Ginkgo biloba leaf extract suppresses intestinal human breast cancer resistance protein expression in mice: Correlation with gut microbiota.

In this study, we investigated the effects of treatment with Gingko biloba leaf extract (GLE) on intestinal transporter expression and gut microbiota composition in mice and the correlation between intestinal transporter expression and gut microbiota composition in mice. When GLE was orally administered to mice, intestinal BCRP expression was significantly suppressed. Pharmacokinetic studies showed that the maximum plasma concentration and area under the curve values of sulfasalazine were increased more than twice by treatment with GLE compared with those in the control group. GLE treatment significantly decreased the populations of Proteobacteria and Deferribacteres at the phylum level. Correlation analysis showed that BCRP expression was positively or negatively correlated with the composition of gut bacteria. In Caco-2 cells, GLE treatment did not affect BCRP expression, but treatment with the lysates of GLE-treated mouse feces significantly suppressed BCRP expression. These findings demonstrate that the suppression of intestinal BCRP expression following GLE treatment may occur through modulation of the gut microbiota composition. Thus, the present study suggests that modulation of gut microbiota composition may cause drug transporter-mediated herb-drug interactions.

Mucoadhesive chitosan hydrogels as rectal drug delivery vessels to treat ulcerative colitis.

Mucoadhesive drug delivery systems stick to mucosal tissues and prolong the local retention time of drugs. Since the colon is covered by a mucosal layer, mucoadhesive rectal formulations may improve treatment of such diseases as hypertension or colon cancer. Ulcerative colitis (UC) is an inflammatory bowel disease characterized by chronic inflammation of the colonic mucosa. It is commonly treated with sulfasalazine (SSZ), which is metabolized by the intestinal flora into the therapeutic 5-aminosalicylic acid (5-ASA) and a toxic by-product sulfapyridine (SP). SSZ can be administered orally or rectally. The latter route avoids unintended absorption of the drug or its degradation products in the upper gastrointestinal tract, but often fails due to limited retention time. Here, we propose a mucoadhesive hydrogel to improve the efficacy of rectal SSZ administration. The gel is made of catechol modified-chitosan (Cat-CS) crosslinked by genipin. After loading the gel with SSZ, we evaluated its efficacy in a mouse model of UC. Compared to oral SSZ treatment, rectal SSZ/Cat-CS delivery was more therapeutic, showed equivalent histological scores, and induced a lower plasma concentration of the potentially toxic SP by-product. These results show SSZ/Cat-CS rectal hydrogels are more effective and safer formulations for UC treatment than oral SSZ. STATEMENT OF SIGNIFICANCE: Ulcerative colitis affects the colon by causing chronic inflammation on the mucosa. One of the most common drugs to treat mild to moderate UC is sulfasalazine, which can be administrated both orally and rectally. Rectal formulations are preferable, since their therapeutic effect happens topically, and they prevent side effects related to absorption of the drug in the small intestine. However, the efficacy of rectal sulfasalazine formulations is decreased by their limited colon residence time. Here we propose a chitosan-catechol mucoadhesive gel that allows delivering sulfasalazine more effectively and safely than oral administration. Our results bring new insights into the field of mussel-inspired catechol hydrogels, showing their potential as drug delivery systems to treat a widespread disease such as ulcerative colitis.

Circulating intestine-derived exosomal miR-328 in plasma, a possible biomarker for estimating BCRP function in the human intestines.

A variant in the breast cancer resistance protein (BCRP) gene, 421C> A is a useful biomarker for describing large inter-individual differences in the pharmacokinetics of sulfasalazine (SASP), a BCRP substrate. However, large intra-genotypic variability still exists in spite of the incorporation of this variant into the pharmacokinetics of SASP. Since miR-328 negatively regulates BCRP expression in human tissues, we hypothesized that exosomal miR-328 in plasma, which leaks from the intestines, is a possible biomarker for estimating BCRP activity in the human intestines. We established an immunoprecipitation-based quantitative method for circulating intestine-derived miR-328 in plasma using an anti-glycoprotein A33 antibody. A clinical study was conducted with an open-label, non-randomized, and single-arm design involving 33 healthy participants. Intestine-derived exosomal miR-328 levels positively correlated (P < 0.05) with SASP AUC0-48, suggesting that subjects with high miR-328 levels have low intestinal BCRP activity, resulting in the high AUC of SASP. Circulating intestine-derived exosomal miR-328 in plasma has potential as a possible biomarker for estimating BCRP function in the human intestines.

