INFLUENCE OF SODIUM TAUROCHOLATE, CHOLESTYRAMINE, AND MYLANTA ON THE INTESTINAL ABSORPTION OF GLUCOCORTICOIOS IN THE RAT

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1 GASTROENTEROLOGY 64: , 1973 Copyright 1973 by The Williams & Wilkins Co. Vol. 64, No. 6 Printed in U.S.A. INFLUENCE OF SODIUM TAUROCHOLATE, CHOLESTYRAMINE, AND MYLANTA ON THE INTESTINAL ABSORPTION OF GLUCOCORTICOIOS IN THE RAT ATHOL J. WARE, M.B.B.S., M.R.A.C.P., AND BURTON COMBES, M.D. Liver-Gastrointestinal Unit, The Department of Internal Medicine, The University of Texas Southwestern Medical School at Dallas, Texas A micellar solution of sodium taurocholate has been shown to increase the solubility of cortisol and prednisolone crystals in vitro. That this enhanced solubility confers little physiological advantage to the rat has been demonstrated in in vivo studies on the intestinal absorption of these compounds. Tritiated crystals of cortisol and prednisolone were delivered intraduodenally to rats with complete biliary diversion receiving an intraduodenal infusion of either 37 mm taurocholate or normal saline. The excretion of the metabolic products of the steroids in bile and urine was used as a measure of the intestinal absorption of the parent compound. Excretion was greater in the first 2 hr after the administration of either cortisol or prednisolone in rats receiving bile salts than in bile salt-deficient animals. By 4 hr, however, no significant differences were found in the excretion values between the two groups of rats. More than 70% of the administered steroid was recovered in bile and urine within 4 hr in animals with a complete bile-salt deficit. Despite the demonstration, in vitro, that both steroid compounds are bound by cholestyramine, no significant decrease in the intestinal absorption of cortisol was observed when either cholestyramine or an antacid containing aluminum hydroxide were administered concomitantly. Therapy with oral preparations of corticosteroids has been employed in patients with a variety of diseases including a group with diverse disorders affecting the liver. This form of therapy has been recom- Received October 26, Accepted January 12, Address reprint requests to: Dr. Athol Ware, Department of Internal Medicine, The University of Texas Southwestern Medical School, 5323 Harry Hines Boulevard, Dallas, Texas This study was supported in part by the United States Public Health Service Research Grant AM and Training Grant Ti AM 5490 from the National Institutes of Health United States Public Health Service. Dr. Combes is recipient of Research Career Program Award 5-K3-AM-18,250 from the National Institutes of Health, United States Public Health Service. The authors wish to express their appreciation to Mr. Marc Hardt for technical assistance and to Mrs. Melba Sanders for preparation of the manuscript. mended in patients with massive hepatic necrosis, viral hepatitis with subacute hepatic necrosis, active chronic hepatitis, primary biliary cirrhosis, acute alcoholic hepatitis, and as a test to distinguish intrahepatic from extrahepatic causes of cholestasis. The value of steroid therapy remains controversial in many of these conditions, and in others, a variable response is seen from patient to patient. The glucocorticoids in clinical use bear a structural similarity to cholesterol, the compound from which they are derived. They are, at best, sparingly soluble in water, and would, presumably, be more soluble in a detergent solution. To our knowledge, the role that bile salts play in the gastrointestinal absorption of these steroid preparations has not been explored. It is conceivable that, in the small bowel, bile-salt micelles are necessary for the adequate solubiliza- 1150

2 June 1973 ABSORPTION OF GLUCOCORTICOIDS IN THE RAT 1151 tion of the steroid compounds. The variable therapeutic effect of these agents in patients with the liver diseases mentioned above could then result from malabsorption of the drug consequent to an intraduodenal bile-salt deficiency. A number of studies on glucocorticoid absorption have been reported in the past. I. 2 These investigations have shown that both cortisol and prednisolone are absorbed rapidly, by passive diffusion, throughout the length of the gastrointestinal tract. These studies, however, utilized steroids in solution and thus provide no information on the process as it occurs after the oral administration of cortisol and prednisolone tablets. Some patients on steroid therapy for liver disease receive, concomitantly, a variety of other drugs whose ability to interfere with the absorption of steroids has not been examined. Cholestyramine 3 and aluminum hydroxide antacids 4 5 are of particular interest in this respect since they are both known to possess drug-binding potential. We