Influence of hydrocortisone on the analgesic effect, toxicity and metabolism of aspirin in mice

Gen. Pharmac. Vol. 28, No. 1, pp. 123-128, 1997 Copyright © 1997 Elsevier Science Inc. Printed in the USA.

ISSN 0306-3623/97 $17.00 + .00 PII S0306-3623(96)00080-8 All rights reserved ELSEVIER

Influence of Hydrocortisone on the Analgesic Effect, Toxicity and Metabolism of Aspirin in Mice L. Tantcheva*, T. Stoytchev and D. Rangelova INSTITUTE OF PHYSIOLOGY, BULGARIAN ACADEMYOF SCIENCES, ACAD. G. BONCHEV STR.,

BL. 23, SOFIA 1113, BULGARIA[FAx: + 359 2 719 109; E-MAIL:[email protected]] ABSTRACT. 1. Hydrocortisone (HC; 80 mg/kg body weight, intraperitoneaUy for 4 days), both alone and in combination with acetylsalicylic acid (ASA; 160 mg/kg body weight, orally, for 4 days), decreased ASA general and specific toxicity via metabolic modulation of drug-metabolizing enzyme systems (intestinal ASA-esterase and hepatic UDP-glucuronyltransferase) and did not change the ASA analgesic effect. 2. ASA alone, given 4 for days, did not change the specific A S A toxicity, but increased its general acute toxicity, which was probably due to alterations in some intestinal and hepatic metabolizing enzyme systems. Copyright © 1997 Elsevier Science Inc. GEN PHARMAC28;1:123-128, 1997. KEY WORDS. Hydrocortisone, aspirin, toxicity, drug metabolism (drug interaction) INTRODUCTION

Influence on analgesic effect of A S A

Glucocorticoids and acetylsalicylic acid (aspirin, A S A ) are among the drugs most widely used in therapy. The ability of glucocorticoids and aspirin to modulate drug toxicity and effects by various pharmacokinetic and pharmacodynamic mechanisms is well known (Castro et al., 1970; Hamrick et al., 1973; Tredger et al., 1973; Ghazal et al., 1975; Hansten, 1975; Kourounakis et al., 1976; Kourounakis and Selye, 1976; Paik et al., 1977; Tantcheva and Stoytchev, 1980; Greim, 1981; Schuetz et al., 1984; Waxman, 1988; Devaux et al., 1992). There are clinical and experimental data supporting both favorable and unfavorable effects of the combined therapy with glucocorticoids and aspirin, but the mechanisms of the drug interactions are not understood (Elliot 1962; Klinenberg and Miller, 1965; Edelman, 1986; Jefferson, 1986; Bauer et al., 1987; Lazor et al., 1987). In previous experiments, some changes in the analgesic effect and toxicity of aspirin as well as some changes in the hepatic and extrahepatic drug-metabolizing enzyme activity were established after combined administration of dexamethasone (DEX) and aspirin in mice and rats (data not shown). The aim of the present work was to study the influence of another glucocorticoid, hydrocortisone, on the analgesic effect and toxicity of ASA, and also the activity of some hepatic and intestinal drugmetabolizing enzyme systems and lipid peroxidation in mice.

For estimation of the analgesic effect of the drugs and of their combination, we used the hot-plate test. Although this test is more specific for narcotic analgesics, it allows for measuring the duration of the analgesic effect after drug administration using the same groups as autocontrols. The animals were placed on a hot surface (39°C) and their sensitivity to thermal irritation (the beginning of jumping or licking of the forepaws measured in seconds) was evaluated in all groups. The effect was examined up to minute 150 after single drug treatment and at hours 24, 25, 26 and 27 after the last dose of the 4-day treatment. A N O V A was used for statistical evaluation of differences between groups.

MATERIALS A N D METHODS

A n i m a l s and drug t r e a t m e n t Male albino mice (18-20 g) were divided into four groups (10 animals per group) as follows: group 1--hydrocortisone (HC; hydrocortisone hemisuccinate; Sopolcort H Polfa; 80 mg/kg intraperitoneally lIP]); group 2--aspirin (ASA; acetylsalicylic acid; Acetysal-Pharmachim, 160 mg/kg, orally); group 3 - - H C plus A S A at the abovementioned doses; and group 4---controls. The compounds were applied at single and repeated doses for 4 days. The ASA solution was prepared in distilled water with some drops of Tween-80 and was introduced through a probe.

