Salicylamide Sulfate Research Articles

Salicylamide sulfate cell entry in perfused rat liver: A multiple-indicator dilution study

The hepatocellular entry of salicylamide sulfate conjugate, which binds to both red blood cells and albumin, was examined with the multiple-indicator dilution technique in the perfused rat liver, with medium containing both 20 red cells and 1 albumin (set A), red cells only (set B), albumin only (set C) and neither red cells nor albumin (set D). [14C]Salicylamide sulfate, 51Cr-labeled red cells (a vascular reference), 125I-labeled albumin, [3H]sucrose or [58Co] ethylenediaminetetraacetic acid (EDTA) (high and low molecular weight interstitial references, respectively) and 3H2O or D2O (a cellular reference) were injected as a bolus into the portal vein. Among all sets of outflow data, the earliest immediate vascular recoveries for the [14C] salicylamide sulfate were lower than those for the vascular reference. For sets A and B, the upslopes precessed those for the 58Co-EDTA curve, then crossed over the 58Co-EDTA curves, with lower magnitude peaks occurring at the same time as those for labeled albumin, whereas for sets C and D, in which red cells were absent, the upslopes of [14C]salicylamide sulfate lagged behind those for labeled albumin and [3H]sucrose, reaching lower magnitude peaks coincidental in time with those for labeled sucrose. The precession of the [14C]salicylamide sulfate curve over 58Co-EDTA or [3H]sucrose in the presence (sets A and B) but not in the absence (sets C and D) of red cells and the absence of precession with albumin alone (set C) suggest that a red cell effect on the upslope is more evident than that for albumin. For all experiments, the downslopes of the sulfate curve crossed over those of the labeled red cells, albumin, 58Co-EDTA and sucrose curves and then the water curve at around the peak. The downslopes of the [14C]salicylamide sulfate were similar to those for labeled water, suggesting rapid cellular influx and efflux of salicylamide sulfate. Quantitative evaluation with a barrier-limited space-variable transit-time model for rapidly equilibrating red cell and albumin binding accounted for the upslope effects on [14C]salicylamide sulfate behavior and demonstrated its relatively high liver cell permeability. Values for the unbound permeability surface area product (0.029 to 0.036 ml sec−1·gm−1) were not different, regardless of the presence or absence of red cells and albumin, and slightly exceeded values for hepatic blood flow (0.0186 ± 0.0016 ml sec−1·gm−1). Comparable influx (0.083 to 0.14 sec−1) and efflux (0.10 to 0.18 sec−1) coefficients were obtained, suggesting rapid cellular entry and efflux of salicylamide sulfate. The ratio of the derived rate constants, which corresponds to the concentration ratio across the membrane, was 0.40 to 0.55. The findings contrast sharply with those for acetaminophen sulfate, which enters liver cells poorly. (Hepatology 1994; 19:229–244).

Salicylamide sulfate cell entry in perfused rat liver: A multiple-indicator dilution study

The hepatocellular entry of salicylamide sulfate conjugate, which binds to both red blood cells and albumin, was examined with the multiple-indicator dilution technique in the perfused rat liver, with medium containing both 20% red cells and 1% albumin (set A), red cells only (set B), albumin only (set C) and neither red cells nor albumin (set D). [14C]Salicylamide sulfate, 51Cr-labeled red cells (a vascular reference), 125I-labeled albumin, [3H]sucrose or [58Co] ethylenediaminetetraacetic acid (EDTA) (high and low molecular weight interstitial references, respectively) and 3H2O or D2O (a cellular reference) were injected as a bolus into the portal vein. Among all sets of outflow data, the earliest immediate vascular recoveries for the [14C] salicylamide sulfate were lower than those for the vascular reference. For sets A and B, the upslopes precessed those for the 58Co-EDTA curve, then crossed over the 58Co-EDTA curves, with lower magnitude peaks occurring at the same time as those for labeled albumin, whereas for sets C and D, in which red cells were absent, the upslopes of [14C]salicylamide sulfate lagged behind those for labeled albumin and [3H]sucrose, reaching lower magnitude peaks coincidental in time with those for labeled sucrose. The precession of the [14C]salicylamide sulfate curve over 58Co-EDTA or [3H]sucrose in the presence (sets A and B) but not in the absence (sets C and D) of red cells and the absence of precession with albumin alone (set C) suggest that a red cell effect on the upslope is more evident than that for albumin. For all experiments, the downslopes of the sulfate curve crossed over those of the labeled red cells, albumin, 58Co-EDTA and sucrose curves and then the water curve at around the peak. The downslopes of the [14C]salicylamide sulfate were similar to those for labeled water, suggesting rapid cellular influx and efflux of salicylamide sulfate. Quantitative evaluation with a barrier-limited space-variable transit-time model for rapidly equilibrating red cell and albumin binding accounted for the upslope effects on [14C]salicylamide sulfate behavior and demonstrated its relatively high liver cell permeability. Values for the unbound permeability surface area product (0.029 to 0.036 ml sec-1.gm-1) were not different, regardless of the presence or absence of red cells and albumin, and slightly exceeded values for hepatic blood flow (0.0186 +/- 0.0016 ml sec-1.gm-1). Comparable influx (0.083 to 0.14 sec-1) and efflux (0.10 to 0.18 sec-1) coefficients were obtained, suggesting rapid cellular entry and efflux of salicylamide sulfate.(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of oral pretreatment with indomethacin on the intestinal first-pass metabolism of salicylamide in rabbits.