Evaluation of the usefulness of breast cancer resistance protein (BCRP) knockout mice and BCRP inhibitor-treated monkeys to estimate the clinical impact of BCRP modulation on the pharmacokinetics of BCRP substrates.

PURPOSE: To evaluate whether the impact of functional modulation of the breast cancer resistance protein (BCRP, ABCG2 421C>A) on human pharmacokinetics after oral administration is predictable using Bcrp knockout mice and cynomolgus monkeys pretreated with a BCRP inhibitor, elacridar. METHODS: The correlation of the changes of the area under the plasma concentration-time curve (AUC) caused by ABCG2 421C>A with those caused by the Bcrp knockout in mice, or BCRP inhibition in monkeys, was investigated using well-known BCRP substrates (rosuvastatin, pitavastatin, fluvastatin, and sulfasalazine). RESULTS: In mice, the bioavailability changes, which corrected the effect of systemic clearance by Bcrp knockout, correlated well with the AUC changes in humans, whereas the correlation was weak when AUC changes were directly compared. In monkeys, the AUC changes pretreated with elacridar resulted in a good estimation of those in humans within approximately 2-fold ranges. CONCLUSIONS: This study suggests that pharmacokinetics studies that use the correction of the bioavailability changes in Bcrp knockout mice are effective for estimating clinical AUC changes in ABCG2 421C>A variants for BCRP substrate drugs and those studies in monkeys that use a BCRP inhibitor serve for the assessment of BCRP impact on the gastrointestinal absorption in a non-rodent model.

Characterization of SAGE Mdr1a (P-gp), Bcrp, and Mrp2 knockout rats using loperamide, paclitaxel, sulfasalazine, and carboxydichlorofluorescein pharmacokinetics.

Transporter gene knockout rats are practically advantageous over murine models for pharmacokinetic and excretion studies, but their phenotypic characterization is lacking. At present, relevant aspects of pharmacokinetics, metabolism, distribution, and excretion of transporter probes [P-glycoprotein (P-gp): loperamide and paclitaxel; breast cancer resistance protein (Bcrp): sulfasalazine; and multidrug resistance-associated protein 2 (Mrp2): carboxydichlorofluorescein] were studied systematically across SAGE P-gp, Bcrp, and Mrp2 knockout rats. In Mdr1a knockout rats, loperamide and paclitaxel oral bioavailability was 2- and 4-fold increased, respectively, whereas clearance was significantly reduced (40-42%), consistent with the expected 10- to 20-fold reduction in paclitaxel excretion. N-Desmethyl-loperamide pharmacokinetics were not altered in any of the three knockouts after oral loperamide. In rats lacking P-gp, paclitaxel brain partitioning was significantly increased (4-fold). This finding is consistent with observations of loperamide central nervous system opioid pharmacology in Mdr1a knockout rats. Sulfasalazine oral bioavailability was markedly increased 21-fold in Bcrp knockouts and, as expected, was also 2- to 3-fold higher in P-gp and Mrp2 knockout rats. The sulfapyridine metabolite/parent ratio was decreased 10-fold in rats lacking Bcrp after oral, but not intravenous, sulfasalazine administration. Carboxydichlorofluorescein biliary excretion was obliterated in Mrp2 knockout rats, resulting in 25% decreased systemic clearance and 35% increased half-life. In contrast, carboxydichlorofluorescein renal clearance was not impaired in the absence of Mrp2, Bcrp, or P-gp. In conclusion, SAGE Mdr1a, Bcrp, and Mrp2 knockout rats generally demonstrated the expected phenotypes with respect to alterations in pharmacokinetics of relevant probe substrates; therefore, these knockout rats can be used as an alternative to murine models whenever a larger species is practically advantageous or more relevant to the drug discovery/development program.

Pharmacokinetic interaction study of sulphasalazine in healthy subjects and the impact of curcumin as an in vivo inhibitor of BCRP.