Effect on acute A S A toxicity after 4.day t r e a t m e n t At hour 24 after the last drug administration, the animals, deprived of food for at least 4 h, received a toxic dose of 2000 mg/kg A S A orally (Goldenthal 1971). The mortality rate of each group was estimated up to day 14. Before acute toxicity testing, the mice were weighed and growth was calculated as the difference between their final and initial weights.

Changes in specific A S A toxicity Some specific toxic effects of A S A were examined at hour 24 after the 4-day treatment, namely: I. Ulcerogenic effect on gastric mucosa. The stomachs were removed, washed with an isotonic solution of NaCI, and the mucosa was carefully examined for ulceration. 2. Changes in bleeding time (in seconds) were estimated through a standard wound on mouse tail. 3. Changes in alkaline serum content. 4. Blood serum was prepared through centrifugation. Alkaline content was determined by the titrimetric method of Nevodov (1984).

Hepatic monooxygenase activity *To whom correspondence should be addressed. Received 27 July 1995; revised 8 January 1996.

At hour 24 after 4-day treatment the animals were killed by decapitation. The livers were perfused with ice-cold 1.15% KC1 and ho-

124

L. Tantcheva et al. 20

8 7

6

0

l

0

0

10

30

60

90

120

150 min

FIGURE 1. Analgesic effect of ASA, H e and A S A + H C after single treatment. (-V-) ASA; (@-) HC; (-D) A S A + H C . The values at 0 min (i.e., before drug treatment) represent the autocontrol for each group.

mogenized. The 10,000g liver supernatant was used for determination of the following parameters: 1. N-demethylase activity with ethylmorphine, amidopyrine and benzphetamine as substrates (EMND, APND and BPND), estimating the HCHO formed (Nash, 1953). 2. Aniline hydroxylase activity (Mazel, 1971). 3. Cytochrome P-450 (cyt P-450) content (Omura and Sato, 1964). 4. Intestinal esterase activity with acetylsalicylic acid as substrate determined in 10% intestinal homogenate (Inowe, 1979). 5. Hepatic esterase activity in 10,000g supernatant (Aldridge, 1953) using diethyl-p-nitrophenyl-phosphate as substrate. 6. UDP-glucuronyltransferase (UDP-GTF) activity (Frei, 1970) with p-nitrophenol as substrate. Lipid peroxidation was determined by the TBA test at 0 min (Buege and Aust, 1978) and expressed in nanomoles of TBA per milligram of protein per minute. Enzyme activity was expressed as the product formed per milligram of protein per minute (in nanomoles) and cyt P-450 content was expressed in nanomoles per milligram of protein. The protein content was determined by the Biuret method. Student's t-test was used for statistical evaluation of differences in the groups. RESULTS An analgesic effect of HC, significant at 10 and 30 min after single administration was found with the hot-plate test (Fig. 1). The combination of HC and A S A exerted a significant analgesic effect at 30 min. ASA alone showed a significant analgesic effect at 60 min. The A S A and the A S A + HC groups tended to increase their sensitivity to thermal irritation between 90 and 150 min. The analgesic effect of HC was more pronounced than the effect of A S A alone or in combination with HC, but some toxic effects of HC were also observed. At the 24, 25, 26 and 27 h after 4-day treatment the analgesic effect of HC and ASA and of their combination was lacking (Fig. 2). After 4-day treatment, acute A S A toxicity (i.e., the mortality) in all groups increased by day 7 after a toxic dose of ASA (2000 mg/

'

24th

25th

26th

27th hour

FIGURE 2. Analgesic effect of ASA, HC and A S A + H C after 4 days of treatment. (C)-) Controls; (-V-) ASA; ( - 0 ) HC; ( l - ) ASA+HC.