The effect of oral pretreatment with indomethacin on the intestinal first-pass metabolism of salicylamide (SAM) was studied in rabbits using in situ intestinal sacs with complete mesenteric venous blood collection. The appearance of both SAM and its metabolites into the mesenteric venous blood was measured directly by cannulating the mesenteric vein of exposed rabbit intestine and collecting all venous blood draining from the absorbing region. By oral pretreatment with indomethacin, the total amounts of SAM absorbed in 20 and 120 min were significantly increased compared to the control. These results indicated the alteration of the permeability in the intestinal mucosa. In 20 min, indomethacin pretreatment resulted in increased appearance of SAM and SAM glucuronide in the mesenteric venous blood. In 120 min, increased appearance of SAM and decreased appearance of SAM sulfate were observed, compared to the control. These findings suggested that the change in the intestinal first-pass metabolism of SAM is probably due to the intestinal mucosal damage by oral pretreatment with indomethacin.

Depression of salicylamide glucuronidation by bacterial lipopolysaccharide.

The effect of lipopolysaccharide (LPS) on the urinary excretion of salicylamide (SAM) (10 mg/head) given orally to rats was examined 24 h after intraperitoneal administration of LPS (0.5 mg/kg) by the direct analysis of urine samples by high-performance liquid chromatography. The cumulative amount of SAM glucuronide excreted in urine in 4 h was decreased by 40-70% in the LPS-administered rats, while that of SAM sulfate was increased by 7-15% compared with the control in the same rats. The amounts of the conjugates of gentisamide (the hydroxylated metabolite of SAM) excreted in urine were decreased by approx. 70% compared with the control. LPS administration provoked a decrease in the activities of aniline hydroxylase of liver and uridine diphosphate (UDP) -glucuronyltransferase of liver, lung and kidney, but did not change the activities of sulfotransferase of liver or UDP-glucuronyltransferase obtained from the small intestine. The mechanism of the decreased urinary excretion of SAM glucuronide in LPS-administered rats is discussed.

Conjugation of salicylamide in the intestinal wall of dogs and rabbits.

The intestinal conjugation of salicylamide (SAM) the formation of SAM sulfate (SAMS) and SAM glucuronide (SAMG) were investigated in dogs and rabbits by using in situ intestinal sac preparation with mesenteric vein cannulation. SAM, SAMS and SAMG in the mesenteric venous blood, sampled at successive intervals after injection of SAM solutions into the sac, were determined. Total recoveries in 1 h were not significantly different in dogs (71%) and rabbits (64%). Both animals, however, showed a quantitative difference in intestinal conjugation. Sulfation predominated over glucuronidation in dog intestine while the formation of the glucuronide was exclusively high in rabbit intestine. Furthermore, the dose-dependent conjugation of SAM was investigated in dog intestine at four doses, 2, 3, 6 and 10 mg/one animal. Upon increasing the doses from 2 to 10 mg, the amounts of SAMS appearing in the blood remained unchanged, those of SAMG increased about two times and those of free SAM increased about six times, while the total recoveries increased parallel to the doses, suggesting saturation kinetics in the formation step of the two conjugates. Simultaneous computer-fitting of the experimental data for 2 and 10 mg doses to a simplified model, containing Michaelis-Menten kinetics in the formation of the conjugates, was examined and good agreement between the observed data and the theoretical values was obtained.