BACKGROUND AND PURPOSE An ATP-binding cassette (ABC) transporter, breast cancer resistance protein (BCRP)/ABCG2, limits oral bioavailability of sulphasalazine. Here we examined the effect of curcumin, the principal curcuminoid of turmeric, on oral bioavailability of microdoses and therapeutic doses of sulphasalazine in humans. EXPERIMENTAL APPROACH Effects of curcumin were measured on the ATP-dependent sulphasalazine uptake by hBCRP-expressing membrane vesicles and on oral bioavailability of sulphasalazine in wild-type and Bcrp(-/-) mice. Eight healthy Japanese subjects received an oral dose of sulphasalazine suspension (100 µg) or tablets (2 g) alone or after curcumin tablets (2 g). Uptake of sulphasalazine was studied in HEK293 cells transfected with the influx transporter (OATP)2B1. KEY RESULTS Curcumin was a potent hBCRP inhibitor in vitro (K(i) 0.70 ± 0.41 µM). Curcumin increased the area under the curve (AUC)(0-8) of plasma sulphasalazine eightfold in wild-type mice at 300 and 400 mg·kg(-1), but not in Bcrp(-/-) mice. Curcumin increased AUC(0-24) of plasma sulphasalazine 2.0-fold at microdoses and 3.2-fold at therapeutic doses in humans. Non-linearity of the dose-exposure relationship was observed between microdoses and therapeutic doses of sulphasalazine. Sulphasalazine was a substrate for OATP2B1 (K(m) 1.7 ± 0.3 µM). Its linear index (dose/K(m)) at the therapeutic dose was high and may saturate OATP2B1. CONCLUSIONS AND IMPLICATIONS Curcumin can be used to investigate effects of BCRP on oral bioavailability of drugs in humans. Besides the limited dissolution, OATP2B1 saturation is a possible mechanism underlying non-linearity in the dose-exposure relationship of sulphasalazine.

Curcumin inhibits the activity of ABCG2/BCRP1, a multidrug resistance-linked ABC drug transporter in mice.

PURPOSE: To evaluate the in vivo efficacy of curcumin as an inhibitor of the multidrug-resistance-linked ATP Binding Cassette (ABC) drug transporter, ABCG2. METHODS: Photoaffinity labeling with [125I]-iodoarylazidoprazosin was used to characterize the interaction of sulfasalazine, a substrate of the mouse ABCG2, with human ABCG2. In addition, the inhibitory effect of curcumin on ABCG2 was evaluated in brain capillaries from rats. Furthermore, the effect of curcumin on absorption of orally administered sulfasalazine in wild-type and abcg2-/- mice was also determined. RESULTS: Sulfasalazine interacted at the drug-substrate site(s) of human ABCG2. Curcumin inhibited ABCG2 activity at nanomolar concentrations at the rat blood-brain barrier in the ex vivo assay. Based on studies in wild type and abcg2-/- mice, we observed that oral curcumin increased Cmax and relative bioavailability of sulfasalazine by selectively inhibiting ABCG2 function. CONCLUSIONS: This study validates our previous in vitro results with human ABCG2 (Chearwae et al., Mol. Cancer Ther. 5:1995-2006, 2006) and provides the first in vivo evidence for the inhibition by curcumin of ABCG2-mediated efflux of sulfasalazine in mice. Based on these studies, we propose that non-toxic concentrations of curcumin may be used to enhance drug exposure when the rate-limiting step of drug absorption and/or tissue distribution is impacted by ABCG2.

Pharmacogenetic characterization of sulfasalazine disposition based on NAT2 and ABCG2 (BCRP) gene polymorphisms in humans.

The role of breast cancer resistance protein (BCRP), an efflux ABC transporter, in the pharmacokinetics of substrate drugs in humans is unknown. We investigated the impact of genetic polymorphisms of ABCG2 (421C>A) and NAT2 on the pharmacokinetics of sulfasalazine (SASP), a dual substrate, in 37 healthy volunteers, taking 2,000 mg of conventional SASP tablets. In ABCG2, SASP AUC(0-48) of C/C, C/A, and A/A subjects was 171 +/- 85, 330 +/- 194, and 592 +/- 275 microg h/ml, respectively, with significant differences among groups. In contrast, AUC(0-48) of sulfapyridine (SP) tended to be lower in subjects with the ABCG2-A allele as homozygosity. In NAT2, AUC(AcSP)/AUC(SP) was significantly higher in rapid than in intermediate and slow acetylator (SA) genotypes. We successfully described the pharmacokinetics of SASP, SP, and N -acetylsulfapyridine (AcSP) simultaneously by nonlinear mixed-effects modeling (NONMEM) analysis with regard to both gene polymorphisms. The data indicate that SASP is a candidate probe of BCRP, particularly in its role in intestinal absorption.