kg) compared with controls (Fig. 3). The toxicity levels in the HC (35%) and the H C + A S A (33%) groups were lower compared with the toxicity in the ASA group (83%); that is, HC decreased the general ASA toxicity when applied alone or in combination with ASA. A decreased body growth was established in the ASA group and, to a lesser extent, in the HC and H C + A S A groups (Table 1). HC tended to diminish the effect of ASA on body growth when administered in combination with ASA (Table 1). The ability of the drugs (administered alone or in combination for 4 days) to provoke gastric irritation or ulcer, to prolong bleeding time and to change alkaline content in blood is shown in Table 1. In the present experiments, however, we failed to find distinct changes in the gastric mucosal epithelium after HC and ASA. Combined administration of HC and ASA did not increase the ulcerogenic effect. The alkaline content was not changed in any treated group as compared with controls (Table 1). Bleeding time was prolonged by ASA when administered alone. Some changes in the activity of hepaic and intestinal drug-metabolizing enzymes were established. The activity levels of the intestinal and liver esterases were significantly increased by ASA (Table 2), whereas HC had no effect on the intestinal esterase activity but de-

100 90 80 70

'~

J

60

I

50' 40

30 201 10

" 7th

I

(

14th

day FIGURE 3. Changes in ASA toxicity after 4 days of treatment with ASA, HC and A S A + H C . (CY-) Controls; ( - ~ ) ASA; (O-) HC; ( I - ) A S A + H C . *p < 0.05.

Hydrocortisone-ASA Interaction

125

TABLE 1. Body growth (BG, g), bleeding time (BT, s), gastric ulcers (GU) and alkaline content (AC, mg%) after 4 days of drug treatment with ASA, HC and the combination of ASA and HC Parameters BG BT GU AC

Controls

ASA

He

ASA + H e

2.83 + 0.34 30.8 -+ 4.3 0/6 272 +_ 19.6

1.48 - 0.47* 63.3 -+ 5.68* 1/6 260 -+ 24.8

1.33 -+ 0.46* 21.9 -+ 2.1 * 0/6 280 -+ 25.3

1.85 _+ 0.37 32.1 - 8.5 1/6 285 -+ 24.4

• p < 0.05 compared to controls.

creased the liver esterase activity. The combination of HC and ASA abolished the effect of ASA on both esterases. ASA significantly increased aniline hydroxylase activity and cyt P-450 content. HC and the combination of HC plus ASA increased aniline hydroxylase activity to a lesser extent than ASA when administered alone. HC did not affect the cyt P-450 content. The hepatic N-demethylases, APND, EMND and BPND, were not changed and only BPND was increased by the combination of HC plus ASA (Table 3). UDP-glucuronyltransferase activity was significantly increased by H C + A S A and was not altered by ASA or HC (Table 3). Lipid peroxidation was significantly decreased at hour 24 after treatment in the HC group and the H C + A S A group but was not changed in the ASA group (Table 3). DISCUSSION The analgesic effect of HC after single administration resembled that of dexamethasone (DEX) (data not shown). The analgesic effect of glucocorticoids (HC and DEX) when administered alone or in combination with A S A could be attributed to their action on prostaglandin synthesis and A S A ' s relationship with the mechanisms of pain response (Gibson and Skett, 1986; McCormack, 1994). Corticosteroid injections were used for chronic pain in patients (Deyo, 1992; Carette et al., 1992). Twenty-four hours after 4-day treatment the analgesic effect of HC and ASA and of their combination disappeared. Probably, the period of 24 h after multiple administration is sufficient for elimination of the drugs and of their effects. There are data in the literature about some toxic and unfavorable effects of prednisolone, triamcinolone and methylprednisolone when administered in combination with ASA, but the mechanisms of interactions between these two groups of drugs are not clear (Vrhovac and Simic, 1978; Edelman, 1986; Bauer et al., 1987; Lazor et al., 1987). The present results, showing that HC decreased ASA toxicity in mice, are at odds with the above-mentioned data. After a toxic oral dose of 2000 mg/kg of ASA no lethal outcome in the control group or in the HC group was recorded by day 7, but on day 14 the mortality rates in the two groups reached 15% and 33%, respectively. The mortality in the A S A group was 50% on day 7 and 83% on day 14, whereas in the group treated with the combination of HC and ASA, it was 20% on day 7 and 53% on day 14. The toxicity of the combination HC plus A S A was lower as compared to

