Targeted adsorption of molecules in the colon with the novel adsorbent-based medicinal product, DAV132: A proof of concept study in healthy subjects.

During antibiotic treatments, active residuals reaching the colon profoundly affect the bacterial flora resulting in the emergence of resistance. To prevent these effects, we developed an enteric-coated formulated activated-charcoal based product, DAV132, meant to deliver its adsorbent to the ileum and neutralize antibiotic residues in the proximal colon. In a randomized, control, crossover study, the plasma pharmacokinetics of the probe drugs amoxicillin (500 mg) absorbed in the proximal intestine, and sulfapyridine (25 mg) metabolized from sulfasalazine in the cecum and rapidly absorbed, were compared after a single administration in 18 healthy subjects who had received DAV132, uncoated formulated activated charcoal (FAC) or water 16 and 8 hours before, concomitantly with the probe drugs, and 8 hours thereafter. The AUC0-96 h of amoxicillin was reduced by more than 70% when it was taken with FAC, but bioequivalent when it was taken with water or DAV132. By contrast, the AUC0-96 h of sulfapyridine was reduced by more than 90% when administered with either FAC or DAV132 in comparison with water. The results show that DAV132 can selectively adsorb drug compounds in the proximal colon, without interfering with drug absorption in the proximal small intestine, thereby constituting a proof of concept that DAV132 actually functions in humans.

The influence of probiotic treatment on sulfasalazine metabolism in rat.

Probiotics are live microorganisms claimed to exert beneficial effects on the host. This study investigated their effect on the metabolism and pharmacokinetics of sulfasalazine (SSZ), a drug whose efficacy depends on metabolism by azoreductase (AR) in the gut microbiota to sulfapyridine (SP) and 5-acetylsalicylic acid (5-ASA). The probiotic strains Lactobacillus acidophilus L10, Bifidobacterium lactis B94 and Streptococcus salivarius K12 possessed AR activity and a corresponding ability to metabolize SSZ. Treatment of male Wistar rats (n = 5) with oral 2 g doses of a mixture of the three probiotics (total dose 1.8 × 109 cfu) every 12 h for 3 days resulted in a significant increase (p < 0.05) in AR activity in ex vivo colon contents with a corresponding increase in SSZ metabolism. Similar probiotic treatment of male Wistar rats (n = 8) followed by an oral 100 mg/kg dose of SSZ produced high plasma levels of SP, but pharmacokinetic parameters of SSZ and SP were not significantly different from control rats given SSZ. These results indicate that probiotic strains possess AR activity and can metabolize SSZ. Treatment with probiotics increases AR activity in the gut microbiota but has no effect on plasma levels of SSZ and SP following a subsequent oral dose of SSZ.

Using pharmacokinetic modeling to determine the effect of drug and food on gastrointestinal transit in dogs.

INTRODUCTION: The gastrointestinal (GI) tract is one of the target organs of adverse drug effects in different phases of drug development. This study aimed to investigate the feasibility of population pharmacokinetic modeling to quantify the rate of gastric emptying (GE) and small intestinal transit time (SITT) in response to drugs that affect GI motility in fed and fasted dogs. Paracetamol and sulfapyridine (sulfasalazine metabolite) pharmacokinetics were used as markers for GE and SITT, respectively. METHODS: In two separate studies, under fed and fasted conditions, six male beagle dogs received a 15min intravenous infusion of vehicle, atropine (0.06mg/kg) or erythromycin (1mg/kg) followed by an intragastric administration of a mixture of paracetamol (24mg/kg) and sulfasalazine (20mg/kg). Food was given just before or at 6h after drug administration in the fed and fasted study, respectively. Blood samples were collected for analysis of paracetamol and sulfapyridine in plasma. Population pharmacokinetic analysis of paracetamol and sulfapyridine in plasma was used to determine the rate of GE and SITT. RESULTS: The quantitative parameter estimates demonstrated a detailed and significant influence of atropine, erythromycin and food on GE and SITT. Compared to fasted conditions food intake delayed GE in pharmacologically treated dogs and SITT was shortened after treatment with vehicle or erythromycin. Atropine substantially delayed GE in fed and fasted conditions but the effect on SITT was evident only under fed condition. Erythromycin, in contrast, increased GE only in fasted conditions, and generally delayed SITT. DISCUSSION: Population pharmacokinetic modeling of paracetamol and sulfapyridine provides a suitable preclinical non-invasive experimental method for quantification of drug- and food-induced changes in the rate of GE and SITT in conscious beagle dogs for use in safety evaluations to predict changes in GI transit and/or to explain the pharmacokinetic profile of drugs under development.

