1850 ± 4.45 ±

p Value*

11

0.11

0.02 0.02

0.23

356

0.06

0.59

0.31

649 ± 115 1.65 ± 0.18

612 ± 163 1.67 ± 0.25

0.28 0.87

0.34 ± 0.06

0.32 ± 0.04 284 ± 80 2.80 ± 0.47

0.01 0.07 0.01

260 ± 62

2.60 ± 0.52

0.01

Data are mean values ± SD (n = 6). CL, Clearance; V,, steady-state volume of distribution; AUC, area under the prednisolone concentration-time curve from zero to infinity; MAT, mean residence time. *Paired t test.

nisolone, the cortisol values returned to baseline, with no difference in the 24-hour cortisol concentrations seen under baseline conditions compared with the days in which prednisolone was administered (Fig. 4). The AUC(0-24) for cortisol was decreased significantly after prednisolone compared with baseline con-

ditions (1523 ± 320 ng hr/ml), as seen in Table II. There was no difference in the AUC(0-24) between prednisolone (568 ± 127 ng hr/m1) and prednisolone plus ketoconazole (542 ± 133 ng hr/ml), suggesting that ketoconazole did not increase the cortisol suppression further by prednisolone. This is supported further by the AUC(0-24) ratios, which remained unchanged

(TIN PHARMAC01, THER

564 Yamashita et al.

MAY 1991

BASELINE

0

0 CC

0

1.11

0

00 0 tn 0

PN PN

+

K

100

TIME (HOURS)

Fig. 4. Plasma concentrations of cortisol in a representative subject during baseline conditions (triangles; top panel) and after prednisolone with (solid circles, solid lines) and without (open circles, broken line) ketoconazole treatment (bottom panel). Symbols show experimental data and lines represent the least-squares regression lines fitted by equations 1 and 4.

after the addition of ketoconazole (0.37 ± 0.05 versus 0.36 ± 0.05). Table II provides the pharmacodynamic parameters obtained by fitting the cortisol data to the dynamic model. There were no significant differences in any of the parameters obtained from modeling of the data with and without ketoconazole. This is also demonstrated in the similarity of the curve fittings of Fig. 4 for the two treatment phases. T cell dynamics. Data are presented for the helper T (CD44) lymphocyte data in Table III and Fig. 5. The data for one subject were excluded because of an inadequate number of T cell samples obtained. The T lymphocytes exhibited a circadian rhythm under baseline conditions, peaking around 4 AM with a nadir in

the morning (8 to 9 AM) (Fig. 5). With steroid administration there is a rapid monoexponential decline in T cell number followed by a prolonged redistribution of lymphocytes. Once prednisolone was eliminated, the lymphocytes returned to their normal baseline rhythm with no significant difference in the 24-hour T cell number between the three phases of study. (Fig. 5; Table III). Both the helper and suppressor T cells exhibited a similar pattern of decline after steroid administration, although the maximum suppression of helper T cells was greater than that of the suppressor T cells (72.4% versus 59.7%). This was also reflected in a decline in the helper/suppressor ratio at times of maximum T cell suppression. Because the helper T lymphocytes appeared to be more sensitive to steroid ad-

VOLUME 49 NUMBERS

Ketoconazole and prednisolone

565

Table II. Pharmacodynamic parameters of cortisol Parameter

Prednisolone

Baseline

Area analysis AUC (ng hr/ml) 24-hr cortisol concentration (ng/ml) AUC ratio

1523 136

± 320 ± 23

568 ± 127 112 ± 22 0.37 ± 0.05

Prednisolone plus ketoconazole 542 ± 133 112 ± 51 0.36 ± 0.05

p Value NS*i-

NSt NST

Model fitting

a (hr-')

11 ± 3.4 0.48 ± 0.12 9.02 ± 2.91

(hr-1) IC50 (ng/ml)

Rm (ng/ml) Rb (ng/ml) tz (hr)

59 ± 7

62 ± 10 43 ± 10 1.66 ± 0.71

± 11 1.92 ± 0.91 41

10 ± 0.50 ± 11.21 ± 62 ± 44 ± 1.57 ±

5.1

NSt

0.15 2.54

NST-

10

NSt NSt NSt

12

0.90

NST.

Data are mean values -± SD (n =- 6). AUC, Area under the cortisol concentration-time curve; a, 3, biphasic slopes; IC,o, 50% inhibitory concentration; Rm, mean cortisol concentration; Rb, concentration amplitude; tz, peak time of circadian function. *Tukey's test: II = III. tOne-way analysis of variance for repeated measures. tPaired t test.

