(a) Absorption, distribution, metabolism, and excretion

The metabolic scheme for sulfasalazine in humans is shown in Fig. 4.1 (Das & Dubin, 1976; NTP, 1997a).

Fig. 4.1

Metabolic pathways of sulfasalazine in humans

Sulfasalazine is not absorbed to any significant extent from the stomach (Das & Dubin, 1976). Slow absorption of small amounts (~10–30%) via the small intestine has been reported before enterohepatic recycling, and with the majority of unchanged drug reaching the colon (Das & Dubin, 1976; Azad Khan et al., 1982).

The sulfasalazine molecule comprises 5-ASA and sulfapyridine moieties, linked by an azo bond, which is cleaved by bacterial azoreductases in the colon, releasing 5-ASA and sulfapyridine (Azad Khan et al., 1982). This cleavage is the rate-limiting step for clearance of sulfasalazine (Das & Dubin, 1976). Most of the 5-ASA is excreted; approximately 50% directly in the faeces, and at least 25% via the kidneys (after absorption and acetylation in the liver) (Das & Dubin, 1976; Azad Khan et al., 1982). In contrast, sulfapyridine is almost completely absorbed. In the liver, sulfapyridine undergoes hydroxylation and/or N-acetylation to 5′-hydroxysulfapyridine, N⁴-acetylsulfapyridine, and N⁴-acetyl-5′-hydroxysulfapyridine subsequently forming glucuronic acid conjugates, before excretion mainly in the urine (Das & Dubin, 1976; Azad Khan et al., 1982).

In studies of serum from 10 healthy male volunteers given single oral doses of 4 g of sulfasalazine, parent drug was detectable at 1.5 hours after dosing, and at maximum concentrations at 3–5 hours in nine subjects, and after 7 hours in one subject (Schröder & Campbell, 1972). Metabolites (sulfapyridine, and acetylated and glucuronidated derivatives) were detected in the serum at 3–5 hours after dosing (Schröder & Campbell, 1972). The pharmacokinetics of rectally administered sulfasalazine have been studied in three healthy male Japanese volunteers (Tokui et al., 2002). Sulfasalazine (6.5 mmol), given as a single suppository, reached maximum plasma concentration (2.5 ± 0.4 µM) in 5 hours (T_max), and an area under the curve (AUC) of 27.4 ± 4.8 µM.h. Parent drug was almost completely hydrolysed in the colon, and the urinary recovery was only approximately 0.2%. Maximum plasma concentration (C_max) of the metabolite N-acetyl-5-aminosalicylic acid was 0.5 ± 0.2 µM, reached in 12 hours, while that of sulfapyridine was 1.2 ± 0.4 µM, reached in 5 hours. 5-ASA was not detected in the serum. Administration of an enema containing 6.5 mmol of 5-ASA, resulted in C_max 5.8 ± 2.0 µM in 1 hour and an AUC of 29.4 ± 11.1 µM.h. In the urine, approximately 0.3% was recovered unchanged. The C_max for the acetylated metabolite, N-acetyl-5-aminosalicylic acid, was 13.3 ± 3.6 µM in 7 hours. More than 10% of 5-ASA was excreted in the urine as acetyl-5-aminosalicylic acid, suggesting that absorption of 5-ASA is favoured when administered rectally (Tokui et al., 2002).

A study to compare the absorption and metabolism of oral preparations of sulfasalazine, mesalazine (5-ASA) and olsalazine (a dimer of 5-ASA) used regularly by patients (n = 12, 13, and 8, respectively) for treatment of ulcerative colitis, showed considerably greater absorption of 5-ASA and less acetylation, in patients receiving mesalazine than in those receiving olsalazine or sulfasalazine (Stretch et al., 1996).

(b) Variation in absorption, distribution, metabolism, and excretion

(c) Genetic polymorphisms

(a) Absorption, distribution, metabolism, and excretion

Experimental studies in Sprague-Dawley rats given diets containing sulfasalazine have shown that most of the sulfasalazine is reductively cleaved by intestinal bacteria to two compounds, 5-ASA (which is poorly absorbed) and sulfapyridine (which is well absorbed) (Peppercorn & Goldman, 1972). After metabolism by mammalian enzymes, 5-ASA and sulfapyridine are excreted mainly in the faeces, and in the urine, respectively (Peppercorn & Goldman, 1972).

In male and female B6C3F₁ mice given sulfasalazine as an intravenous dose at 5 mg/kg bw, plasma concentrations of the parent drug rapidly declined with a mean elimination half-life of 0.5 hour in males and 1.2 hour in females (Zheng et al., 1993). The sex-specific differences in clearance rate were reflected in AUC_{sulfasalazine} values (9.21 µM.h^-1 and 21.39 µM.h^-1, in males and females, respectively).