the toxicity of A S A . T h e mortality rate decreased from 50% in the ASA group to 20% in the H C + A S A group on day 7 and from 83% to 53%, respectively, on day 14; that is, after 4-day treatment, HC decreased the general toxicity of ASA when applied alone or in the combination with ASA. Body growth was significantly decreased in ASA-treated mice as well as in ASA-treated rats (data not shown). HC tended to diminish the effect of ASA on body growth when administered in combination with ASA. The decreased body weight in mice and rats could be related to inadequate feeding of animals after oral daily drug treatment with ASA, or might be considered a result of the catabolic effect of HC and HC+ASA. A specific effect of ASA on body growth should not be excluded. ASA increased bleeding time, a finding supported by the literature (Williams et al., 1993; Brune, 1994; Dabaghi et al., 1994), but HC did not change it. The combination of HC and ASA abolished the effect of ASA. The ability of ASA and the glucocorticoids to produce gastric ulcer is well known (Cole et al., 1992; Talley, 1992; Levine, 1994; Torrado et al., 1995; Weil et al., 1995). Distinct changes in the gastric mucosal epithelium after HC or ASA were not found, probably because of adaptation to the 4-day drug administration. Similar are the data of Wallace et al. (1995) and Konturec et al. (1994), demonstrating that the rat stomach adapts to multiple oral administration of aspirin. In our experiments, combined administration of the two substances did not increase the ulcerogenic effect. The lack of changes in the serum alkaline content in the ASAtreated mice and in the HC+ASA-treated mice suggests the presence of regulatory mechanisms of the blood system, compensating for the formation of acid products after 4-day repeated drug treatment. According to Vrhovac and Simic (1978) corticosteroids increase A S k concentrations in the tissues but they also increase ASA elimination. In summary, HC administered together with ASA decreased mortality rate, tended to decrease the effect of ASA on body growth, normalized the ASA-prolonged bleeding time and did not change the ulcerogenic effect of ASA and the alkaline content of blood serum. These findings speak for a decreased general and specific toxicity of ASA by HC. Park et al. (1994) established a prevention ofsa-

TABLE 2. Intestinal esterase activity (IEA) and liver carboxylesterase activity (LCEA) after 4 days of treatment with ASA, HC and the combination of ASA and HC Esterases lEA LCEA

Controls

ASA

HC

ASA + HC

0.102 _ 0.007 0.905 - 0.085

0.160 - 0.023* 1.658 _+ 0.264*

0.093 -+ 0.001 0.662 +_ 0.077*

0.080 ± 0.015 1.174 -+ 0.063*

* p < 0.05 compared to controls.

126

L. Tantcheva et al. TABLE 3. Monooxygenase activity and UDP-glucuronyltransferase activity in mouse liver 10,000g supernatant, after 4 days of treatment with ASA, HC and the combination of ASA and HC Parameters APND EMND BPND AH Cyt P-450 UDP

Controls 1.932 2.037 1.047 0.154 0.043 0.840

+- 0.567 -+ 0.427 -+ 0.075 -+ 0.018 -+ 0.005 +- 0.064

ASA 1.697 1.827 1.410 0.332 0.080 0.901

HC

-+ 0.105 + 0.381 -+ 0.222 -+ 0.059* -+ 0.011" -+ 0.06

2.254 1.854 1.161 0.238 0.041 0.937

-+ 0.168 -+ 0.072 + 0.132 -+ 0.011" -+ 0.006 -+ 0.08

Combination 2.503 1.860 1.392 0.282 0.050 1.099

-+ 0.309 -+ 0.078 -+ 0.130" -+ 0.088* -+ 0.009 -+ 0.058*

* p < 0.05 compared to controls.