Determination of sulphasalazine and its main metabolite sulphapyridine and 5-aminosalicylic acid in human plasma by liquid chromatography/tandem mass spectrometry and its application to a pharmacokinetic study.

A simple and sensitive liquid chromatography/positive-ion electrospray ionization mass spectrometry (LC-ESI-MS/MS) method has been developed for the simultaneous determination of sulphasalazine (SASP) and its main metabolite sulphapyridine (SP) and 5-aminosalicylic acid (5-ASA) with 100 µL of human plasma using dimenhydrinate as the internal standard (I.S.). The API-3000 LC-MS/MS was operated under the multiple reaction-monitoring mode (MRM) using the electrospray ionization technique. Protein precipitation process was used to extract SASP, SP, 5-ASA and I.S. from human plasma. The total run time was 9.0 min and the elution of SASP, SP and 5-ASA was at 4.8 min, 2.5 min and 2.0 min, respectively. The separation was achieved with a mobile phase consisting of 0.2% formic acid, 2 mM ammonium acetate in water (mobile phase A) and 0.2% formic acid, 2 mM ammonium acetate in methanol (mobile phase B) by using gradient elution on a XBP Phenyl column (100 mm × 2.1 mm, 5 µm). The developed method was validated in human plasma with a lower limit of quantitation of 10 ng/mL for SASP, SP and 5-ASA, respectively. A linear response function was established for the range of concentrations 10-10,000 ng/mL (r>0.99) for SASP and 10-1000 ng/mL (r>0.99) for SP and 5-ASA. The intra and inter-day precision values for SASP, SP and 5-ASA met the acceptance as per FDA guidelines. SASP, SP and 5-ASA were stable during stability studies, i.e., long term, auto-sampler and freeze/thaw cycles. The method was successfully applied for the evaluation of pharmacokinetics of SASP, SP and 5-ASA after single oral doses of 250 mg SASP to 10 healthy volunteers.

Phenotyping with sulfasalazine - time dependence and relation to NAT2 pharmacogenetics.

OBJECTIVE: N-acetyltransferase 2 (NAT2) genotype-phenotype relation with sulfasalazine as probe drug by means of detailed genotype analysis and kinetic data evaluation. BACKGROUND: Though phenotype analysis of sulfasalazine metabolism has been described before, genotype investigations in this regard are scarce. The influence of different single point mutations on the metabolism of the sulfasalazine metabolite sulfapyridine (SP) should give more insight into the functionality of different alleles especially with those still under discussion. METHODS: In two bioavailability studies performed under comparable conditions with 24 healthy subjects of both genders equally distributed, plasma levels of SP and acetylsulfapyridine (Ac-SP) were determined after oral intake of enteric coated formulations of sulfasalazine (500 mg and 1,000 mg, respectively). The resulting metabolic ratios were calculated. NAT2 genotype was analyzed in parallel for all subjects deducing haplotype set as well as putative functional phenotype as (homozygous or heterozygous) rapid acetylator (RA) or slow acetylator (SA) and correlated with the PK results. RESULTS AND DISCUSSION: RA genotype in the overall study population was seen with 45.5% (including 6.8% homozygous wildtype *4/*4) and SA genotype with 54.5%. Compared to RA genotype, apparent terminal elimination half-life of SP as well as of Ac-SP was prolonged in the SA genotype population, C(max) and AUC values of SP were higher whereas average C(max) value of Ac-SP was lower (with AUC only some tendency to lower values). In general, phenotype-genotype correlation was good with only few exceptions. Strongest functional effect on enzyme activity was noticed in slow acetylators carrying the 341T > C mutation, followed by 590G > A mutation whereas the influence of 857G > A was considerably less pronounced. Homozygous 803A > G mutation (lysine > arginine shift) did not reveal enzyme activity reduction.

Effects of NAT2 polymorphism on SASP pharmacokinetics in Chinese population.