Table III. Pharmacodynamic parameters for helper T lymphocytes Parameter

Baseline

Prednisolone

Area analysis AUC (cells hr/mm3) 24-hr T cell no. AUC ratio

23151 ± 9997 721 ± 242

16583 ± 4226 817 ± 286

0.76 ± 0.14

Prednisolone plus ketoconazole 17075 ± 3189 899 ± 192 0.81 ± 0.27

NS* NS*

0.30

NSt NSt

Model fitting k, IC50 (cells/mm3)

Rm (cells/mm3) Rb (cells/mm3) tz (hr)

870 ± 256 409 ± 282 20 ± 9

0.73 ± 109 ± 910 ± 440 ± 21

±

0.35 81

227 242 11

p Value

0.83 ± 78 ± 963 ± 319 ±

39 391 122

22 ± 10

NSt

NS* NS* NS*

Data are mean values ± SD (n = 5). AUC, Area under the T lymphocyte cell count-time curve; kt, helper T cell decline rate constant; IC50, 50% inhibitory concentration: Rm, mean cortisol concentration; Rb, concentration amplitude; tz, peak time of circadian function. *One-way analysis of variance for repeated measures. tPaired t test.

ministration, only the data for these cells were used in modeling. The helper T lymphocyte AUC(0-24) values were reduced after steroid administration, although this difference did not reach statistical significance (baseline, 23,151 ± 9997; prednisolone, 16,583 ± 4226; and prednisolone plus ketoconazole, 17,075 ± 3189 cells hrimm3). Ketoconazole did not appear to alter the helper T cell AUC(0-24) ratio (0.76 ± 0.14 versus 0.81 ± 0.27). The pharmacodynamic parameters obtained from modeling of the helper T lymphocyte data did not differ between treatments, as evidenced by insignificant changes in the rate constants of decline and the IC50

values. However, there were fewer data points and more variability in the T lymphocyte data than in the other measures. Blood basophils. The pharmacodynamic data for the basophils (WBH) are presented in Table IV and Fig. 6. One subject in the blood histamine analysis was excluded because of abnormally low values during all three phases of the study. Fig. 6 shows the baseline WBH pattern, which typically is flat with occasional variability. After administration of prednisolone, there is a decline in WBH that remains suppressed until elimination of prednisolone. Unlike cortisol, the decline in WBH was less dramatic and

GUN PHARMAC01, TETER

566

Yamashita et al.

MAY 1991

900

500

BASELINE C-)

IX

100

CO

Z

-J

900

L.L1

LU

_1 LLJ

500

100 0

20

10

TIME (HOURS) Fig. 5. Helper T lymphocyte profile in a representative subject during baseline conditions (triangles; top panel) and after prednisolone, with (solid circles, solid lines) and without (open circles, broken line) ketoconazole treatment (bottom panel). Symbols show experimental data and lines represent the least-squares regression lines fitted to the T cell model.

appeared incomplete in most subjects. This is evidenced further by the WBH AUC(0-24) values in Table IV that were reduced after prednisolone administration (baseline, 370 ± 123; prednisolone, 253 ± 82; and prednisolone plus ketoconazole, 275 ± 59 ng hr/ml), although only significantly between baseline and prednisolone treatments. The extent of suppression of WBH by prednisolone when ketoconazole was absent and present is similar, as demonstrated by the AUC(0-24) ratios (0.69 ± 0.10 versus 0.78 ± 0.22; p > 0.05). The pharmacodynamic parameters for WBH are also presented in Table IV and Fig. 6. Although the kr° appeared to decrease when ketoconazole was

added (4.22 ± 2.01 versus 2.97 ± 2.36), this did not reach statistical significance. The kh and the IC50 were similar between treatments. As a result of the incomplete suppression of WBH, the data may not have fitted the pharmacodynamic model as well, resulting in the large variability in these IC50 values.