lycilate ototoxicity by glucocorticoid treatment. Surprising enough was our finding that the combination of HC and ASA normalized body growth. We suggest that some changes in the drug-metabolizing enzymes involved in ASA metabolism could explain the observed pharmacologic and toxic effects. ASA, as an ester compound, is rapidly transformed into salycilic acid and the rate of this reaction correlates with the hydrolytic activity of the enzymes in blood, gastrointestinal tract, liver and other tissues (Martin, 1971; La Du and Snady, 1971; Dean et al., 1989; Brune, 1994). In the present experiments, the activity of the intestinal and liver esterases was significantly increased after 4-day treatment with ASA, suggesting an accelerated hydrolyzation of ASA to salicylic acid. The more rapid biotransformation of ASA into salycilic acid in the ASA-treated mice could explain the higher general toxicity in this group. Accumulation of ASA (salicylic acid, respectively) after 4-day administration could further increase the ASA toxicity in this group. H e alone did not change intestinal esterase activity, but decreased liver esterase activity. The combination of HC and A S A abolished the effect of ASA on both esterases; that is, HC prevents the effects of ASA on the esterase activity. This could explain the lower ASA toxicity in the group treated with ASA plus HC. Klinenberg and Miller (1965) have shown decreased free salicylic acid concentrations following HC administration to patients. Most clinical and experimental data suggested decreased levels of free salicylic acid when ASA was applied together with glucocorticosteroids (DEX, prednisolone, triamcinolone, hydrocortisone, methylprednisolone) (Eliot, 1962; Muirden and Barraclough, 1976; Graham et al., 1977; Edelman, 1986; Bauer et al., 1987; Koren, 1987; Lazor et al., 1987). The mechanism underlying the effect of corticosteroids on salicylic acid pharmacokinetics has not yet been fully understood. Some investigators have considered the possibility that glucocorticolds increase renal salicylic acid clearance, thus decreasing salicylic acid levels (Klinenberg and Miller, 1965; Webel et al., 1972; Graham et al., 1977; Lazor et al., 1987, Muirden et al., 1976). Others have suggested a modulating effect of glucocorticoids and other steroids on salicylic metabolism (Kourounakis et al., 1976; Kourounakis and Selye, 1976; Gambertoglio et al., 1980; Gupta, 1982; Tucker, 1992; Kraml et al., 1995; Dean et al., 1989). The present experiments have provided some support to the second suggestion without excluding the first possibility. We obtained data to show that HC administered together with ASA decreased ASAenhanced esterase activity, thus decreasing the metabolism of ASA to salicylic acid. After its biotransformation to salicylic acid by esterases ASA is eliminated like a conjugated product with either the amino acids glycine or glucuronic acid (Scott et al., 1968; Bowman and Rand,

1980; Parke, 1968; Brune, 1994). At low doses ofASA, glycine conjugation is the main metabolic pathway, but on increasing ASA dose or multiple administration, as in our experiments, ASA conjugation switches to glucuronide formation because of saturation of glycine conjugation (Gibson and Skett, 1986). After the highest doses of ASA, the glucuronidation system also becomes saturated and salicylic acid becomes a major excretory product. This probably occurred in our experiments with the toxic dose of ASA. The activity of UDP-glucuronyltransferase was increased significantly by the combination of HC plus ASA, but was not altered by ASA or HC. The increased UDP-glucuronyltransferase activity could be the reason for the more rapid elimination of ASA and for the decreased ASA toxicity. Thus two metabolic mechanisms (i.e., the decreased hydrolysis of ASA to salicylic acid and the increased conjugation of salicylic acid by UDP-glucuronyltransferase) could underlie the decreased salicylic acid levels in blood and the decreased ASA toxicity caused by HC. A small quantity of hydroxyl metabolites and unchanged ASA has also been shown to be excreted (Martin, 1971; Gibson and Skett, 1986). Our results showed some changes in monooxygenase enzyme activity in mouse liver after 4-day treatment. ASA significantly increased aniline hydrozylase activity and cyt P-450 content. ASA's ability to alter drug effects and toxicity by different mechanisms is well known (Hansten, 1975; Vrhovac and Simic, 1978; Bowman and Rand, 1980; Scheler, 1980; Jefferson, 1986; Mansuy, 1987), about which the present data provided additional information. A stimulated formation of salicylic oxidative derivatives as result of the inducing effect of ASA should not be excluded. However, the relationship between increased ASA toxicity and the changes in its metabolic profile is a matter of speculation. The changes in monooxygenase enzyme activity produced by ASA alone (i.e., the increased aniline hydroxylase activity and cyt P-450 content) in mice pose the question of the compatibility of ASA in combination with other analgesic drugs, which can serve as substrates of hepatic monooxygenases (e.g., amidopyrine, acetaminophen, etc.) in man. There are some data about the inducing and autoinducing effects of some analgesics-antipyretics (pyrasolone derivatives) (Bien et al., 1985) on drug-metabolizing enzyme activity in rat liver, but we failed to find any information about the enzyme-inducing action of ASA. HC and the combination of HC plus ASA increased aniline hydroxylase activity but did not change cyt P-450 content. Benzphetamine N-demethylase activity was significantly increased by the combination of HC plus ASA. In the literature glucocorticoid's ability to alter drug metabolism and toxicity via induced synthesis of specific cyt P-450 isozyme is well known (Tantcheva and Stoytchev, 1980; Wood et al., 1983; Wrighton et al., 1985; Park et al., 1986; Devaux et al., 1992; Jonsson et al., 1995). The ability of glucocorti-

