Effects of Indomethacin on the Pharmacokinetics and Pharmacodynamics of Prednisolone in Rats

VARUN GARG AND WILLIAM J. JUSKO~ Received July 20, 1993, from the Depatfment of Pharmaceutics, School of Pharmacy, State University of New York at Buffalo, Buffalo, NY 14260. Accepted for publication December 27, 1993@.

Abstract 0 The effect of indomethacin on the disposition of prednisolone and the induction of tyrosine aminotransferase (TAT) was examined in male Sprague-Dawley rats. Rats were pretreated with either indomethacin (5 mg/kg, intraperitoneally) or phosphate buffered saline (control) twice dally for 6 days followed by a single dose of prednisolone. Blood samples were collected after prednisolone administration. In separate animals, hepatic TAT activity (pharmacologic effect) was measured 4 h after the prednisolone dose. In addition, the effect of indomethacin on the in vitro protein binding of prednisolone was examined in pooled rat and human plasma. The clearance andapparent volume of distribution of prednisolone in the control and indomethacin- treated animals were similar, averaging 4.71 versus 4.05 Llhlkg and 1.37 versus 1.33 L/kg, respectively. The elimination half-life was 0.48 h in both groups. Indomethacin also did not affect the protein binding of prednisolone in rat or human plasma. However, indomethacin pretreatment increased the hepatic TAT activity induced by pred- nisolone. These studies indicate that indomethacin may affect the pharmacological effects of prednisolone without influencing its pharmacokinetics.

Both glucocorticoids and nonsteroidal anti-inflammatory drugs (NSAIDS) are used to treat a wide variety of inflammatory conditions. However, there is little information on the potential interaction when these two classes of drugs are given together. Previous studies suggest that NSAIDS may act as steroid-sparing agentslq by being able to reduce the therapeutic doses of steroids in the treatment of rheumatic disorders. This steroid-sparing effect may be due to changes in either the pharmacokinetics or the pharmacodynamics of the steroid in the presence of the NSAID. For example, Vukoson et aI.* reported that in- domethacin treatment [5 mg/kg, intraperitoneally (ip), twice daily for 1, 2, or 3 days] of rats inhibited several microsomal enzymes and led to an increase in the corticosterone half-life. Further, these effects were directly related to the number of days of indomethacin treatment. Penning et aL5S6 found that indomethacin and many other NSAIDS were potent inhibitors of 3a-hydroxysteroid dehydrogenase in the rat liver cytosol. Because this enzyme is involved in the metabolic pathway of glucocorticoids, its inhibition by NSAIDS may cause the reduction in the metabolic clearance of concurrently adminis- tered corticosteroids. Rae et al.7 found that both indomethacin and naproxen increased free plasma prednisolone concentrations significantly in arthritis patients without affecting the total prednisolone concentrations. Our recent studies with a new NSAID, tenidap (which is also highly bound to plasma proteins and is structurally similar to indomethacin), however, did not reveal any significant changes in the pharmacokinetics or pharmacodynamics of prednisolone in humans.*

Feldmang reported that NSAIDs, including indomethacin, are capable of binding to glucocorticoid receptors in uitro. Thus, indomethacin may influence the receptor-mediated pharmaco- dynamic effect of prednisolone and other corticosteroids.

The purpose of the present study was to examine the effects of indomethacin on the pharmacokinetics and pharmacody-

*Abstract published in Advance ACS Abstracts, February 15,1994.

namics of prednisolone in rats. We studied the effect of indomethacin on the induction of the enzyme tyrosine ami- notransferase (TAT) as a pharmacodynamic index.lOJ1 We also investigated the effect of indomethacin on the binding of [3H]- dexamethasone to rat cytosolic receptors and protein binding of prednisolone in rats and in pooled human plasma.

Experimental Section Pharmacokinetic Study-Male Sprague-Dawleyrats weighing 0.27-

0.34 kg were used in the study. The rats were divided into two groups of six each and were kept in a 12-h light-dark cycle with free access to food and water. Each animal received either indomethacin (5 mg/kg, ip, Sigma, St. Louis, MO) or the vehicle [phosphate buffered saline (PBS), pH 7.41 for indomethacin (1 mL/kg, ip) every 12 h for 5 days (an extension of the Vukoson et aL4 regimen). On the fifth day, before the tenth dose, the rats were anesthetized with a mixture of ketamine and xylazine and the jugular vein was cannulated. On the sixth day, 30 min after the eleventh dose of indomethacin, each rat received prednisolone (10 mg/kg, as prednisolone sodium succinate, Sigma, St. Louis, MO) dissolved in normal saline intravenously (iv) through the jugular vein. Blood (0.3-0.6 mL) was collected at selected intervals up to 4 hand was replaced by an equal volume of blood from a donor rat. Plasma was obtained by centrifugation and kept frozen at -20 "C until analysis.