In male and female B6C3F₁ mice given sulfasalazine by gavage at various doses (67.5, 675, 1350, or 2700 mg/kg bw), the bioavailability of the parent drug was approximately 17% (range, 16–18%) at the lowest dose (67.5 mg/kg bw), and was lower (range, 3–9%) at the higher doses. Both sulfapyridine and N⁴-acetylsulfapyridine were identified in the plasma. Sulfapyridine was eliminated more slowly than the parent compound, and thus accumulated in all mice given multiple doses of sulfasalazine. The differential between plasma concentrations of sulfapyridine and sulfasalazine varied, however, between males and females: the AUCs for sulfapyridine, at all four doses of sulfasalazine, were higher than those of parent drug by 21–32 times in males and by 5–25 times in females, while maximum plasma concentrations were higher than those of the parent drug by 6–8 times in male mice, and up to 4 times in females. Plasma concentrations of N⁴-acetylsulfapyridine were very low compared with those of sulfapyridine. [This indicated slow acetylation of sulfapyridine by B6C3F₁ mice, comparable to that found in humans with the slow-acetylator phenotype.] The pharmacokinetic pattern in B6C3F₁ mice given multiple oral doses of sulfasalazine (i.e. daily doses for three consecutive days) was similar to that in mice given a single oral dose, but accumulation of sulfapyridine was evident, producing greater AUC_{sulfapyridine} values in the multiple-dose study than in the single-dose study (Zheng et al., 1993).

In a study by the NTP (1997a), male F344/N rats were given sulfasalazine as an intravenous dose at 5 mg/kg bw, and pharmacokinetic parameters were compared with those in the study in male B6C3F₁ mice by Zheng et al. (1993). The rats retained sulfasalazine longer than mice; the AUC for sulfasalazine in rats was double that in mice, while values for systemic clearance and apparent volume of distribution in mice were double those in rats. The rate of elimination of sulfasalazine was similar in rats and mice; plasma elimination rate constants were 1.47 per hour and 1.28 per hour, respectively, and elimination half-lives, 0.53 hour and 0.54 hour, respectively. In F344/N rats given a low oral dose of 67.5 mg/kg bw, sulfasalazine and its metabolites were undetectable; however, after a higher dose (675 mg/kg bw), plasma concentrations of parent compound were detectable within 12 hours (NTP, 1997a).

(b) Role of transporter proteins

BCRP is a member of the ATP-dependent efflux transporters, which includes P-glycoprotein or multi-drug resistance protein 1 (MDR1/ABCB1) and multidrug resistance-associated protein 2 (MRP2/ABCC2). These transporter proteins have significant roles in the processes of drug absorption, distribution, and clearance, and are expressed at the apical membrane of cells in the liver, kidney, brain, placenta, colon, and intestine. From the latter location, on the villus tip of the apical brush-border membrane of intestinal enterocytes, they actively cause efflux of drugs from gut epithelial cells back into the intestinal lumen.

In experiments in Bcrp^−/− [Abcg2^−/−] knockout mice given sulfasalazine, the AUC for sulfasalazine was greater than in wildtype mice by 13-fold after an intravenous dose (5 mg/kg bw) and 111-fold after an oral dose (20 mg/kg bw) (Zaher et al., 2006). This treatment in the mdr1a [Abcb1a] knockout mouse did not significantly change the AUC for sulfasalazine. Furthermore, studies in wildtype mice treated with an inhibitor of Bcrp (gefitinib) before an oral dose of sulfasalazine resulted in a 13-fold increase in the AUC of plasma sulfasalazine compared with nontreated controls. This work thus demonstrated that Bcrp has a key role in controlling [i.e. maintaining a low (Dahan & Amidon, 2010)] oral bioavailability of sulfasalazine (Zaher et al., 2006).

In Caco-2 cells, sulfasalazine normally exhibits a basolateral-to-apical permeability that is 19 times higher than apical-to-basolateral permeability, indicative of net mucosal secretion (Dahan & Amidon, 2009). In this study of three ATP-dependent efflux transporters (in Caco-2 cells and rat jejunum), specific inhibitors of BCRP and of MRP2 were shown to disrupt the normal direction of sulfasalazine permeability. The presence of both MRP2 and BCRP inhibitors produced an efflux ratio of 1, indicating no efflux of sulfasalazine. Inhibitors of P-glycoprotein had no effect on the movement of sulfasalazine. The results thus suggested that efflux transport of sulfasalazine is mediated by BCRP and MRP2 (Dahan & Amidon, 2009). A more recent study of sulfasalazine absorption has shown that curcumin is a potent inhibitor of human BCRP. Curcumin not only increased the plasma AUC_0–8h eightfold in wildtype mice (but not in Bcrp^–/– mice), but also increased the plasma AUC_0–24h by twofold at microdoses of sulfasalazine and by 3.2-fold at therapeutic doses in humans (Kusuhara et al., 2012).