Hydrocortisone-ASA Interaction coids to change the reactivity of the organism to other drugs, so as to modulate drug effects, is also clearly documented (Ghazal et al., 1975; Kourounakis and Selye, 1976; Paik et al., 1977; Maines et al., 1995). The variety of catatoxic, syntoxic and permissive mechanisms emphasize the effects of glucocorticoids on drug toxicity and drug metabolism. Our data demonstrate only one aspect of these possibilities. In the present experiments, H C diminished A S A toxicity-probably by modulation of A S A m e t a b o l i s m ~ e c r e a s i n g liver esterase activity. According to Lazor et al. (1987), short-term administration of glucocorticoids does not influence salicylic metabolism in patients. Our experiments with H C on mice (4 days, 80 mg/ kg body weight, IP) suggested a catatoxic effect of this glucocorticold, probably via modulation of drug-metabolizing enzyme activity or some other independent mechanism (synthoxic or permissive) on A S A toxicity. It is well known that lipid peroxidation processes are involved in the metabolism of arachidonic acid to prostaglandins. The normal values of lipid peroxidation at 24 h after A S A treatment, confirmed by other investigators (Kirkova et al., 1995), could explain at least partly the lack of analgesic effect of A S A tn this case. Why, however, the decreased lipid peroxidation was accompanied by an increased sensitivity to hot-plate irritation in the groups treated with H C and H C + A S A is not yet clear. CONCLUSIONS We suggest that the increased general toxicity of A S A after 4-day A S A treatment of mice is not connected with changes in the specific toxic effects of the drug (acidosis and ulcerogenic effect) but rather with metabolic modulation of intestinal and hepatic drugmetabolizing enzyme activities. H C in combination with A S A could decrease the general and specific toxcity of A S A in mice via pharmacologic and metabolic modulation, which is due to the H C glucocorticoid activity and to its enzyme-modulating effects. SUMMARY 1. Hydrocortisone (HC) administered in mice at a single dose of 80 mg/kg body weight, intraperitoneally, or in combination with acetylsalicylic acid ( A S A 160 mg/kg body weight, orally) did not increase the A S A analgesic effect upon hot-plate test. Twentyfour hours after 4-day treatment with the same doses the analgesic effect of HC, A S A and their combination disappeared. 2. The general toxicity of A S A increased and the bleeding time was prolonged after 4-day A S A treatment. 3. H C (80 mg/kg body weight, intraperitoneally, 4 days) in combination with A S A decreased the general toxicity of A S A and normalized the bleeding time. 4. Two metabolic mechanisms, decreased hydrolysis of A S A to salicylic acid (decreased intestinal A S A esterase activity) and increased glucuronidation (increased UDP-glucuronyltransferase activity), could possibly play a role in the decreased A S A toxicity, caused by HC. References Aldridge N. N. (1953) Enzyme hydrolysing diethyl-p-nitrophenyl phosphate E-600 and its identity with the A-estemse of mammalian sera. Biochem. J. 53, 117-124. Bauer P. A., Shore A. and Ikeman R. L. (1987) Transient fall in serum salicylate levels following intraarticular injection of steroid in a patient with rheumatoid arthritis. Arthit. Rheum. 30, 345-347. Bien E. J., Gebert J. and Skorka G. (1985) Biphasic effect of pyrazolone de-

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