Pharmacodynamic Study-Two groups of 14 rats each were treated with indomethacin or its vehicle, as in the pharmacokinetic studies, except that no cannulation was performed. On the sixth day, 30 min after the last indomethacin (or PBS) dose, the rats were injected with prednisolone sodium succinate at 5 mg/kg (equivalent to prednisolone at 3.7 mg/kg) or saline. Thus, a total of four groups of seven animals each were used. Four hours after the prednisolone or saline treatment, the rats were sacrificed and their livers removed. The livers were immediately placed in ice-cold solution of 0.14 N KC1 and 1-g portions were sliced off to measure the TAT activity by the rate of change in absorbance.12 The TAT activity was normalized to hepatic protein content, which was determined by the method of Lowry et al.I3

The in vitro glucocorticoid activity of indomethacin was measured as its capacity to displace [3H]dexamethasone from its receptor binding. For this purpose, the method of Boudinot et al.I4 was followed and receptor binding assessed at 4 "C. Displacement curves were fitted with the Hill equation using PCNONLIN (Statistical Consultants Inc., Lexington, KY).

In Vitro Protein Binding Studies-These studies were carried out in both pooled human and rat plasma. In the case of human plasma, prednisolone (Sigma, St. Louis, MO) concentrations ranging from 5 to 4000nglmL were made up in triplicate for control and treatment groups. Indomethacin (5 pg/mL) was added to each sample in the treatment group. In the pooled rat plasma (obtained from Sprague-Dawley rats), the same range of prednisolone concentrations was used, but in- domethacin at 30 pg/mL was added to each sample in the treatment group. Protein binding determinations were made by ultrafiltration. Trace amounts of purified, 3H-labeled prednisolone (Amersham, Ar- lington Hts., IL) were added to each 500-pL sample. Then, 50 pL of the sample was counted in a liquid scintillation counter (model 1900CA, Packard, Downers Grove, IL) and 400 pL of the remaining sample was used for ultrafiltration at 37 "C with the Centrifree system (Amicon, Danvers, MA). In Vivo Protein Binding-Aliquots of plasma from each sample

were pooled and the final concentration was determined by HPLC (see below). Pooled untreated rat plasma was used to generate concentrations in the range 5-4000 ng/mL. In the case of indomethacin-treatedanimals, the dilutions were made from the plasma of rats that received

0 1994, American Chemical Society and American Pharmaceutical Association

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indomethacin but no prednisolone. Protein binding was determined by ultrafiltration as described above.

Analysis of Plasma-Plasma was analyzed for prednisolone, pred- nisone, and corticosterone by a previously published16 HPLC assay. Briefly, 400 ng of betamethasone (internal standard) was added to each sample and extraction was carried out with methylene chloride. After drying, the samples were reconstituted in the mobile phase and injected onto a silica column (Zorbax Sil, DuPont, Wilmington, DE). Detection was carried out by UV absorbance at 254 nm. This method is linear in the range 10-2000 ng (intra- and interday CV was <lo% for all compounds).

Pharmacokinetic Analysis-The pharmacokinetic parameters of total prednisolone were obtained by noncompartmental methods as well as a two-compartment model. The area under the curve (AUC) of concentration versus time extrapolated to infinity and the area under the moment curve (AUMC) were calculated by Lagrange polynomial interpolation.16 The apparent volume of distribution at steady-state (Vd,,) and systemic clearance (CL) of prednisolone were calculated with the following equations:

I . ,,ol

Time, min Flgure 1-Plasma concentration-time profile of prednisolone (O), pred- nisone (0), and corticosterone (A) in a representative animal (indomethacin treated) after iv administration of prednisolone (1 0 mg/kg).

Div C L = - A UC

Di;A UMC Vd,, =

( A UC)’

In eqs 1 and 2, Di, is the ivdose of prednisolone. The two-compartmental parameters [viz., the distributional clearance (CLd), the volumes of the central ( VC) and tissue ( VT) compartments, and the elimination half- life (tllz)] were obtained by nonlinear fitting of the data with PCNON- LIN. The protein binding of prednisolone in rat and human plasma was characterized by the following equation17:

(3)

In eq 3, DB and DF are molar concentrations of bound and free drug, NT andNAare thenumberofbindingsitesonthe transcortin (T) andalbumin (A) molecules, KT and K A are the affinity constants, and PT and PA are the molar concentrations of the proteins in the plasma. The binding parameters were obtained by fitting with PCNONLIN.