have explored the effect of bile salts on the solubilization of prednisolone and cortisol in vitro, and have examined the effect of bile-salt deprivation on the gastrointestinal absorption of these compounds in the rat. We have also investigated the influence exerted on the absorption of cortisol by cholestyramine and a commercial antacid (Mylanta, Stuart Pharmaceuticals, Pasadena, Calif.) containing aluminum and magnesium hydroxides. Materials and Methods Preparation of Tritiated Steroid Crystals Tritiated cortisol and prednisolone (dissolved in benzene-ethanol 9: 1 v/v), each with a specific activity of 5,500 mc per mg, were obtained from Schwarz Bioresearch Inc., Orangeburg, New York. About 150 mg of nonradioactive cortisol and prednisolone, in crystalline form (Steraloids Inc., Pauling, N. Y.) were dissolved in ethanol. A small volume (0.1 ml) of the appropriate tritiated steroid was mixed well into these solutions. The steroid crystals were precipitated by the addition of excess ice-cold water. The supernatant fluids were decanted and the crystals were then air-dried. The specific activity of each of the final crystalline steroid powders was of the order of 220,000 dpm per mg. Confirmation of the specific attachment of the tritium label to the steroid molecule was obtained by partition chromatography on a celite column 6 using a partitioning solution of isooctane-tertiary butanol-water in the ratio of 250: 125 : 225 v/v/v. The recrystallized cortisol and prednisolone preparations both gave a single peak of identity for radioactivity and steroid concentration as determined spectrophotometrically at 240 m. In Vitro Studies Solubility studies. Different amounts of the cortisol and prednisolone crystals (range 0.5 mg to 10 mg) were added to 3-ml samples of normal saline and agitated in a Dubnof shaking water bath at 37 C for 24 hr. In other studies, the steroid was added to 3-ml samples of a 37 mm solution of sodium taurocholate in normal saline and treated similarly. Each sample was then filtered through no. 1 What man filter paper and the radioactivity of 100-liter aliquots of the filtrates was counted. The quantity of the steroid dissolved in 3 ml of solvent was calculated from the known specific activity of the steroid crystals. The results were expressed as milligrams of steroid dissolved per milliliter of solvent at 37 C. In vitro cholestyramine binding. Five milliliters of saline were added to a series of 5- mg samples of cortisol and prednisolone crystals. After incubation for 1 hr at 37 C, each sample was filtered and the radioactivity of 1oo-liter aliquots of each filtrate was counted. The filtrates were then added to increasing amounts of cholestyramine (range 100 mg to 1 g). These solutions were thoroughly mixed and then centrifuged at 10,000 rpm for 10 min in an International centrifuge (model CM). The radioactivity of aliquots of the supernatant fluids was again determined. The concentration of steroid remaining in solution after the addition of cholestyramine was calculated and expressed as milligrams of steroid dissolved per milliliter of solvent. In Vivo Studies Preparation of animals. Adult male Sprague Dawley rats (Simonson, Calif.), weighing between 200 and 350 g were maintained under light ether anesthesia while Teflon catheters (internal diameter, inches) were placed in a femoral vein, the common bile duct, the urinary bladder, and via a gastrotomy into the second part of the duodenum. Teflon catheters were chosen when preliminary experiments

3 1152 WARE AND COMBES Vol. 64. No.6 demonstrated a variable adsorption of the steroids to polyethylene tubing; adsorption was not found to occur with Teflon catheters. The animals were then placed in restraining cages and were maintained on an intravenous infusion of 5% glucose in distilled water and an intraduodenal infusion of normal saline (±37 mm taurocholate). Both infusions were delivered at a rate of 1.5 ml per hr. Urine and bile were collected and the rectal temperatures of the rats were maintained between 37 C and 39 C by heating pads placed around the cages. Experiments were conducted 72 and 96 hr after surgery only if the animal remained vigorous and healthy. Preliminary experiments conducted on the 1st and 2nd postoperative days were found to give extremely variable results when the steroids were administered intraduodenally. Presumably, intestinal recovery from the surgical trauma was not uniformly complete until the 3rd day. Administration of the steroids. 