Further studies of the absorption characteristics of sulfasalazine in the isolated mouse intestine, have indicated that both influx and efflux transporters are involved in the intestinal absorption of sulfasalazine (Tomaru et al., 2013). OATP2B1 is a multispecific organic anion influx transporter. Like BCRP, it is localized at the brush-border membrane of intestinal epithelial cells, and mediates uptake of many endogenous and xenobiotic substrates from the lumen. Like BCRP, sulfasalazine is a substrate for OATP2B1 (Kusuhara et al., 2012; Tomaru et al., 2013). The study by Kusuhara et al. (2012) was inspired by the finding that pharmacokinetic data (plasma AUC for parent drug) from human subjects given sulfasalazine, at either a microdose (100 µg suspension) or a therapeutic dose (2 g as tablets), demonstrated nonlinearity between doses in the AUC of plasma sulfasalazine (Kusuhara et al., 2012). Investigations of sulfasalazine transport were performed in three systems in vitro, namely: (i) ATP-dependent uptake of sulfasalazine by membrane vesicles expressing human BCRP; (ii) oral bioavailability of sulfasalazine in vivo, in wildtype and Bcrp^–/– mice; and (iii) uptake of sulfasalazine in HEK293 cells transfected with the influx transporter OATP2B1 (SLCO2B1). The results indicated that sulfasalazine is a substrate for OATP2B1, and that saturation of the influx transporter OATP2B1 at the therapeutic dose is a possible mechanism underlying nonlinearity in the dose–exposure relationship for sulfasalazine (Kusuhara et al., 2012).

The nonsteroidal anti-inflammatory drug indomethacin (an inhibitor of the MRP family that includes MRP2) has been shown to change, in a concentration-dependent manner, the direction of membrane permeability to sulfasalazine in Caco-2 cells; high concentrations of indomethacin substantially reduced efflux of sulfasalazine. Efflux was not however abolished, due to the contribution of BCRP to the control of absorption. Additionally, an indomethacin-induced increase in sulfasalazine permeability through the gut wall was also shown in the rat jejunal perfusion model. The concomitant intake of indomethacin and sulfasalazine may lead to increased absorption of sulfasalazine in the small intestine, reducing its concentration in the colon, and potentially altering its therapeutic effect (Dahan & Amidon, 2010).

(a) Mutagenicity

Sulfasalazine was not mutagenic in assays for gene mutation in bacteria, including a variety of strains of Salmonella typhimurium, Escherichia coli, and Klebsiella pneumoniae, in a variety of protocols, with or without metabolic activation (Voogd et al., 1980; Zeiger et al., 1988; Iatropoulos et al., 1997). In addition, treatment with sulfasalazine did not result in an increase in mutations conferring 6-thioguanine resistance in mouse lymphoma L5178Y cells, with or without metabolic activation (Iatropoulos et al., 1997).

(b) Chromosomal damage

Mackay et al. (1989) reported positive results in a test for induction of sister chromatid exchange in cultured human lymphocytes treated with sulfasalazine at a concentration of 20–160 μg/mL in the absence of metabolic activation. In contrast, Bishop et al. (1990) observed no increase in the frequency of sister chromatid exchange in Chinese hamster ovary cells treated with sulfasalazine at up to 1000 μg/mL, with or without metabolic activation. An increase in the formation of micronuclei in cultured human lymphocytes after treatment with sulfasalazine (effective concentration range, 40–160 μg/mL) in the absence of metabolic activation was reported by Mackay et al. (1989). No significant increases in the frequency of chromosomal aberration were observed in cultured Chinese hamster ovary cells (Bishop et al., 1990), or cultured human lymphocytes (Iatropoulos et al., 1997), treated with sulfasalazine (concentration, up to 1000 or 100 μg/mL, respectively). Thus the results of tests for chromosomal damage in vitro after treatment with sulfasalazine were generally negative, although sporadic positive results were reported.

In vivo, consistent with results reported in assays in vitro, no increases in the frequency of chromosomal aberration were observed in male mice or male and female rats treated with sulfasalazine by gavage at doses of up to 4000 mg/kg bw per day (Bishop et al., 1990; Iatropoulos et al., 1997; NTP, 1997a).