The unpaired t test was used to evaluate the differences in the pharmacokinetic parameters between the two groups. For the phar- macodynamicstudy, analysis of variance (ANOVA), followed by Tukey’s testla were used to compare the mean activity of TAT between the four groups. The sample sizes were chosen to provide 80% power to detect a 30% difference at a = 0.05 (two-tailed).

The plasma concentration-time profile of prednisolone, its metabolite prednisone, and corticosterone (an endogenous corticosteroid in rats) in a representative animal is shown in Figure 1. In general, plasma concentrations of prednisolone were several-fold higher than those of prednisone and corticosterone. Plasma corticosterone decreased after prednisolone adminis- tration similar to the suppression of cortisol in humans. In some animals, prednisone and corticosterone could be detected only in the initial phase. Hence, the disposition of these steroids could not be evaluated.

The mean plasma prednisolone concentrations in the control and indomethacin-treated animals are shown in Figure 2, and the pharmacokinetic parameters are listed in Table 1. One animal in the control group showed extremely high concentra- tions of prednisolone in the plasma and was excluded from data analysis. There was a slight increase in the AUC in the indomethacin-treated group, but the difference was not statis- tically significant. There were no significant differences in the

1 O . 2 L O I 120 180 240

0 60

Time, min Flgure 2-Plasma concentration-time profile of prednisolone in control (0) and indomethacin-treated (0) rats showing mean values and SDs.

Table 1-Pharmacoklnetic Parameters of Prednlsolone In Control or Indomethacln-Treated Rats’

~ ~~ ~~

Control Treated Parameter (n = 5) (n = 6)

~~

AUC, ng.h/mL 2216f459 _____ 2526 f 377 CL, L/h/kg 4.71 f 0.96 4.05 f 0.61 Ch, Lihikg 2.11 f 0.90 1.78 f 0.60 vc, L/kg 0.50 f 0.23 0.63 f 0.14 VT, Lfkg 0.65 f 0.04 0.63 f 0.16 v,, L/kg 1.37 f 0.21 1.33 f 0.12 f l l 2 r h 0.48 f 0.12 0.48 f 0.15

a Results are expressed as mean f SD.

values of the other pharmacokinetic parameters between the two groups. The pharmacokinetic parameters obtained were similar to those reported in previous s t u d i e ~ ~ ~ ~ ~ with Wistar rats.

The effects of indomethacin on the in vivo and in vitro protein binding of prednisolone in the rat plasma are shown in Figures 3 and 4. The results in pooled human plasma are shown in Figure 5. It is apparent that the binding profiles of prednisolone in rat and human plasma are quite different. However, as evident from these figures and from the fitted parameter values in Table 2; indomethacin did not cause any significant alteration in prednisolone binding either in rat or human plasma.

The effect of indomethacin on the hepatic TAT activity in prednisolone-treated and untreated rats, 4 h after the pred- nisolone or saline dose, is shown in Figure 6. Based on previous work in our lab,lo TAT activity was expected to reach a maximum at 4 h after the prednisolone dose. In the absence of prednisolone

148 / Journal of Pharmaceutical Sciences Vol. 83, No. 5, May 1994

Table 2-1 n Vltro Proteln Blndlng Parameters of Prednlsolone In Pooled Human and Rat Plasman a e

Fp g e .I

3 2 a U

8 $ a

Prednisolone Concentration, ng/ml Figure 3-In vivo plasma protein binding of prednisolone in control (0) and indomethacln-treated (e) rats.