BACKGROUND: Sulfasalazine (SASP) pharmacologic actions are widely applied in clinical therapy. The role of N-Acetyltransferase 2 (NAT2) in the pharmacokinetics of SASP and its metabolites has not been clarified. We investigated the effects of genetic polymorphism of NAT2 on pharmacokinetic profiles of SASP and its two metabolites, sulfapyridine (SP) and N-acetylsufapyridine (AcSP). METHODS: Eighteen subjects were recruited and divided into 3 groups by NAT2 genotype: wild type (w/w), heterozygous variant (w/m), homozygous variant (m/m). After taking 1000mg SASP tablets, the plasma concentrations of SASP, SP and AcSP were measured with HPLC method and pharmacokinetic parameters were calculated by using the computing program 3P97. RESULTS: The AUC(0)(-)(72) and Cmax of SP in m/m subjects were significantly higher than those in w/m and w/w subjects, with the values of 172.57+/-49.42, 103.38+/-39.85, 71.37+/-17.52mg h/l, and 9.65+/-2.34, 6.10+/-1.79, 4.55+/-1.38mg/l, respectively. In contrast, the AUC(0)(-)(72) of AcSP was significantly lower in m/m subjects. The Cmax of AcSP in w/w, w/m and m/m subjects was 12.67+/-3.32, 9.07+/-2.29 and 4.22+/-0.93mg/l, respectively, with significant differences among groups. However, there was no significant difference in any pharmacokinetic parameter of SASP among groups. CONCLUSION: Different NAT2 genotypes, leading to functional heterogeneity of NAT2, may affect pharmacokinetics of SP and AcSP. Therefore, genotyping NAT2 gene before administration would be important in SASP therapy.

Evaluation of in vivo dissolution behavior and GI transit of griseofulvin, a BCS class II drug.

Mean plasma concentration-time profile of griseofulvin, a BCS class II drug, orally administered as powders into rats, was predicted based on GITA model. However, it was very difficult to predict the individual plasma profile because of large inter-individual difference. As the absorption of griseofulvin would be rate-limited by the dissolution process, we tried to analyze the in vivo dissolution kinetics of griseofulvin by focusing on gastric emptying and intestinal transit as physiological factors influencing the in vivo dissolution kinetics. After oral administration of griseofulvin, theophylline and sulfasalazine into rats, gastric emptying and intestinal transit were simultaneously estimated by analyzing the absorption kinetics of theophylline and observing the appearance of sulfapyridine in plasma, respectively. Gastric emptying kinetics was not significantly correlated with absorption or dissolution behavior of griseofulvin. On the other hand, the cecum-arriving time reflecting the intestinal transit was significantly correlated with both AUC and total dissolved amount of griseofulvin. T(max) of griseofulvin also increased with the increase of cecum-arriving time. These results clearly indicate that the longer residence time could lead to the higher dissolution and absorption of griseofulvin and that the variance of intestinal transit could be responsible for the inter-individual difference of the in vivo absorption behavior.

Kinetic phenotypic diagnosis of N-acetylation polymorphism in patients based on ratio of urinary metabolites of salicylazosulfapyridine.

We found that N-acetylation polymorphism can be evaluated from the disposition kinetics of sulfapyridine (SP) and 5-aminosalicylic acid (5-ASA) and their acetylated metabolites generated by N-acetyltransferase (NAT2) after oral administration of salicylazosulfapyridine (SASP). In 126 Japanese subjects, the homozygote of NAT2*4 was the most frequent (40%), followed by heterozygotes of NAT2*4 and mutant genes (28% NAT2*4/*6A, 15% NAT2*4/*7B, and 2% NAT2*4/*5B). Combinations of mutant genes accounted for 16%. When the relationship between the molar ratio of N-acetyl-SP (Ac-SP)/SP or N-acetyl-5-ASA(Ac-5-ASA)/5-ASA in serum and five genotypes of polymorphic NAT2* was examined in patients who received multiple doses of SASP, the molar ratios of Ac-SP/SP, rather than Ac-5-ASA/5-ASA tended to decrease according to the classification of genotype. We calculated the pharmacokinetic parameters in healthy subjects with various genotypes of polymorphic NAT2* after a single p.o. administration of SASP, according to a model of the SP metabolic pathways. The molar ratios of Ac-SP/SP in serum and urine were simulated using these parameters, and the molar ratio of Ac-SP/SP in urine at 4 days after the first administration could be categorized into ranges that were specific to various NAT2* genotypes. Thus, we were able to predict the N-acetylation polymorphic genotypes of patients by measuring the molar ratio of Ac-SP/SP in urine, after administration of SASP.

Small bowel motility and colonic transit are altered in dogs with moderate renal failure.

Although gastrointestinal complications are common in patients with renal disease, the effects of renal dysfunction on bowel motility and gut transit times are not well known. We assessed gastrointestinal electromyographic activity, gastric emptying rate, orocolonic transit time, oroanal transit time, and xylose absorption before and after surgically inducing a 66% decrease in glomerular filtration rate in dogs. Moderate renal failure induced no gross or microscopic gastrointestinal lesions but caused a 16-42% increase in gastrointestinal motility indexes. We found a 24% decrease in the propagation velocity of the myoelectrical migrating complex in the duodenojejunal segment, a 30% decrease in phase I duration in duodenal and jejunal regions, a 20% increase in the total irregular electrical activity of the small intestine, and a 22% increase in duration of the meal response in the duodenum and jejunum. Renal failure did not change xylose absorption, gastric emptying rate, and orocolonic transit time but decreased colonic transit time by 38%. The mean weight of feces was increased. These results indicate that moderate renal failure alters duodenojejunal motility and decreases colonic transit time.