DISCUSSION Pharmacokinetics. Steroid-specific drug interactions have been documented in the literature. Troleandomycin, a macrolide antibiotic that inhibits microsomal enzyme activity, reduces the metabolism of other corticosteroids such as methylprednisolone without altering the metabolism of prednisolone.8 Similarly, ci-

VOLUME 49 NUMBER 5

Ketoconazole and prednisolone

567

40

/

30

20

1

0

BASELINE 0

TIME

(HOURS)

Fig. 6. Whole blood histamine concentrations in a representative subject during baseline conditions (triangles; top panel) and after prednisolone, with (solid circles, solid lines) and without (open circles, broken line) ketoconazole treatment (bottom panel). Symbols show experimental data and lines represent the least-squares regression lines fitted by equation 6.

metidine, which inhibits the oxidative metabolism of many drugs through inhibition of the cytochrome P450 system, does not alter prednisolone clearance.25 There is evidence that ketoconazole also exhibits specificity with respect to inhibition of drug metabolism, as reflected in its ability to inhibit both aminopyrine and caffeine demethylation, but to varying degrees.26 In addition, the oxidative clearance of chlordiazepoxide is reduced significantly by ketoconazole, but minimal effects on theophylline and antipyrine metabolism are observed.27-29 On the basis of this information it is not surprising to see steroid-specific drug interactions between ketoconazole and different corticosteroids. We518 previ-

ously found that ketoconazole markedly inhibits the disposition of methylprednisolone. The interaction between ketoconazole and prednisolone has been less clear. Zurcher et al.6 reported that ketoconazole inhibited the clearance of prednisolone in 10 subjects, resulting in a 50% increase in prednisolone concentrations. Conversely, Ludwig et al.7 found no change in prednisolone clearance or cortisol suppression after the addition of ketoconazole. We presently find no difference in the clearance of prednisolone after the administration of ketoconazole. The reasons for these opposing results are unclear. Differences between the two studies included use of different assays and slightly different subject ages.3° More important per-

PLIARMA('01.1t I ER

568

Yamashita et al.

MAY 1991

Table IV. Pharmacodynamic parameters for blood basophils measured as whole blood histamine Parameter

Baseline

Prednisolone

Prednisolone plus ketoconazole

Area analysis AUC (ng hr/m1) 24-hr histamine concentration

370 ± 123 13.04 ± 2.5

253 ± 82 13.15 ± 3.7

275 ± 59 14.15 ± 2.3

p Value NS*1-

NSt

(ng/m1)

AUC ratio

0.69 ± 0.10

0.78 ± 0.22

NSf

0.26 ± 0.09 4.22 ± 2.01 75 ± 47

0.16 ± 0.12 2.97 ± 2.36 64 ± 49

NSf.

Model fitting kh

(hr- I)

kr° (ng/hr) IC50 (ng/ml)

NSf NS*

Data are mean values ± SD (a = 5). AUC, Area under the WBH concentration-time curve; lc,. first-order rate constant for migration of basophils from blood to the extravascular compartment; kr", zero-order rate of return of basophils from the extravascular compartment; IC50, 50% inhibitory concentration. *Tukey's test: I = Ill. i'One-way analysis of variance for repeated measures. *Paired t test.

haps, Zurcher et al. employed a dose of prednisolone (0.8 mg/kg) that was significantly higher than the present dose. However, drug interactions usually are more profound with a higher interactant:drug ratio. Pharmacodynamics. The effects of ketoconazole on the pharmacodynamics of prednisolone have not been well described. The AUC ratio has been used as a measure of the suppression of cortisol. The smaller the ratio, the greater the extent and duration of the suppression. When administered with methylprednisolone, ketoconazole appeared to extend the suppression of cortisol (AUC ratio, 0.45 ± 0.19) beyond that produced by methylprednisolone alone (AUC ratio, 0.39 ± 0.19; p < 0.01).18 However, this effect was not observed with prednisone (AUC ratio for prednisone alone, 0.45 ± 0.03; AUC ratio for prednisone and ketoconazole, 0.40 ± 0.1; p > 0.05) in the four subjects studied by Ludwig et al.' This study confirms such findings in that ketoconazole did not further suppress the concentrations of cortisol over and above that produced by prednisolone alone. Furthermore there did not appear to be any differences in the suppression of WBH and T cells when ketoconazole was added. The degree of suppression for cortisol (60%), however, was greater than that of WBH (26%) and the T lymphocytes (22%). In addition, there was greater variability in the histamine and T cell data compared with the cortisol data, which reduced the power to detect significant differences within this data set. The T lymphocyte (both helper and suppressor) response to steroid administration was similar in magnitude and time course to that reported previously by other investigators.31'32 In contrast, the incomplete

