Small bowel absorption of sulfasalazine and its hepatic metabolism in human beings, cats, and rats

GASTROENTEROLOGY 77:260-264,1979

Small Bowel Absorption of Sulfasalazine and Its Hepatic Metabolism in Human Beings, Cats, and Rats KIRON M. DAS, J. ROY CHOWDHURY, and JOHN W. FARA

B. ZAPP,

Division of Gastroenterology and Liver Disease, Department of Medicine, Albert Einstein College of Medicine, New York, and Department of Physiology, State University of New York, Stony Brook, New York

To elucidate the role of the small bowel and liver in sulfasalazine (SASP) metabolism, we performed studies in patients, cats, and rats. The role of the small bowel in absorption and metabolism of SASP was determined by the amount of administered SASP excreted in ileostomy effluents, and the concentration of serum and urinary SASP and its metabolites, sulfapyridine and 5-amino salicylic acid. Seventy-five to ninety percent of the drug was excreted in ileostomy effluents of 6 patients as SASP, and only 5% of the dose was sulfapyridine. In cats, ileostomy and portal venous cannulations revealed that 20-30% of administered SASP is absorbed from the small bowel without being metabolized. The role of the liver in SASP metabolism was established in vivo and in vitro. SASP metabolites were measured in bile, serum, and urine of 2 patients with a choledochal T-tube and in serum and bile in six cats and four rats. Twenty to fifty percent of the absorbed drug was excreted in bile as SASP and no detectable sulfapyridine appeared in the bile. SASP concentration in peripheral blood and urine ranged between 3 and 12 pg/mJ, and no significant amount of sulfapyridine metabolites were detected in the bile, serum, or urine of animals with an ileostomy. In vitro experiments with liver from cats and rats Received August 1.1976.Accepted February 26,1979. Address requests for reprints to: Kiron M. Das, M.D., Department of Medicine, Albert Einstein College of Medicine, 1366 Morris Park Avenue, Bronx, New York 10461. Dr. Fara’s present address is the Department of Radiology and Radiological Science, The Johns Hopkins Hospital, Baltimore, Maryland 21205. This work was presented in part at the Fifth Annual Meeting of the American College of Clinical Pharmacology, Philadelphia, May, 1976. This work was supported in part by a grant from the Rowe11 Laboratories, Baudette, Minnesota. 0 1979 by the American Gastroenterological Association 001~5065/79/060260-05$02.00

showed nearly complete reduction of SASP into metabolites. These studies reveal that SASP is not metabolized by the liver in vivo, and after absorption from the small bowel, 25-50% of the SASP is excreted unchanged in the bile, and the rest enters the circulation. Jmplications of these results in the treatment of patients with Crohn’s disease of the small intestine are discussed.

The role of sulfasalazine in the management of inflammatory bowel disease is established,‘-” and studies have outlined the pharmacokinetics of SASP and correlated the relationship between the serum concentrations of SASP and its metabolites and the clinical states of ulcerative colitis and Crohn’s disease.4-s Because colonic bacteria are important in hydrolyzing SASP,’ partial or complete absence of the colon affects metabolism and absorption of the drug.” SASP is hydrolyzed into 5-aminosalicylic acid (5ASA), which is not absorbed and is largely excreted in the stool, and sulfapyridine (SP), which is primarily absorbed from the colon.4.5 Only l-10% of SASP is excreted in urine unchanged. This work was intended to determine the role of the small intestine and liver in SASP absorption and metabolism. Materials

and Methods

Patients: Group A-Four patients with idiopathic ulcerative colitis and 2 patients with Crohn’s disease of the colon had pancolectomy and permanent ileostomy 4-6 mo before study. Serial samples of blood were collected at 1,3,5,X?, 24, and 43 hr intervals before and after taking 2 g of SASP. Urine and ileostomy effluents were collected for two consecutive 24-hr intervals. Group B-Two patients with indwelling T-tubes in the common bile duct for 7-10 days after choledocholithiasis were given an identical

August 1979

SULFASALAZINE-SMALL BOWEL ABSORPTION AND HEPATIC METABOLISM

dose of SASP. Both patients had normal serum bilirubin and transaminase. Alkaline phosphatase was slightly elevated (cl50 mu/ml, normal 30-85 mu/ml). Aliquots of serum and urine were collected as in group A for 48 hr. Bile obtained from the T-tube drainage was collected for 48 hr. Informed consent was obtained from all patients. Group G-Three of the investigators acted as normal control subjects and ingested 2 g of SASP after an overnight fast. Serial blood samples and 48-hr urine samples were obtained and processed like those of the patients.