Analysis of Salicylamide and Its Metabolites in Blood and Urine by HPLC

Abstract Sensitive liquid chromatographic assays for salicylamide and its metabolites in urine and plasma were developed to facilitate pharmacokinetic studies of the drug's metabolism. The drug and its hydroxylated metabolite, gentisamide, were extracted and concentrated prior to separation on a small-bore reverse-phase column. Conjugated metabolites were assayed separately using reverse-phase ion-pair chromatography. An accurate method of assay calibration in the absence of pure metabolite standards was developed using radioactively-labelled parent drug. In addition one of the metabolites, salicylamide sulfate, was isolated by ion-pair extraction and purified. A significant species difference in salicylamide metabolism was observed. In the dog the drug is almost exclusively (90%) metabolized to its sulfate conjugate, while in humans the glucuronide conjugates of salicylamide (50%) and gentisamide (15%) predominate over salicylamide sulfate (30%).

Determination of Salicylamide and Five Metabolites in Biological Fluids by High-Performance Liquid Chromatography

Two high-performance liquid chromatographic (HPLC) assay procedures were developed for the determination of salicylamide and its metabolites in serum, urine, and saliva. One method involves reverse-phase ion-pair chromatography and UV detection, and is used to determine salicylamide, salicylamide glucuronide, and salicylamide sulfate. The other method, with a different mobile phase and without the ion-pairing reagent, is used to determine gentisamide (the hydroxylated metabolite of salicylamide), gentisamide glucuronide, and gentisamide sulfate. The assays are performed by direct injection of the sample after protein precipitation with ethanol containing the internal standard. Increased sensitivity for the determination of low concentrations of salicylamide is obtained by organic extraction of this drug from serum or saliva. Calibration curves for the conjugates of salicylamide and gentisamide were obtained, in the absence of authentic standards, by partial enzymatic hydrolysis, using the decrease of the conjugate peaks and the concomitant increase of free salicylamide or gentisamide concentrations to determine peak area ratio–concentration relationships. Application of the HPLC assay procedures to the determination of salicylamide excretion products in the urine of three normal human subjects resulted in 98.6% (range: 97.1–100.1%) recovery of a 1-g oral dose of the drug. All five metabolites of salicylamide were found in urine, but only salicylamide glucuronide, salicylamide sulfate, and gentisamide glucuronide were found consistently and in appreciable quantities. Salicylamide and all of its metabolites except gentisamide sulfate were found in human and rat serum, and unconjugated salicylamide as well as gentisamide were found in human saliva.

Biopharmaceutical studies of drugs. III. Serum concentration level of o-ethoxybenzamide and N-(beta-hydroxybutyryl)-p-phenetidine after simultaneous administration to rabbit (author's transl)

The concentrations of o-ethoxybenzamide (EBA), N-(β-hydroxybutyryl)-p-phenetidine (HBP) and metabolites in the rabbit serum were investigated when EBA was administered orally with HBP and EBA or HBP administered alone. 1) The concentration of EBA in the serum increased when 100 mg/kg of EBA was administered concurrently with 28.5, 33.3 and 62.5 mg/kg of HBP as compared with the administration of 100 mg/kg of EBA alone. The co-administration of EBA and HBP also decreased the concentration of salicylamide (SAM) sulfate in the serum. 2) The concentration of HBP in the serum increased when 28.5, 33.3 and 62.5 mg/kg of HBP and 100 mg/kg of EBA were administered together, while that of N-(β-hydroxybutyryl)-p-aminophenol (HBA) glucuronide was decreased. 3) The deethylation of EBA was inhibited competitively by the addition of HBP, and also the deethylation of HBP inhibited competitively by the addition of EBA in 9000×g supernatant fraction of the rabbit liver. 4) The area under the serum concentration-time curve (AUC, 0-8 hr) of SAM sulfate approached a limiting value when 300 mg/kg of EBA was administered orally, while the AUC values of SAM glucuronide, EBA, SAM total and SAM total plus EBA increased with increasing doses of EBA. 5) The deethylation of HBP approached a limiting value when 300 mg/kg of HBP was administered orally, while the AUC values of HBP and HBA total plus HBP total (HBP plus HBP glucuronide) increased with increasing doses of HBP.

Age-related differences in salicylamide and acetaminophen conjugation in man

Following a concomitant oral dose of salicylamide and acetaminophen (5 mg/kg of each) the urinary excretion of glucuronide and sulfate conjugates of the drugs were followed in children (ages seven to ten years) and adults. No significant difference were observed between the two age groups in the half-lives for appearance of salicylamide conjugates in urine. Age-related changes in the metabolic pathways, however, were observed. The mean percentage of dose excreted as salicylamide sulfate was significantly higher in children (78%) than in adults (36%). In contrast, salicylamide glucuronide was the major excretory product in adults. Similar age-related differences were observed for acetaminophen conjugation. Pharmacokinetic analysis indicated that the deficiency in glucuronide conjugation of these drugs in children is accompanied by a higher rate of sulfate formation.