suppression of WBH observed in this study differed from the results of Kong et al.,19 who, after administration of 10, 20, and 40 mg doses of methylprednisolone, demonstrated complete suppression in all but the lowest dose. Other investigators, however, reported less than complete suppression of WBH after 35 mg doses of oral prednisone33 and 20 mg doses of intravenous methylprednisolone.34 This suggests that individual pharmacodynamic parameters representative of the immune system demonstrate different patterns of sensitivity to different doses of corticosteroids. In addition, comparing the cortisol IC50 values to those from Kong et al.,19 our prednisolone values (mean, 9.02 ng/ml) are more than twice those for methylprednisolone (mean, 3.61 ng/ml). These findings raise questions regarding the accepted 4:5 potency ratio for the relative actions of methylprednisolone/prednisolone. It is also notable that the IC50 values for cortisol suppression (9.02 ng/ml = 25 nmol/L) after prednisolone administration are in the same order of magnitude as the dissociation constant reported for prednisolone binding in rat liver (61 nmol/L) at 37° C.35 The pharmacodynamic parameters obtained from fitting the cortisol, T lymphocyte, and basophil data to the appropriate models did not appear to change with the addition of ketoconazole. This suggests that, at the dosage levels used, ketoconazole does not alter the pharmacodynamics of prednisolone or possess an independent effect on cortisol, T cell, or WBH suppression. The existence of steroid specificity in the inhibition of corticosteroid clearance by ketoconazole has importance in the selection of corticosteroids for clinical use, with particular relevance in the treatment of patients with leukemia, as well as other immunocom-

VOLUME 49 NUMBER 5

promised patients who are likely to be receiving both ketoconazole and corticosteroids. Avoidance of additional immunosuppression through the choice of corticosteroid may minimize complications of combination therapy with ketoconazole. This study has demonstrated that ketoconazole does not have an effect on the pharmacokinetics and pharmacodynamics of low single therapeutic doses of prednisolone. Hence, when combined therapy with ketoconazole and a corticosteroid is required, prednisolone may be preferable to methylprednisolone. The technical and computer assistance of Mr. Jeffrey Wald and Ms. Nancy Pyszczynski is greatly appreciated. Flow cytometric measurements were performed by Dr. Adrian Vladutiu, Department of Pathology/Clinical Laboratories, Buffalo General Hospital. Clinical assistance was provided by Dr. Rita Sloan.

References Gustayson LE, Benet LZ. Pharmacokinetics of natural and synthetic glucocorticoids. In: Anderson DC, Winter JSD, eds. Adrenal cortex. Cornwall: Butterworth, 1985:235-81. Boekenoogen SJ, Szefler SJ, Jusko WJ. Prednisolone disposition and protein binding in oral contraceptive users. J Clin Endocrinol Metab 1983;56:702-9. Brooks PM, Buchanan WW, Grove M, Downie WW. Effects of enzyme induction on metabolism of prednisolone. Ann Rheum Dis 1976;35:339-43. Wilkinson CF, Hetnarski K, Yellin TO. Imidazole derivatives: a new class of microsomal enzyme inhibitors. Biochem Pharmacol 1972;21:3187-92. Glynn AM, Slaughter RL, Brass C, D'Ambrosio R, Jusko WJ. Effects of ketoconazole on methylprednisolone pharmacokinetics and cortisol secretion. CLIN PHARMACOL THER 1986;39:654-9. archer RM, Frey BM, Frey FJ. Impact of ketoconazole on the metabolism of prednisolone. CLIN PHARMACOL TIIER 1989;45:366-72. Ludwig EA, Slaughter RL, Savliwala M, Brass C, Jusko WJ. Steroid-specific effects of ketoconazole on corticosteroid disposition: unaltered prednisolone elimination. Drug Intell Clin Pharm 1989;23:858-61. Szefler SJ, Ellis EF, Brenner M, et al. Steroid-specific and anticonvulsant interaction aspects of troleandomycin steroid therapy. J Allergy Clin Immunol 1982; 69:455-60. Pont A, Williams PL, Loose DS, et al. Ketoconazole blocks adrenal steroid synthesis. Ann Intern Med 1982;97:370-2. Bradbrook ID, Gillies HC, Morrison PJ, Robinson J, Rogers HJ, Spector RG. Effects of single and multiple doses of ketoconazole on adrenal function in normal subjects. Br J Clin Pharmacol 1985;20:163-5.