Animal Experiments After overnight fast, six cats (weighing 2.7-3.4 kg) were anesthetized with i.p. and i.v. sodium pentobarbital (20-30 mg) in 5% dextrose and water. Surgical preparation and experiments were performed in the anesthetized cats without administration of any further anesthetics. A terminal complete ileostomy, ligation of the cystic duct, cannulation of the common bile duct, insertion of a catheter into the duodenum through a gastrostomy, ligation of the pylorus, and cannulation of the portal vein, femoral artery and vein were performed. Portal venous blood was shunted via a drop counter to measure blood flow and was returned immediately to the hepatic end of the portal vein. Equal volumes of 10% dextrose were administered i.v. to replace blood volume after sampling. The surgical procedure took 30 min, after which the abdomen was closed, and SASP (Rowe11 Laboratories, Inc., Baudette, Minn.; 50 mg/kg) dissolved in 25 ml of normal saline was immediately introduced via the duodenal tube. Portal and peripheral venous blood samples and bile were collected at 0 hr and every half hr for 3-4 hr. Arterial systolic pressure was monitored through the femoral arterial catheter and remained at 80-110 mm Hg during the study. The experiments were terminated if systolic blood pressure dropped below 80 mm Hg. In two cats, similar experiments were performed, and 20 mg of SASP in 10 ml of sterile normal saline were infused into the portal vein over 2.5 hr. In these animals, only the femoral artery and vein and the common bile duct were cannulated. Serial peripheral blood and bile samples were collected at 0.5 hr intervals during infusion, and for 1 and 2 hr after stopping infusion. Twelve male Wistar rats weighing 300-500 g were obtained from Marland Farms (Peekskill, N.Y.). Under ether anesthesia, the terminal ileum and gastric antrum were ligated, and a polyethylene cannula was placed in the proximal duodenum. SASP, 50 mg/kg, was introduced through the cannula into the duodenum. Because presence of a biliary fistula might influence the blood level of SASP/metabolites and because collection of 1-2 ml of peripheral venous blood for four to five times might influence renal perfusion and urinary excretion, all 12 rats were divided into three groups for collection of bile, urine, and blood. In five rats, the bile duct was cannulated (“Intramedic” PElO), and bile was collected for three consecutive hourly periods and in two rats for 12 additional hours. Four other rats had catheters placed into the urinary bladder via a stab wound, and complete urine collections were obtained for similar intervals as those used for

281

the bile collections and for an additional period of 12-24 hr. In the remaining three animals with similar gastrointestinal tract preparation, 1 ml of blood was collected from a jugular vein catheter at 2, 4, 6-6, and 24 hr after introduction of SASP into the small intestine. Baseline samples of bile, urine, and blood were obtained before introduction of the drug. Samples of serum, bile, urine, and ileostomy effluents were frozen at -20°C until analyzed.

Assay SASP

of Hepatic Azoreductase as a Substrate

Activity

with

To prepare cat and rat liver fractions containing microsomes and cytosol, animals were decapitated under ether anesthesia. The liver was quickly removed, perfused with ice-cold potassium chloride (0.9%), minced, and homogenized in 4 ml of potassium chloride (0.9%) per g of wet tissue in a Potter-Elvehjem homogenizer equipped with a motor driven Teflon pestle. The homogenate was centrifuged at 9000 g for 20 min, and the supernatant was used for assay of azoreductase activity, as described by Fouts et al.g The incubation mixture contained SASP 3 x lo-'mol in 0.1 M phosphate buffer, pH 7.6, NADPH 1 X lo4 mol, FAD 1 x 10e3 mol, and the liver preparation (microsome + cytosol) equivalent to 0.5 g liver. The volume was brought up to 5 ml by adding phosphate buffer 0.1 M, pH 7.6. After 80 min incubation in a Dubnoff shaking incubator at 37OC in a nitrogen atmosphere, the reaction was stopped by adding 5 ml trichloroacetic acid (10%). The mixture was centrifuged at 900 g for 10 min, and SASP and SP were extracted from the supernatant with amylacetate and methyl isobutyl ketone respectively+” and quantitated as described below.