Dapsone and sulfapyridine.

Dapsone and sulfapyridine are structurally related compounds with anti-microbial and anti-inflammatory effects. Dapsone remains the most important drug for leprosy and is useful in the prophylaxis of Pneumocystis pneumonia in patients with HIV disease. The medical treatment of choice for dermatitis herpetiformis is dapsone; and sulfapyridine also can be used for those patients who are intolerant of dapsone. Other neutrophilic disorders also may respond to these drugs. Toxic side effects of both dapsone and sulfapyridine are mediated through the hydroxylamine metabolite. These include hemolysis, methemoglobinemia, and agranulocytosis. Careful monitoring for possible adverse reactions includes frequently performing complete blood counts and regular blood chemistry profile determinations.

Mechanisms of transport and structure-permeability relationship of sulfasalazine and its analogs in Caco-2 cell monolayers.

PURPOSE: To investigate the mechanisms involved in transport of sulfasalazine in Caco-2 cells. METHODS: Permeability coefficients of sulfasalazine and its analogs across Caco-2 cell monolayers were measured as a function of direction of transport, energy and concentration dependence, and in the presence of inhibitors of various cellular efflux pumps and transporters. RESULTS: Permeability coefficients of sulfasalazine across Caco-2 cell monolayers were approximately 342-, 261-, and 176-fold higher from basolateral to apical direction (BL-->AP) than from apical to basolateral direction (AP-->BL) at 100, 200, and 500 microM, respectively. Carrier permeability coefficient, non-saturable membrane permeability coefficient, and Michaelis constant were estimated to be 1.4x10(-5) cm/s, 1.9x10(-8) cm/s, and 369 microM, respectively. The efflux of sulfasalazine was completely blocked at 4 degrees C and in the presence of an uncoupler of oxidative phosphorylation. Using cellular efflux inhibitors, the permeability of sulfasalazine was shown to depend on multidrug resistance-associated protein and anion sensitive transport mechanisms. Structure-permeability studies showed that the affinity of sulfasalazine for the cellular efflux pumps and transporters in Caco-2 cells depended strongly on the carboxylic acid functional group. CONCLUSIONS: The permeability of sulfasalazine across Caco-2 cell monolayer is very low due to its strong interaction with multiple cellular efflux pumps and transporters. This may partially explain its low absorption in vivo.

Mechanistic studies on genotoxicity and carcinogenicity of salicylazosulfapyridine an anti-inflammatory medicine.

Salicylazosulfapyridine (SASP), which has been in clinical use for over 50 years, was reported by the National Toxicology Program to increase rat (F344 strain) urinary bladder and mouse (B6C3F1 hybrid) liver tumours under ad libitum (AL) feeding conditions, while under a feed restriction (FR) regimen, these tumours were not increased. The present investigations were undertaken to assess the implications of these results for the safety of SASP in humans. SASP and its 2 major metabolites, 5-aminosalicylic acid (ASA) and sulfapyridine (SP) were tested for in vivo induction of micronuclei in mouse bone marrow cells with or without prefolic treatment and for in vivo formation of DNA adducts in rat and mouse liver and urinary bladder. None exhibited mutagenicity or DNA reactivity. SASP and SP have induced sister chromatid exchanges and micronuclei (MN) in cultured human lymphocytes in the absence of liver activation enzymes and in B6C3F1 mice (but not in rats) MN in bone marrow and peripheral RBC. Treatment with folate reduces the frequency of MN. Perhaps the short (28 days) RBC lifespan in mouse underlies the sensitivity of this species. Thus, SASP without folate supplementation is an aneuploidogen. In a 2-year study in AL fed SASP-treated (high dose 337.5 mg/kg) rats, urinary pH was increased and urinary specific gravity was reduced at 60 weeks. At the end, this SASP group showed urothelial hyperplasia and papillomas in the urinary bladders of male rats primarily. In the FR high dose SASP group, the hyperplasia was reduced from 82% to 14%. At the end of 2 years, the incidence of multi-organ leukemia was reduced in both AL and FR high dose SASP groups. Thus, SASP caused intraluminal bladder changes in the rat (especially males) consisting of chronic urothelial stimulation, concretions, hyperplasia which resulted in neoplasia. In the mouse, because of species differences in liver ratios (mouse > rat) and, increasing (3 times higher) liver perfusion in the mouse, compared to the rat, there was hepatocellular toxicity and resulting preneoplasia and neoplasia within 2 years. These findings occurred in all AL SASP groups (flat curve without dose response); but were reduced under FR conditions. In this species, the multiorgan lymphoma incidence was reduced in both AL and FR high dose SASP groups. Thus, SASP and its major metabolites are not genotoxic. Folate deficiency associated with SASP administration is probably responsible for aneuploidy in lymphocytes and erythrocytes. SASP does not induce neoplasia directly in either livers, urinary bladders or other organs. Accordingly, SASP is judged to pose no carcinogenic risk to humans.