The influence of pyrogen-induced fever on salicylamide metabolism in man.

Salicylamide is metabolized in man by biotransformation to salicylamide glucuronide, salicylamide sulfate, and gentisamide glucuronide. The metabolites are quantitatively and rapidly excreted in urine. Study of the metabolism of this drug in volunteers during episodes of pyrogen-induced fever shows a significant reduction in the half-life (t(1/2)) of the excretion of the drug metabolites. The proportion of the drug transformed to its major metabolite, salicylamide glucuronide, is significantly reduced by fever, with concomitant increase in the proportion of one or both of the other metabolites. Thus, the pattern of urinary metabolites of salicylamide is altered. The shortened t(1/2) of the metabolite excretion is probably due to increased hepatic and renal blood flow known to accompany pyrogen-induced fever. This concept was supported by the observation that when two subjects were placed in a high-temperature environmental chamber, a condition in which hepatic and renal blood flows are known to diminish, the t(1/2) of salicylamide metabolite excretion actually increased. No simple explanation exists to explain the changed metabolite pattern noted during febrile periods. It is most likely to be due to complex interactions between the direct or indirect effects of the pyrogens and the factors affecting the hepatic biotransformation of drugs.

EFFECT OF BILIRUBIN ON DRUG CONJUGATIONS IN CHILDREN

The formation of glucuronides of salicylate, salicylamide, and acetaminophen in two sisters with congenital unconjugated hyperbilirubinemia responsive to phenobarbital was normal at widely different (6 to 37 mg per 100 ml) serum bilirubin concentrations. The conjugation of salicylate with glycine, and the formation of salicylamide sulfate and acetaminophen sulfate were also normal. This shows that unconjugated hyperbilirubinemia per se does not affect the biotransformation of the three drugs tested. The clinical and pharmacokinetic characteristics of the children in this study are consistent with a recently proposed mechanism involving a deficiency in hepatic anion-binding protein (rather than glucuronyl transferase) as one cause of unconjugated hyperbilirubinemia.

Drug Biotransformation Interactions in Man III: Acetaminophen and Salicylamide

Acetaminophen (1 and 2 g.), salicylamide (1 g.), and 1 g. acetaminophen followed 1.5 hr. later by 1 or 2 g. salicylamide were administered in aqueous solutions to healthy human subjects. Urine was collected every 15-30 min. for the first 4-5 hr. and then at longer intervals. The urine samples were analyzed for acetaminophen, acetaminophen sulfate, acetaminophen glucuronide, salicylamide, salicylamide sulfate, and salicylamide glucuronide. The excretion rates of acetaminophen sulfate and acetaminophen glucuronide decreased immediately after administration of salicylamide. Acetaminophen decreased the formation of salicylamide sulfate. Coadministration of L-cysteine (a source of sulfate) prevented this effect and also increased the excretion of acetaminophen sulfate. The results indicate a competitive inhibition in the formation of acetaminophen sulfate and salicylamide sulfate and suggest also a competitive inhibition in the formation of acetaminophen glucuronide and salicylamide glucuronide.

Drug Biotransformation Interactions in Man I: Mutual Inhibition in Glucuronide Formation of Salicylic Acid and Salicylamide in Man

Co-administration of salicylamide (SAM) and sodium salicylate to healthy adult humans resulted in a pronounced decrease in the formation of the glucuronides of these two drugs. This is thought to be due to a limited capacity for glucuronide conjugation in man and/or to the uncoupling action of salicylate on oxidative phosphorylation. Salicylate may inhibit also the formation of SAM sulfate; it has no effect on the excretion rate constants of SAM glucuronide and SAM sulfate in the doses used in the investigation. The results of this study indicate that concomitant use of certain drugs which are readily available to the public may lead to appreciable interactions in biotransformation and to enhanced pharmacologic effects.

Synthesis of Salicylamide Sulfate and its Effect on Calcium Ion Solubility

Salicylamide sulfate has been synthesized and found to be chromatographically identical to a metabolic product appearing in the urine of patients on salicylamide therapy. This compound, when incubated with suspensions of calcium hydroxide, was found to increase the aqueous solubility of calcium ion beyond that expected from a salt effect alone. An explanation of the effect of salicylamide in halting calcium stone growth has been suggested.