Pharmacokinetic-pharmacodynamic modeling of the immunomodulating agent susalimod and experimentally induced tumor necrosis factor-alpha levels in the mouse.

The main objective of this study was to explore the concentration-effect relationship between the immunomodulating agent susalimod and lipopolycaccharide (LPS)-induced elevated serum levels of the proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha). Bacterial LPS (1 mg/kg) was given i.p. along with different doses of susalimod (0, 25, 50, 100, and 200 mg/kg) to female CD-1 mice. Blood samples were drawn at different time points (15-300 min), and serum was analyzed with respect to susalimod and TNF-alpha. The concentration-effect relationship was explored by modeling the data from all dose levels simultaneously using specially written program models, i.e., a three-compartment pharmacokinetic model, including biliary excretion, and an indirect mechanistically based pharmacodynamic model. The models, which were successfully fitted to the experimental data, showed that LPS induced the TNF-alpha synthesis during approximately 70 min and that during this time course, the synthesis rate was governed by the serum phamacokinetics of susalimod. Because the results supported the assumption that the maximum inhibitory effect was equal to full inhibition of the synthesis, the in vivo potency (IC(50)) of susalimod could be estimated to 293 microM. In conclusion, susalimod decreased the LPS-induced TNF-alpha mouse serum levels in a concentration-related manner. The compound is suggested to inhibit the synthesis of TNF-alpha. The integrated pharmacokinetic-pharmacodynamic model estimated the in vivo potency of susalimod in the mouse to be 293 microM.

Massive sulfasalazine and paracetamol ingestion causing acidosis, hyperglycemia, coagulopathy, and methemoglobinemia.

CASE REPORTS: Reports of acute toxicity following sulfasalazine ingestion are rare. A case of an acute ingestion of sulfasalazine 50 g and paracetamol 50 g resulting in severe lactic acidosis, seizures, coagulopathy, hyperglycemia, ketosis, and methemoglobinemia is reported. Despite the ingestion of a large amount of paracetamol with serum paracetamol 5486 nmol/L (844 mg/L), significant hepatotoxicity did not occur. The patient recovered fully following administration of intravenous N-acetylcysteine, methylene blue, sodium bicarbonate, and supportive therapy.

Activity of melphalan in combination with the glutathione transferase inhibitor sulfasalazine.

Glutathione (GSH) transferases (GST), a family of detoxification enzyme proteins, are suggested to play an important role in tumor cell resistance to melphalan. The GST-activity inhibitor ethacrynic acid has been shown to increase the antitumor activity of melphalan in vitro as well as in vivo. In this study we determined the activity and toxicity of melphalan in combination with another GST-activity inhibitor, sulfasalazine, an agent used to treat ulcerative colitis. We entered 37 previously treated patients with advanced cancer of different histologies on sulfasalazine given at the individually calculated maximum tolerated dose (MTD) and melphalan given at doses beginning at 20 mg/m2. The main toxicity arising from this combination was nausea and vomiting, whereas increased myelosuppression was not observed. A partial response was seen in 2/4 of the ovarian cancer patients only. Plasma sulfasalazine levels varied between 2.5 and 47.1 micrograms/ml. Although reductions in GSH/GST levels were observed in peripheral mononuclear cells of certain patients following sulfasalazine treatment, there was no correlation between the extent of reduction and the plasma sulfasalazine level. A larger patient population must be studied to determine the usefulness of this combination.

Interference of circulating azathioprine but not methotrexate or sulfasalazine with measurements of interleukin-6 bioactivity.