1. Intravenous injection. About 2 mg of the steroid crystals,were dissolved in 0.2 ml of 95% ethanol. Normal saline was added to bring the final volume to 2 ml. A bolus of 1 to 1.5 ml of this solution (i.e., 1 to 1.5 mg of steroid) was delivered to the animal via the femoral vein catheter over 30 sec. The intravenous infusion of 5% glucose in water was then continued at the previous rate. A 100-JLliter aliquot of the solution was counted in duplicate and the dose of steroid administered to the rat was calculated as the product of the volume and the specific activity of the injected solution. 2. Intraduodenal injection. A sample (2 to 3 mg) of the steroid crystals was accurately weighed, placed in a flask, and suspended in 0.5 ml of normal saline. This suspension was taken into a small syringe. The flask was rinsed with 0.5-ml saline and this rinsing was also drawn into the syringe. The contents of the syringe were then delivered, as a bolus, into the duodenum via the gastrotomy catheter. An additional 0.5 ml of saline was used to rinse the syringe and this rinsing was also delivered into the duodenum. The intraduodenal infusion of normal saline (±37 mm taurocholate) was then continued at the previous rate. The flask and syringe were each washed with 1 ml of 95% ethanol to dissolve any residual crystals, and 100-JLliter aliquots of these washings were counted. The quantity of steroid remaining in the vessels were calculated from the known specific activity of the steroid crystals and the volume and radioactivity of the washings. The quantity of steroid delivered to the animal was derived by subtracting the amount of steroid left in the flask and syringe from the weight of the original sample. The amount delivered was generally between 1 and 1.5 mg and was comparable to the dose given in the intravenous experiments. Urine and bile were collected in tared bottles at 2-hr intervals after administration of the steroid. The radioactivity of 100-JLliter aliquots of each fluid was counted for each collection period. The quantity of steroid excreted was determined by assuming a specific gravity of 1 for both bile and urine, deriving the product of the volume of the sample and its radioactivity per unit volume, and dividing this by the known specific activity of the steroid crystals. The quantity of steroid excreted was expressed as a percentage of the administered dose. Experimental design. The following sets of experiments were performed. 1. Bile salt-depleted. Steroids were delivered intraduodenally to rats in whom biliary diversion had been present for at least 72 hr and to whom no replacement bile salts had been given for at least 20 hr. This period of time was sufficient to totally deplete the intestine of bile salts if replacement bile salts had previously been given to the animal. 2. Bile salt-repleted. Steroids were delivered intraduodenally to rats who had received an intraduodenal infusion of a 37 mm taurocholate solution at a rate of 1.5 ml per hr for the preceding 20 hr. After administration of the steroid, the bile salt infusion was continued at the same rate throughout the collection period. 3. Intravenous administration of steroids was effected in both bile salt-depleted and bile salt-replete animals. 4. Cholestyramine suspension (1 g in 10 ml of normal saline) was injected into the duodenum in 1-ml boluses immediately before, immediately after, and then at 20-min intervals for 1 hr after the intraduodenal administration of cortisol to bile salt-depleted rats. 5. Mylanta suspension was injected into the duodenum in 1-ml boluses before, after, and at 20-min intervals after the intraduodenal administration of cortisol to bile salt-depleted animals. Preliminary studies demonstrated that, irrespective of the route of administration, 95% of the steroid excreted in 24 hr was recovered in the first 4 hr after its administration (fig. 1.). Less than 1% of the administered dose was recovered between 24 and 48 hr after the compound was given. The collection period in these experiments was therefore limited to 4 hr and it was deemed valid to use the same animal for different experiments on the 3rd and 4th postoperative days. Studies involving bile-salt de-

4 June 1973 ABSORPTION OF GLUCOCORTICOIDS IN THE RAT '" i'! z lij 80 a:rj) "'0 0.0 <10 ll! 60 5 rj)!!! i= l5 40 "'<I z '" a: '" x '" 00 I I ji Totol--... e-ii.1' ,.P- _ f/- _-0 fl' Urine, b,./j, I>. _ I>. -I>. _.