Estimation

of SASP

and its Metabolites

The methods are described in detail elsewhere.‘%” Briefly, SASP was extracted from serum and urine with amylacetate and quantitated by measuring the absorbance at 455 nm after the final extraction into NaOH (0.5 M). SP and its metabolites, namely, acetylated SP, SP glucuronide, and acetylated SP glucuronide, were extracted from serum, urine, bile, and ileostomy effluent with methyl isobutyl ketone. Pure acetylated SP and SP glucuronide were used as reference substances. They were obtained from Pharmacia (Uppsala, Sweden). SASP in bile and ileostomy effluent was reduced to SP by treatment with titanium trichloride (TiCl,) (15%), and SP formed was extracted with methyl isobutyl ketone. Ninety-five percent of SASP and SP were extractable by the methods. Parallel extraction of unreduced samples gave the values for SP which was already present and not derived from reduction of SASP by TiCl,. SP metabolites were converted to SP by acid hydrolysis of acetyl SP and /$glucuronidase treatment of SP glucuronides. Total SP (free + acetyl SP + SP glucuronide) was diazotized by Bratton-Marshall reactionXz; azopigments formed were quantitated from absorbance at 544 nm. Standard curves were established using pure SASP and SP (Rowe11 Laboratories, Baudette, Minn.), and the curves were linear for both compounds through

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TIME

Vol. 77,No. 2

(hours) TlMEjhours)

USP 2C

Figure 1. Mean (zt SEM) serum sulfasalazine (SASP) concentration in the control subjects (A-A), patients with ileostomy (W), and patients with a T-tube in the common bile duct (0-O) at various intervals up to 48 hr after a single 2-g dose of sulfasalazine by mouth.

Figure 2. Mean (k SEM) serum total sulfapyridine (SP) concentration in the control subjects (A-A), patients with ileostomy (OX)), and patients with a T-tube in the common bile duct (0-O) at various intervals up to 48 hr after a single 2-g dose of sulfasalazine by mouth.

similar range of concentrations (Z-80 pg/ml). All human sera, urine, and ileostomy effluents from Group A patients were also analyzed for 5-ASA by spectrofluorometry at an excitation wavelength of 310 nm, and a fluorescence wavelength of 430 nm after extraction with methyl isobutyl ketone.13

33% compared with 55% of the administered dose in control subjects. The decreased proportion of administered dose excreted in urine of the patients with bile fistula suggests an enterohepatic circulation of SASP. Bile was collected via a T-tube, and, therefore, complete collection was not possible. Bile contained mostly SASP (2-20 pg/ml) and 0.2 pg/ml of SP metabolites. Serum 5-ASA concentrations in the three groups of subjects were cl.5 pg/ml. 5-ASA excreted in the urine of patients with ileostomy was cl%, and their ileostomy effluents contained 2-4% of the administered dose of 5-ASA.

Results Patients Figures 1 and 2 show serum SASP and total SP (free SP and SP formed from its metabolites) concentrations in the two groups of patients and in controls. Serum SASP concentrations in the three groups after administration of 2 g of SASP were similar. Serum total SP concentration in the 2 patients with intact colon and a biliary fistula was also comparable with that in control subjects. Patients with ileostomy had little detectable SP metabolites in the serum. Urinary excretion of SASP did not differ significantly in the three groups of subjects (Table 1); however, total SP excretion was less than 2% of the administered dose in patients with ileostomy reflecting the low serum concentration of this component of the drug. In these patients, 75-90% of the administered drug was recovered unchanged in the ileostomy effluents (Table 1). Urinary excretion of total SP in the 2 patients with bile fistulas was reduced to Table

1.

Mean 24-Hour Excretion (2 g) Administration

Animals Simultaneous measurement of mesenteric venous blood flow and drug concentrations enabled us to measure absorption and small intestinal metabolism in cats. Approximately 20-30% of the intraduodenal dose of SASP was absorbed from the small intestine during the 3-4-hr experimental period (Table 2), of which 20% was recovered in bile as SASP, and no detectable SP appeared in the bile. The rest of the absorbed SASP entered the peripheral circulation; the level of SASP in peripheral blood and urine ranged between 3 and 12 pg/ml. SP metabolites in serum or urine were trace (<2 pg/ml). When SASP was infused directly into the portal

of SASP and Its Metabolites

of SASP in 6 Patients

Group A: patients with ileostomy (6) (X of admin. 24 hr urinary

dose)

and 2 Patients

Effluent After a Single Dose with Biliary Fistula

Group B: patients with biliary fist& (2) (% of admin.

dose)

Group C: normal volunteers (3) (% of admin.

excretion 4 2

SASP

Total SP Biliary drainage via T-tube SASP Total SP lleostomy effluent SASP

Total SP -

in Urine, Bile, and Ileostomy

with an lleostomy

= Not applicable.