Bioassays are currently used to measure the presence of functionally active cytokines in biological fluids. These assays may be influenced by the presence of other substances, either cytokine specific or not, in such fluids. In the present study, we analyzed whether some currently used disease-modifying antirheumatic drugs (DMARDs) could interfere with the measurements of circulating interleukin-6 (IL-6) bioactivity in the B9 hybridoma assay. When sera from healthy controls and patients treated with various DMARDs, such as azathioprine (AZA), methotrexate (MTX), intramuscular gold, and sulfasalazine (SASP), were tested in the IL-6 bioassay, an inhibitory effect was observed only with sera from patients treated with AZA. Addition of exogenous AZA, 6-mercaptopurine (6-MP), and MTX to the IL-6 bioassay resulted in a dose-dependent inhibition of the B9 cell proliferation induced by IL-6, AZA being most potent on a molar basis. Concentrations of AZA and 6-MP compatible with serum concentrations achieved in RA patients were able to inhibit the bioassay, but this was not the case for MTX. Exogenous SASP and its metabolites did not modify the IL-6-induced B9 cell proliferation. This study shows that circulating AZA (or its metabolites) exert an inhibitory effect in the IL-6 bioassay. This method is therefore not suitable to measure IL-6 concentrations in patients treated with AZA. Interference of drugs must be ruled out when bioassays are used to evaluate cytokine levels in biological fluids.

Sulfasalazine alters the character of dimethylhydrazine-induced colorectal carcinoma in rats.

The etiologic variables involved in the increased incidence of colorectal carcinoma in patients with chronic ulcerative colitis have not been defined. Sulfasalazine is the most commonly used medication in the treatment of ulcerative colitis. It is not known whether the pharmacologic treatment of ulcerative colitis alters the incidence of cancer in man, but a drug related to sulfasalazine has been shown to reduce the incidence of colorectal carcinoma in rats. In this study we examined the effect of sulfasalazine on the development of 1,2-dimethylhydrazine-induced (DMH) colorectal carcinogenesis in rats. Daily oral ingestion of sulfasalazine in doses equivalent to human daily doses resulted in serum salicylate levels in those animals that were comparable to human therapeutic serum salicylate levels. Sulfasalazine administration did not significantly effect the incidence of DMH-induced colorectal tumors. However, sulfasalazine treated animals were found to have significantly smaller tumors and to show a trend towards multiple, flat, sessile, frequently microinvasive tumors compared to the fewer, larger, exophytic tumors observed in animals treated only with DMH. These results demonstrate that in the doses given, sulfasalazine treatment alters the character of DMH-induced colorectal tumors without significantly effecting tumor incidence.

Colitis ulcerosa en niños: niveles de salazosulfapiridina y sulfapiridina durante el tratamiento / Ulcerative colitis in children: levels of salicylazosulfapyridine and sulfapyridine during the treatment

La salazosukfapiridina (SASP) tiene una acción efectiva en la colitis ulcerosa (CU), al desdoblarse en el ciego en sulfapiridina (SP) y 5 aminosalicilato (5 ASA), siendo este último el que actúa por contacto sobre la mucosa colónica. El objetivo fué conocer los niveles séricos efectivos de la droga con eventual presencia o no de efectos colaterales, y también verificar fluctuaciones de los mismos en el intervalo de dosificación. Se estudiaron 10 niños de 6 a 16 años con CU, a quienes se les suministraba SASP de 0.50 a 2 gramos por día y cada 12 horas. Los niveles de SASP y SP en sangre se realizaron a las 6 y 12 horas de administrada la droga, y en materia fecal se cuantificó en recolección de 24 horas. Estos dosajes se realizaron por el método de Hansson y Sandberg. Los niveles lasmáticos de SP fueron a las 6 horas de 6.8 a 36.3 microng/ml (x 17.7-9.0 microng/ml) y a las 12 horas de 5.7 a 25.0 microng/ml (x 14.1 -7.2 microng/ml los de SASP fueron de 2.1 a 53.4 microng/ml (x 15.5-15.4microng/ml) a las 6 horas, y 3.9 a 70.7 microng/ml (x 14.0 -20.4 microng/ml) a las 12 horas. La excreción en materia fecal de SASP durante 24 horas fué de 17.4 a 236 mg., observándo-se una correlación significativa (r: 0.88) con la dosis administrada calculada en gramos por metro cuadrado de superficie corporal en 24 horas. Los niveles de SP y SASP no se correlacionaron con la dosis. Los niveles plasmáticos de SASp y SP no tuvieron diferencia significativa entre las 6 y 12 horas. Los niveles de SP no alcanzaron los mínimos ee toxicidad, no detectándose manifestaciones clíncias ni humorales adversas. Estos hallazgos sugieren que conociendo los niveles en plasma de la droga, la dosis puede ser modificada con mayor amplitud

Sulphasalazine and sulphapyridine serum levels in children to mothers treated with sulphasalazine during pregnancy and lactation.