'' r a B

C .I

U P)

e a U

8 z &

4

I 1

0 1030 zm 3030 4aX)

Prednisolone Concentration, ng/ml Figure 4-In vitro plasma protein binding of prednisolone in control (0) and indomethacin-treated (e) pooled rat plasma (mean and SD, n = 3 determinations).

a E

ep 8 .f 3 e &

0 1000 2000 3000 4wo

Prednisolone Concentration, ng/ml Figure 5-In vltro plasma protein binding of prednisolone in control (0) and indomethacin-treated (e) pooled human plasma (mean and SD, n = 3 determinations).

administration, TAT activity (measured as the rate of change in absorbance at 331 nm per mg of liver protein, Abs/min/mg) was not changed significantly by indomethacin (no drug: 0.027 f 0.007, only indomethacin: 0.032 f 0.01 Abs/min/mg). The low prednisolone dose used in this experiment (3.7 mg/kg) was sufficient to raise TAT activity significantly to 0.066 f 0.01 Abs/ min/mg. In rats that received indomethacin followed by prednisolone, TAT activity was further increased to 0.102 f 0.04 Abs/min/mg. Thus, indomethacin increased the predniso- lone - induced hepatic activity of TAT but not the baseline activity. Indomethacin also did not displace [3H] dexamethasone from glucocorticoid receptor sites.

Parameter Control (n = 3) Treated (n = 3) Human Plasma

N r S ( X 10' M) 0.52 f 0.07 0.43 f 0.06 KT (X lo-' M-') 8.53 f 1.60 10.04 f 1.90 NAKAPA 0.86 f 0.02 0.91 f 0.02

Rat Plasma Nrs (X to6 M) 3.84 f 2.3 2.58 f 1.2 Kr (X lo-' M-') 0.46 f 0.23 0.66 f 0.30 NAKAPA 0.40 f 0.36 0.42 f 0.14

* Results are expressed as mean f SD.

Untreated

1M

PSLN

IM + PSLN C

0 0.1 0.2

TAT Activity, Absorbancelmidmg

Figure 6-Activity of TAT in untreated, indomethacin (1M)-treated, prednisolone (PSLNhtreated, or indomethacin and prednlsolone (IM + PSLNhtreated rats. Groups marked with different letters are significantly different (p < 0.05).

Discussion

The results of this study indicate that indomethacin does not change the in vivo pharmacokinetics of prednisolone in rats. This is in contrast to the results obtained by Vukoson et a1.4 who found that the half-life of corticosterone increased after in- domethacin treatment. These authors also found that in- domethacin treatment of rats led to the inhibition of several microsomal enzymes that may be responsible for corticosteroid metabolism. Differences in the metabolic pathways and rate- limiting steps between the two corticosteroids may be a possible reason for this discrepancy.

In a previous study in arthritis patients, Rae et al.7 found that following indomethacin or naproxen pretreatment, the plasma concentrations of free prednisolone increased whereas the total prednisolone concentrations remained unchanged. These au- thors concluded that indomethacin and naproxen displace prednisolone from its binding proteins in the plasma. However, because prednisolone is a low-extraction drug in humans, protein binding displacement is unlikely to cause any major change in its free concentration in the plasma. Our study shows that indomethacin does not alter the plasma protein binding of prednisolone either in rats or in humans. Similarly, tenidap, a new nonsteroidal drug with high protein binding, also did not change the protein binding of prednisolone in humans.8 The larger free prednisolone concentration found after indomethacin or naproxen administration could be due to a decrease in the intrinsic clearance of prednisolone. Other factors in the human studies could be an increase in bioavailability due to improved absorption, inter- and intraindividual differences in prednisolone disposition, or decreased renal function resulting from chronic dosage of NSAIDS. Also, Rae et al.7 examined plasma concen- trations only at 1,2,4, and 6 h after dosing and did not examine the complete disposition profile of prednisolone. The RIA used in their study may not have been specific enough.

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The increase in the prednisolone - induced hepatic TAT activity by indomethacin pretreatment indicates that indometha- cin may have a synergistic effect on the receptor-mediated pharmacologic effects of corticosteroids. However, because there was no increase in TAT activity in the prednisolone untreated rats, indomethacin is unlikely to have any significant intrinsic glucocorticoid activity as proposed by Feldmamg This is also supported by the results of our in oitro receptor binding studies. The mechanism by which indomethacin could influence the pharmacodynamics of prednisolone is not clear. It is possible that indomethacin, and possibly other NSAIDS, cause an upregulation of the glucorticoid receptors. It remains to be seen if indomethacin changes other pharmacologic effects of pred- nisolone and whether such changes are caused by single doses of indomethacin.

In conclusion, our studies indicate that indomethacin may have a synergistic effect on the receptor-mediated effects of prednisolone without influencing its pharmacokinetics. Further studies in arthritis patients or animal models of inflammation are needed to clarify the mechanisms of the steroid-sparing effect of NSAIDS.

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Acknowledgments

This work was supported in part by Grant No. GM 24211 from the National Institutes of Health. We thank Ms. Yuen Yi Hon for providing technical assistance in the TAT studies.

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