65 5

3 33

5 55

9

Trace (C 0.2) -

-

dose)

SULFASALAZINE-SMALL BOWEL ABSORPTION AND HEPATIC METABOLISM

August 1979

Table 2.

Mesenteric Venous Outpow and the Concentration of SASP and Its Metabolites at Various Intervals Venous Blood After the Administration of 50 mg/kg SASP into the Duodenum of Six Cats

in Portal

Bileb

Time of sample

SMV outflow

SMV blood vol

SMV

(min)

(ml/min)

(flow X time) (ml)

SASP” (pg/ml)

Peripheral VB SASP” @g/ml)

SASP” (mg)

-

-

-

0

6 f 1.3

-

283

30 60

a k 2.8 7.8f 2.8

138f21 163 i52

4 + 1.0 12.6zk4.3

5 f 2.3

0.8 f 0.3

'90 120 180 240

5.1f 1.5 6.6 f 0.5 6.3+-0.6 6.2 f 0.7

164f38 ZOO+15 19lf18 170 f 35

9.6 zt1.3 10.6k 2.1 14.2f 2.2 8.6+ 1.7

6.3f 1.8 6 f 3.1 4.5 zk2.1

1.5f 0.6 1.4* 0.3 1.8f 0.2

Results are expressed as mean f SEM. SMV = superior mesenteric vein: BV = venous blood. a No detectable SP metabolites were observed in any of these samples. b Total biliary outputs during the different time periods are shown.

of two cats, biliary excretion of the intact drug rose to 5O-60% of the administered amount without detectable SP metabolites. Peripheral blood SASP concentration rose simultaneously to 16-32 pg/ml without any detectable metabolites. The findings in cats were confirmed by results in different sets of rat experiments. Total amount of SP metabolites recovered in the bile and urine of rats was ~2% of the administered dose. These results indicate that hepatic azoreduction of sulfasalazine does not occur in vivo to any significant extent.

vein

In

Vitro Hepatic Azoreduction

When 30 pM SASP was incubated anerobitally in. the presence of 1 PM NADPH, 1 mM FAD, and liver microsome + cytosol (equivalent to 0.1 g wet weight/ml) from cats and rats, about 90% of SASP was reduced to free SP and 5-ASA.

Discussion The present study indicates that about onequarter to one-third of administered SASP is absorbed from the small intestine without being metabolized either in the lumen or small intestinal mucosa. Although the azoreductase system is known to exist in the liver,9.14 SASP does not appear to be a substrate for this system in vivo in species we have studied. SASP absorbed from the small bowel is excreted unchanged in bile; the remainder enters the general circulation and is excreted by the kidney. The mechanism responsible for absence of reduction of SASP in vivo but significant azoreduction in vitro is unknown. Because azoreduction in vitro is anerobic, appropriate conditions may not exist in vivo. In addition, the concentration of NADPH in liver cells does not approach 1 PM as used in the assay. In vivo hepatic glucuronidation and biliary excretion of SASP may be preferential to reduction. The method of SASP estimation in these studies

cannot differentiate free SASP and glucuronidated SASP. Following the same methods as demonstrated for the azodye amaranth,15 we have recently observed in vitro that azoreduction of SASP by rat liver microsomes is mediated by two independent systems: One depends on cytochrome P-450 and the other requires flavins. The in vivo hepatic azoreductase system does not reduce SASP into SP and 5ASA. The mode of action of metabolites (&ASA and SP) may be related to local antiinflammatory effects of 5ASA or SP, antiprostaglandin effects of 5-ASA,‘” or suppression of cytotoxicity of lymphocytes by SP as demonstrated in vitro.” Indeed, in patients with active ulcerative colitis, the systemic immunologic abnormalities such as an increase of circulating complement receptor positive B cells and activated monocytes reversed to normal after successful treatment with SASP a10ne.17 A recent study has suggested that rectal administration of 5-ASA, but not SP, promotes remission of colitis.‘” SASP seems to work less well when there is fecal stasis proximal to the lesion which also suggests that its action is loca1.19 Fecal bacteria hydrolyze the drug and make the metabolites locally available in the colon.’ These results probably explain the findings of the National Cooperative Crohn’s disease study group which showed that SASP is effective mainly in patients with Crohn’s disease of the colon.3 The clinical dispute regarding the efficacy of SASP in small intestinal Crohn’s disease may be explained by lack of metabolism of SASP in the small intestine, and enterohepatic circulation of the absorbed drug without azoreduction by the liver. As a result, “active metabolites” cannot reach the small intestine, the site of inflammation. Further investigation is needed to evaluate the efficacy of SP and/or 5-ASA administered orally instead of SASP in patients with small intestinal Crohn’s disease. Aspirin or indomethacin may be better candidates than 5-ASA because, although many congeners of salicylate have antiinflammatory

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DAS ET AL.

activity, 5-ASA has never been investigated for this property.‘” This is even more relevant in patients with Crohn’s disease who have an ileostomy. The best method of delivery of the “active metabolites” to the disease site is also to be evaluated.