Serum concentrations of sulphasalazine and sulphapyridine were measured during the first week of life in 15 children whose mothers had been on sulphasalazine during pregnancy. The serum concentrations of sulphapyridine and sulphasalazine were similar in the children and their mothers at delivery. The elimination rate of the drugs in the newborn children was slow but the concentrations were not so high that a bilirubin displacing effect could be expected. In eight mothers who were breast-feeding and taking sulphasalazine, analyses were done of mothers' serum, breast-milk and serum from their children. The results showed that the amount of sulphasalazine and sulphapyridine transferred to the child via the breast-milk is negligible with regard to the risk of kernicterus. It is concluded that a woman in need of sulphasalazine treatment can continue the medication throughout pregnancy and lactation without risk of development of kernicterus in her child. Only term infants without haemolytic disease were included in the study. Thus our conclusion is not necessarily valid for the prematurely born child or the child with haemolytic disease.

Disposition of disodium azodisalicylate in healthy subjects. A possible new drug for inflammatory bowel disease.

The disposition of disodium azodisalicylate and salicylazosulfapyridine was studied in 6 healthy volunteers. After a single oral dose (1.0 g disodium azodisalicylate; 2.3 g salicylazosulfapyridine) maximum serum concentrations of the intact compound ranged between 1.4 and 6.8 mumol/L and 32 and 114 mumol/L, respectively. Mean residence time and serum half-life of disodium azodisalicylate were considerably longer than those of salicylazosulfapyridine, probably because of a higher apparent volume of distribution. Both compounds were largely split by colonic bacteria and comparable amounts of the active moiety, (acetyl-)5-aminosalicylic acid, were recovered in feces. During long-term ingestion of disodium azodisalicylate (1.0 g/day) it took 6-19 days to reach a steady state. Serum concentrations of disodium azodisalicylate at steady state were low: 2.2-8.4 mumol/L. The serum half-life was 6-10 days. It is concluded that the disposition of disodium azodisalicylate is similar, in important respects, to that of salicylazosulfapyridine. Disodium azodisalicylate, therefore, deserves therapeutic trial.

Albumin reserve for binding of bilirubin in maternal and cord serum under treatment with sulphasalazine.

Twelve pregnant patients with ulcerative colitis or Crohn's disease who were being treated with sulphasalazine (SASP) were studied together with their newborn, full-term babies. An earlier study had shown almost identical concentrations of SASP and its metabolic sulphapyridine (SP) in maternal and cord serum. As controls, blood samples were collected from 25 healthy women and their newborn infants. Sera from mothers and infants were analysed for the vacant amount of high-affinity bilirubin binding site on albumin (reserve albumin) by the MADDS method. The mean reserve albumin concentration of the SASP-treated mothers was 9% lower than that of the controls, which is probably insignificant. No difference was observed in the corresponding two groups of infants. In vitro studies showed that neither SASP nor SP in therapeutic plasma concentrations had a significant bilirubin-displacing capacity. It seems that SASP preferentially is bound to other sites on albumin than to the high-affinity binding site for bilirubin. The risk of kernicterus in the full-term newborn does not seem to be increased by treatment of the mother with sulphasalazine.

Sulphasalazine treatment during breast feeding.

The steady-state concentrations of sulphasalazine and sulphapyridine in serum and mother's milk have been measured in 12 nursing patients who were being treated with sulphasalazine. The results show that sulphasalazine passes only in negligible amounts or not at all into milk. The concentration of sulphapyridine in milk was about 40% of the serum concentration in the mother. We therefore conclude that sulphasalazine may safely be given during nursing.