A comparison of orocaecal transit times assessed by the breath hydrogen test and the sulphasalazine/sulphapyridine method in healthy beagle dogs.

Orocaecal transit time (OCTT) was assessed in six healthy beagles by means of the breath hydrogen test (BH2T) and the sulphasalazine/sulphapyridine method (SLZ) after the administration of a test meal of canned food mixed with sulphasalazine. Orocaecal transit time was defined as the time taken from the oral administration of the test meal to the time when the first portion of the meal reached the colon. In five of the dogs the OCTTs assessed by the BH2T were shorter than those measured by the SLZ method by 30, 15, 45, 30 and 45 minutes. However, the median OCTT assessed by the BH2T (135 minutes, range 120 to 195 minutes) was not significantly different from that measured by the SLZ (180 minutes, range 150 to 210 minutes) and was highly correlated with it (r = 0.94, P = 0.016). The sixth dog maintained baseline hydrogen and plasma sulphapyridine readings throughout the monitoring period and the OCTT could not be measured.

The influence of multiple oral doses of zileuton on the steady-state pharmacokinetics of sulfasalazine and its metabolites, sulfapyridine and N-acetylsulfapyridine.

The effects of zileuton (Abbott-64077) on the pharmacokinetics of sulfasalazine (SASP) and its metabolites, sulfapyridine (SP) and N-acetylsulfapyridine (ASP), were studied in a randomised double-blind placebo-controlled study enrolling 14 healthy male volunteers. All subjects received SASP 1 g every 12 hours for 8 days and zileuton 800mg or placebo administered twice daily from day 4 to day 8 inclusive. Coadministration of zileuton did not significantly affect the area under the plasma concentration-time curve, the maximum (Cmax) or minimum (Cmin) plasma concentration and the time to Cmax of SASP, SP or ASP. Likewise, zileuton did not modify the terminal elimination half-life of SASP. It is concluded that coadministration of zileuton 1.6 g/day has no significant effects on the pharmacokinetics of SASP 2 g/day or its metabolites, SP and ASP.

Disposition kinetics and dosage regimen of sulfapyridine in buffalo (Bubalus bubalis).

The disposition kinetics and dosage regimen of sulfapyridine were studied in buffalo calves following a single intravenous dose of 100 mg/kg. Distribution half-life (t1/2 alpha) elimination half-life (t1/2 beta) and Vd (area) was 0.181 +/- 0.008 h, 13.4 +/- 0.52 h and 0.59 +/- 0.03 L kg-1, respectively. Total body clearance, which represents the sum of all clearance processes, and tissue/plasma (T/P) ratio were calculated to be 31.1 +/- 2.28 ml kg-1 h-1 and 2.25 +/- 0.09, respectively. A satisfactory intravenous dosage regimen of sulfapyridine in buffalo would be 104 mg/kg followed by 75 mg/kg at 24 h intervals.

Novel oxidative pathways of sulphapyridine and sulphadiazine by the turtle Pseudemys scripta elegans.

The sulphonamides sulphapyridine and sulphadiazine show novel hydroxy metabolites in the turtle Pseudemys scripta elegans. In the excreta of the turtles the monohydroxy metabolites 4-hydroxy- and 5-hydroxysulphapyridine and the dihydroxy metabolite 4,5-dihydroxysulphapyridine were detected. Of sulphadiazine only dihydroxy metabolites 4,5- and 4,6-dihydroxysulphadiazine were detected. About 70-90% of the dose of sulphapyridine was recovered, while this figure varied between 48 and 69% for sulphadiazine.

Comparison of the metabolism of four sulphonamides between humans and pigs.

Pigs are unable to form N1-glucuronides of sulphadimethoxine and sulphamethomidine, while humans are able to do so. Pigs and humans are able to oxidise sulphapyridine and form the O-glucuronide. The double conjugate N4-acetylsulphapyridine-O-glucuronide is formed in humans but not in pigs. Sulphadiazine is mainly acetylated in both humans and pigs. A hypothesis about N1-glucuronidation is presented.