References 1

2. 3.

4.

5.

6.

Dissanayake AS, Truelove SC: A controlled therapeutic trial of long term maintenance treatment of ulcerative colitis with sulphasalazine (salazopyrin). Gut 14:923-926,1973 Das KM, Sternlieb I: Salicylozosulfapyridine in inflammatory bowel disease. Am J Dig Dis 20:971-976,1975 Summers RW, Sessions JT, Switz DM, et al: National Cooperative Crohn’s disease study: response of sub-groups to drug treatment (abstr). Gastroenterology 74:1100,1978 Schroder H, Campbell DES: Absorption, metabolism, and excretion of salicylazo-sulfapyridine in man. Clin Pharmacol Ther 13:506-551,1972 Das KM, Eastwood MA, McManus JPA, Sircus W: The metabolism and salicylazosulphapyridine in ulcerative colitis. I. The relationship between metabolites and the response to treatment in inpatients. II. The relationship between metabolites and the progress of the disease studied in outpatients. Gut 14:631-641,1973 Das KM, Dubin R: Clinical pharmacokinetics of sulfasalazine. Clin Pharmacokinetics 1:406-425,1976 Peppercorn MA, Goldman P: The role of intestinal bacteria in the metabolism of salicylazosulfapyridine. J Pharmacol Exp Ther 181:555-562,1972 Das KM, Eastwood MA, McManus JPA, Sircus W: The role of the colon in the metabolism of salicylazosulphapyridine. Stand J Gastroenterol9:137-141,1974 Fouts JR, Kamm JJ, Brodie BB: Enzymatic reduction of prontosil and other azodyes. J Pharmacol Exp Ther 120:291300,1957

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Sandberg M, Hansson KA: Determination of salicylazosulfapyridine in biological materials. Acta Pharm Suet lO:lO7-111, 1973 11.Hansson KA, Samdberg M: Determination of sulphapyridine and its metabolites in biological materials after administration of salicylazosulphapyridine. Acta Pharm Suet 10:87-92, 1973 12. Bratton AC, Marshall Jr, EK: A new coupling component for sulfanilamide determination. J Biol Chem 128:537-550,1939 13. Hansson KA: Determination of free and acetylated 5-amino salicylic acid in serum and urine after administration of salicylazosulphapyridine. Acta Pharm Suet 10:153-155,1973 14. Hernandez PH, Gillette JR, Maze1 P: Studies on the mechanism of action of mammalian hepatic azoreductase. I. Asoreductase activity of reduced nicotinamide adenine dinucleotide phosphate-cytochrome C reductase. Biochem Pharm 16:1859-1975,1967 15. Fujita S, Peisach J: The mechanism of stimulation of microsomal azoreduction by riboflavin, FMN and FAD. Fed Proc 37:305,1978 16. Sharon P, Ligumsky M. Rachmilewitz D, et al: Role of prostaglandins in ulcerative colitis. Enhanced production during active disease and inhibition by sulfasalazine. Gastroenterology 75:638-640.1978 17. Holm G, Perlmann P: The effect of antimetabolites on the cytotoxicity by human lymphocytes. In: Advance in Transplantation. Copenhagen, Munksgaard, 155-161.1968 18. Rubinstein A, Das KM, Melamed J, et al: Comparative analysis of systemic immunological parameters in ulcerative colitis and idiopathic proctitis: effects of sulfasalazine in vivo and in vitro. Clin Exp Immunol33:217-224,1978 19. Azad Khan AK, Piris J, Truelove SC: An experiment to determine the active therapeutic moiety of sulfasalazine. Lancet 2:892-895.1977 20. Cowan GO, Das KM, Eastwood MA: Further studies of sulfasalazine metabolism in the treatment of ulcerative colitis. Br Med J 2:1057-1059.1977 N 21. Goldman P, Peppercorn MA: Drug therapy-sulfasalazine. Engl J Med 293:20-23,1975