The results of assays for micronucleus formation in male and female mice treated with sulfasalazine were uniformly positive when multiple treatments (at least two) were employed (Bishop et al., 1990; Witt et al., 1992a, NTP, 1997a). Further investigation of the nature of the induced micronuclei revealed that the majority were kinetochore-positive, suggesting that the micronuclei contained whole chromosomes rather than fragments, and were primarily due to aneuploidy events rather than chromosome breakage (Witt et al., 1992a). This observation was consistent with the negative results in assays for chromosomal aberration with sulfasalazine in vitro and in vivo (Mitelman et al., 1980; Bishop et al., 1990; Iatropoulos et al., 1997; NTP, 1997a). The negative results of one test in male mice given a single dose of sulfasalazine at 4000 mg/kg bw underscored the need for multiple treatments to induce an observable increase in micronucleus formation (NTP, 1997a).

In addition to the necessity for multiple treatments, sulfasalazine may also demonstrate selective activity in mice; the results of a study on micronucleus formation in bone marrow of male rats given three doses of sulfasalazine (highest dose, 3000 mg/kg bw) were judged to be equivocal (NTP, 1997a); in this assay, an initial trial gave a positive response at the highest dose of 2700 mg/kg bw, but a second trial, with a highest dose of 3000 mg/kg bw, gave negative results. This apparent preferential activity in mice was consistent with the observation that mice have a greater systemic exposure than rats to sulfapyridine, the active moiety, after administration of similar doses (Zheng et al., 1993).

In one study, no evidence for genotoxicity was obtained for sulfasalazine when tested for the induction of micronuclei in mouse bone marrow, with or without pretreatment with folate. Likewise, no evidence for formation of DNA adducts was detected by ³²P-postlabelling in rat and mouse liver and urinary bladder (Iatropoulos et al., 1997). [The nuclease P1 enrichment procedure was used in the ³²P-postlabelling method. Assuming that N-hydroxylation of sulfapyridine occurred in vivo, adducts derived from this metabolite would probably be lost.]

(a) 5-ASA

5-ASA has not shown activity in any assay for genotoxicity in vitro or in vivo. It does not induce mutations in any of a variety of Salmonella typhimurium strains, with or without metabolic activation or in Klebsiella pneumoniae in the absence of metabolic activation (Voogd et al., 1980). No induction of sister chromatid exchange, micronucleus formation, or chromosomal aberration has been reported in human lymphocytes or Chinese hamster ovary cells in vitro (Mackay et al., 1989; Witt et al., 1992b). In vivo, no increase in the frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow of male mice treated with 5-ASA (dose range, 125–250 mg/kg bw per day for 3 days) by intraperitoneal injection (Witt et al., 1992b).

(b) Sulfapyridine

Sulfapyridine has been reported to induce sister chromatid exchange in Chinese hamster ovary cells and cultured human lymphocytes in the absence of metabolic activation (Mackay et al., 1989; Witt et al., 1992b); no increase in the frequency of sister chromatid exchange was noted in the presence of metabolic activation in Chinese hamster ovary cells (Witt et al., 1992b). Sulfapyridine did not induce chromosomal aberration in Chinese hamster ovary cells, with or without metabolic activation (Witt et al., 1992b). Mackay et al. (1989) reported that sulfapyridine did not induce micronucleus formation in cultured human lymphocytes in the absence of metabolic activation, at concentrations that reached 400 µg/mL. Sulfapyridine induced a strong, dose-related increase in the frequency of micronucleated polychromatic erythrocytes when administered either as multiple intraperitoneal injections (Witt et al., 1992b) or by gavage (Witt et al., 1992a). As with sulfasalazine, the majority of micronucleated erythrocytes induced by sulfapyridine in mice were shown to contain kinetochores (Witt et al., 1992a), implying that sulfapyridine-induced micronucleus formation resulted from failure of mitotic chromosomal segregation, rather than chromosome breakage.

(c) Metabolites of 5-ASA and sulfapyridine

Mackay et al. (1989) also tested four acetylated and/or hydroxylated metabolites of sulfapyridine and 5-ASA for their ability to induce sister chromatid exchange and micronucleus formation in cultured human lymphocytes. N⁴-acetylsulfapyridine was capable of inducing both sister chromatid exchange and micronucleus formation, while N⁴-acetyl-5′-hydroxysulfapyridine only induced sister chromatid exchange. 5′-Hydroxysulfapyridine and N⁴-acetyl-5-aminosalicylic acid did not induce either sister chromatid exchange or micronucleus formation at the concentrations tested.