// I> B 11-;;;- TIME IN HOURS FIG. 1. Representative experiment showing the time pattern of tritium excretion after the intraduodenal administration of tritiated cortisol in the presence of bile salts. pletion and repletion were alternated for these consecutive day experiments. Approximately equal numbers of each type of experiment were performed on both the 3rd and 4th postoperative days. Counting Methods The counting solution used throughout these studies was constituted by mixing 1 liter of Triton-X, 2 liters of toluene, 450 ml of 0.25 N hydrochloric acid, 14 g of 2, 5-diphenyloxazole and 0.2 g of 1, 4-bis-2-(5 phenyloxazolel-benzene. Aliquots (100 /Lliters) of the samples were added to 11 ml of this mixture. All samples were counted in duplicate for 10 min in a Beckman LS-100 liquid scintillation counter; internal standards were used to correct for the effects of quenching. Statistical Analysis Student's t-test and analysis of variance were used to determine the statistical significance of differences found in the results of these experiments. Results In Vitro Studies The solubility of cortisol crystals in normal saline at 37 C was found to be 0.28 mg per ml. In a 37 mm solution of taurocholate, this value was increased to 0.48 mg per ml. The solubility of the prednisolone crystals was found to be 0.41 mg per ml in saline at 37 C and 0.70 mg per ml in a 37 mm solution of taurocholate. The effect of increasing amounts of cholestyramine on the concentration of both cortisol and prednisolone in solution in normal saline is shown in figure 2. Both steroids were almost totally removed from solution when 250 mg or more of cholestyramine were added to 5 ml of solution. In Vivo Studies There were no significant differences between the results of studies performed 72 hr and 96 hr after surgery, within any of the experimental circumstances, for either steroid. The results of the different types of experiments which were done on the 3rd or 4th postoperative day have therefore been grouped together and are presented III table l. Cortisol. The mean total excretion of cortisol and its metabolites in 4 hr after its intravenous injection was 92.4% of the administered dose. In 2 hr, 77.5% of the injected dose had been recovered. This pattern of excretion was uninfluenced by the state of bile-salt repleteness of the animal. Introduction of the cortisol crystals into the duodenum in the presence of bile salts was followed by prompt absorption of the steroid which was reflected by a mean 2-hr excretion of 73.9% and a mean 4-hr excretion of 83.6% of the administered radioactivity. z o i= ::J...J g o a:_ rj).. LL E Oz z- a: '" z o '" _ Prednisolone Cortisol e, \ ---'U"==a OO B AMOUNT OF CHOLESTYRAMINE ADDED TO SUPERNATE IN mg FIG. 2. The in vitro binding capacity of choles tyramine for cortisol and prednisolone.

5 1154 WARE AND COMBES Vol. 64,No. 6 TABLE 1. Mean steroid excretion expressed as a percentage of the administered dose ± SE Compound and route of administration No. 2-hr bile 2-hr urine 2-hr total 4-hr bile 4-hr urine 4-hrtotal Cortisol Intravenous 8 66 ± ± ± ± ± ± 2.2 Intraduodenal with bile ± ± ± ± ± ± 3.1 salts Intraduodenal without 9 54 ± ± ± ± ± ± 3.0 bile salts Intraduodenal with My ± ± ± ± ± ± 6.9 lanta (no bile salts) Intraduodenal with cho ± ± ± ± ± ± 3.6 lestyramine (no bile salts) Prednisolone Intravenous ± ± ± ± ± ± 1.3 Intraduodenal with bile ± ± ± ± ± ± 5.2 salts Intraduodenal without ± ± ± ± ± ± 3.4 bile salts A mean of 82.9% of the administered dose was recovered in 4 hr after the intraduodenal injection of cortisol in the absence of bile salts. This was not different from the mean 4-hr excretion in the presence of bile salts (83.6%). The mean 2-hr excretion of cortisol in the bile saltdeprived animals, however, was only 64% and this value was significantly lower (P < 0.05) than the corresponding value for cortisol excretion in the presence of bile salts (73.9%). The concomitant administration of either Mylanta or cholestyramine did not significantly alter the excretion of cortisol at 2 or 4 hr from that obtained in bile salt-deprived animals not given these drugs. Within 4 hr, more than 70% of the administered dose of radioactivity was recovered from those animals who had received either drug. Prednisolone. The mean 4-hr excretion of prednisolone and its metabolites was 96.8% and the mean 2-hr excretion was 88.5% of the administered dose after intravenous infusion of this compound. The rate of excretion of prednisolone was more rapid than that of cortisol for comparable doses of each compound given intravenously. This difference resulted from a greater urinary clearance of the prednisolone excretory product in the first 2 hr. These findings are in agreement with previous reports of differences in disposition and excretion of cortisol and prednisolone in man. 7 No significant difference was found in the mean 4-hr excretion values after the intraduodenal administration of prednisolone in the presence of (82.7%), and the absence of bile salts (73.9%). At 2 hr, however, there was a significant difference (P < 0.02) with 68% of the administered dose being recovered in the presence of bile salts and only 55.3% being excreted in the bile salt-deficient animals. Thus, after the intraduodenal injection of both cortisol and prednisolone, significantly less of each compound was excreted in 2 hr in the bile salt-deprived animals. By 4 hr, however, no difference could be demonstrated in the quantity of the compound recovered between animals who were bile salt-deficient and those who were replete with bile salts. Discussion The metabolic and excretory pathways of glucocorticoids have been well delineated in both mans-to and rat. li Previous studies 11 have shown (and ours have confirmed) that within 24 hr, and over a wide range of doses, almost all of the steroid administered to the rat is metabolized and excreted. This remains so, irrespective of the route by which the compound is administered. 12 In the rat, the bile is the