Ketoconazole and prednisolone

569

Loose DS, Kan PB, Hirst MA, Marcus RA, Feldman D. Ketoconazole blocks adrenal steroidogenesis by inhibiting cytochrome P-450-dependent enzymes. J Clin Invest 1983;71:1495-9. Couch RM, Muller J, Perry YS, Winter JSD. Kinetic analysis of inhibition of human adrenal steroidogenesis by ketoconazole. J Clin Endocrinol Metab 1987; 65:551-4. Senior DS, Shaw JTB. In vitro effects of fluconazole (UK-49,858) and ketoconazole on mouse lymphocyte proliferation and on Candida blastospore destruction by human polymorphonuclear leukocytes. Int J Immunopharmacol 1988;10:169-73. Odds FC, Webster CE. Effects of azole antifungals in vitro on host/parasite interactions relevant to Candida infections. J Antimicrob Chemother 1988;22:473-81. Loose DS, Stover P, Feldman D. Ketoconazole binds to glucocorticoid receptors and exhibits glucocorticoid antagonist activity in cultured cells. J Clin Invest 1983;72:404-8. Badcock NR, Bartholomeusz FD, Frewin DB, Sansom LN, Reid JG. The pharmacokinetics of ketoconazole after chronic administration in adults. Eur J Clin Pharmacol 1987;33:531-4. Portner M, Mollmann H, Rohdewald P. Glucocorticoid receptors in human synovial tissue and relative receptor affinities of glucocorticoid-21-esters. Pharm Res 1988; 5:623-7. Kandrotas RJ, Slaughter RL, Brass C, Jusko WJ. Ketoconazole effects on methylprednisolone disposition and their joint suppression of endogenous cortisol. CLIN PHARMACOL THER 1987;42:465-70. Kong AN, Ludwig EA, Slaughter RL, et al. Pharmacokinetics and pharmacodynamic modeling of direct suppression effects of methylprednisolone on serum cortisol and blood histamine in human subjects. CLIN PHARMACOL THER 1989;46:616-28. Rose JQ, Jusko WJ. Corticosteroid analysis in biological fluids by high-performance liquid chromatography. J Chromatogr 1979;162:273-80. Metzler CM, Weiner DL. PCNONLIN. Lexington, Kentucky: Statistical Consultants, Inc., 1986. Rose JQ, Yurchak AM, Jusko WJ. Dose-dependent pharmacokinetics of prednisone and prednisolone in man. J Pharrnacokinet Biopharrn 1981;9:389-417. Rocci ML, Johnson NF, Jusko WJ. Serum protein binding of prednisolone in four species. J Pharm Sci 1980;69:977-8. Hollander M, Wolfe DA. Nonparametric statistical methods. New York: John Wiley, 1973:219-24. Sirgo MA, Rocci ML, Ferguson RK, Eshelman FN, Vlasses PH. Effects of cimetidine or ranitidine on the conversion of prednisone to prednisolone. CLIN PHARMACOL THER 1985;37:534-8. Meredith CG, Maldonado AL, Speeg Ky. The effect of ketoconazole on hepatic oxidative drug metabolism in

CLIN PLIARMACOL TILER

570

Yamashita et al.

the rat in vivo and in vitro. Drug Metab Dispos 1985; 13:156-62. Brown MW, Maldonado AL, Meredith CG, Speeg Ky. Effect of ketoconazole on hepatic oxidative drug metabolism. CLIN PHARMACOL THER 1985;37:290-7. Blyden GT, Abernethy DR, Greenblatt DJ. Ketoconazole does not impair antipyrine clearance in humans. Int J Clin Pharmacol Ther Toxicol 1986;24:225-6. Daneshmend TK, Warnock DW, Ene MD, et al. Multiple-dose pharmacokinetics of ketoconazole and their effects on antipyrine kinetics in man. J Antimicrob Chemother 1983;12:185-8. Jusko WJ. Ketoconazole effects on corticosteroid disposition. CLIN PHARMACOL THER 1990;47:418-9. Ludwig EA, Slaughter RL, Gannon PM, DiMasi J, Middleton E, Jusko WJ. Effect of methylprednisolone on T-helper and T-suppressor cell populations [Abstract]. Pharmacotherapy 1988;8:138 .

MAY 1991

Slade JD, Hepburn B. Prednisone-induced alterations of circulating human lymphocyte subsets. J Lab Clin Med 1983;101:479-87. Saavedra-Delgado AM, Mathews KP, Pan PM, Kay DR, Muilenberg ML. Dose-response studies of the suppression of whole blood histamine and basophil counts by prednisone. J Allergy Clin Immunol 1980;66: 464-71. Reiss WG, Slaughter RL, Ludwig EA, Middleton E, Jusko WJ. Steroid dose sparing: pharmacodynamic responses to single versus divided doses of methylprednisolone in man. J Allergy Clin Immunol 1990;85: 1058-66. Boudinot FD, D'Ambrosio R, Jusko WJ. Receptormediated pharmacodynamics of prednisolone in the rat. J Pharmacokinet Biophann 1986;14:469-93.