6 June 1973 ABSORPTION OF GLUCOCORTICOIDS IN THE RAT 1155 major pathway of steroid elimination, and more than 90% of an administered dose can be recovered in bile and urine within 4 hr of its intravenous delivery.12 The apparent absence of any corporeal sequestration and the rapid clearance of exogenous steroids, once absorbed, validate our assumption that the excretion of steroid metabolites after the intraduodenal administration of the glucocorticoid reflects the amount of steroid absorbed from the gastrointestinal tract. Our in vitro studies have demonstrated that while cortisol and prednisolone are both reasonably soluble in water, their solubility is markedly enhanced by the presence of taurocholate micelles. Our in vivo studies have shown, however, that this enhanced solubility provides little physiological advantage. The gastrointestinal aborption of these compounds is quite adequate even in the total absence of bile salts. The excretion of the steroid metabolites (and hence, presumably, the absorption of the steroids themselves) was in fact not different after 4 hr, whether bile salts were present within the lumen of the intestine. Both steroid compounds are, apparently sufficiently water-soluble to allow for the absorption in 4 hr of more than 75% of a large pharmacological dose without the aid of the increased solubilizing potential of bile salts. The higher 2-hr excretion values in bile salt-replete animals does suggest, however, that steroid absorption is more prompt in the presence of bile salts, presumably because of their enhanced solubility under these circumstances. The presence of either Mylanta or cholestyramine was shown not to decrease the absorption of cortisol in this experimental model. This is somewhat surprising in view of the in vitro demonstration that cortisol is bound to cholestyramine. This paradox may be explained by postulating a weak and reversible bond between the two compounds. Additional studies are required to better delineate these forces as they apply to cortisol and other steroids. Extrapolation from our in vivo studies would suggest that, in the clinical setting of a patient with liver disease receiving oral corticosteroid therapy, the absorption of the steroid should not be sufficiently depressed by a bile-salt deficit in the intestinallumen, to interfere with its therapeutic purpose. The concomitant administration of cholestyramine or Mylanta would not be expected to interfere significantly with the absorption of cortisol under these circumstances, although similar information with regard to prednisolone therapy is not yet available. REFERENCES 1. Schedl HP, Clifton JA: Small intestinal absorp tion of steroids. Gastroenterology 41: , Stahl TJ, Tapley DF: Transport of adrenal cortical steroids by rat intestine in vitro. Endocrinology 73: , Gallo DG, Bailey KP, Sheffner AL: The interaction between cholestyramine and drugs. Proc Soc Exp Bioi Med 120:60-65, Paul HE, Harrington CM: Absorption characteristics of Aureomycin and Terramycin on aluminum hydroxide gel and on a bismuth subsalicylate preparation. J Am Pharm Assoc (Sci Ed) 41:50, Grote IW, Woods M: Studies on antacids: Adsorption effects of various aluminum antacids upon simultaneously administered anticholinergic drugs. J Am Pharm Assoc (Sci Ed) 42: , Siiteri PK: The isolation of urinary estrogens and determination of their specific activities following the administration of radioactive precursors to humans. Steroids 2: , Sandberg AA, Siaunwhite WR: Differences in the metabolism of prednisolone-c 14 and cortisol-c". J Clin Endocrinol Metab 17: , Hellman L, Bradlow HC, Adesman J, et al: The fate of hydrocortisone-4-c 14 in man.. J Clin Invest 33: , Peterson RE, Wyngaarden JB, Guerra SL, et al: The physiological disposition and metabolic fate of hydrocortisone in man. J Clin Invest 34: , Migeon CJ, Sandberg AA, Decker HA, et al: Metabolism of 4-C "-cortisol in man: Body distribution and rates of conjugation. J Clin Endocrinol 16: , Wyngaarden JB, Peterson RE, Wolff AR: Physiological disposition of radiometabolites of hydrocortisone-4-c 14 in the rat and guinea pig. J Bioi Chern 212: , Hyde PM, Williams RH: Absorption and metabolism of hydrocortisone-4-ci4. J BioI Chern 227: ,1957