Effect of Taurolithocholate on In Vivo Sulfation and Glucuronidation of Acetaminophen in Rats

Taurolithocholate produces a prompt, complete, and reversible cessation of bile flow in rats. This is associated with impaired hepatic oxidative drug-metabolizing activity. The purpose of this study was to examine the effects of taurolithocholate-induced cholestasis on in vivo conjugation. The pharmacokinetics of acetaminophen and the two major processes specifically responsible for its elimination, namely, the formations of acetaminophen sulfate and acetaminophen glucuronide, were used to assess hepatic conjugating activity. A 30-mg/kg bolus of acetaminophen was administered intravenously to rats 2 hr (acute cholestasis) or 20 hr (postcholestasis) after intravenous pretreatment with sodium taurolithocholate, 5 µmol/100 g body weight. Acute cholestasis increased the total clearance of acetaminophen 20%, the partial clearance to acetaminophen sulfate 12%, and the partial clearance to acetaminophen glucuronide 85%. Postcholestasis, these parameters had significantly decreased compared to those during acute cholestasis and were comparable to control values. The results show that cholestasis does not impair acetaminophen conjugation in the rat.     

Sulfation predominates the pharmacokinetics,metabolism, and excretion of forsythin in humans: major enzymes and transporters identified

Forsythin extracted from Forsythiae Fructus is widely used to treat fever caused by the common cold or influenza in China,Japan and Korea.The present study aimed to analyze the pharmacokinetics,metabolism and excretion routes of forsythin in humans and determine the major enzymes and transporters involved in these processes.After a single oral administration,forsythin underwent extensive metabolism via hydrolysis and further sulfation.In total,3 of the 13 metabolites were confirmed by comparison to reference substances,i.e.,aglycone M1,M1 sulfate(M2),and M1 glucuronide(M7).Hydrolysis was the initial and main metabolic pathway of the parent compound,followed by extensive sulfation to form M2 and a reduced level of glucuronidation to form M7.In addition,the plasma exposure of M2 and M7 were 86-and 4.2-fold higher than that of forsythin.Within 48 h,~75.1%of the administered dose was found in urine,with M2 accounting for 71.6%.Further phenotyping experiments revealed that sulfotransferase 1A1 and UDP-glucuronosyltransferase 1A8 were the most active hepatic enzymes involved in the formation of M2 and M7,respectively.The in vitro kinetic study provided direct evidence that M1 showed a preference for sulfation.Sulfated conjugate M2 was identified as a specific substrate of organic anion transporter 3,which could facilitate the renal excretion of M2.Altogether,our study demonstrated that sulfation dominated the metabolism and pharmacokinetics of forsythin,while the sulfate conjugate was excreted mainly in the urine.     

葛根总黄酮生物粘附性缓释片体外组织粘附力及释放度研究

目的 :探讨葛根总黄酮缓释片的体外释放度及其释药动力学 ,测定、比较生物粘附性缓释片和普通片与家兔离体胃、小肠组织的粘附力。方法 :采用转篮法 ,以0 1mol/LHCl900ml为溶出介质 ,转速100r/min测定其累积释放度 ,并分别用单指数模型、威布尔分布函数、Higuchi方程、零级释放模型对释放度进行释药动力学模拟 ,根据各模型拟合的AIC计算值与实测值的绝对误差和相对误差确定缓释片的最佳拟合模型 ,同时以自制粘附力测定装置测定、比较生物粘附性缓释片及普通片与家兔离体胃、肠组织的粘附力。结果 :缓释片以单指数模型拟合最佳 ,生物粘附性缓释片与家兔离体胃、肠组织的粘附力明显大于普通片 (P<0 01) ,且与小肠的粘附力大于胃 (P<0 05)。结论 :缓释片在体外释药动力学符合单指数模型 ;生物粘附性缓释片在胃、肠道具有较强的粘附作用。     

Enteric Excretion of Baicalein,a Flavone of Scutellariae Radix,via Glucuronidation in Rat: Involvement of Multidrug Resistance-Associated Protein 2

PURPOSE: Baicalin (BG) and its aglycone, baicalein (B), are strong antioxidants and have various pharmacological actions. The purpose of this study was to evaluate efflux of BG from rat intestinal mucosal cell following glucuronidation of B absorbed after oral administration of B. METHODS: The absorption and excretion of BG and B were evaluated in rats using the in situ jejunal loop technique and in vitro jejunal everted sac experiments. BG and B levels were determined by high-performance liquid chromatography with electro-chemical detection to ensure selectivity and high sensitivity. RESULTS: A large amount (30.4% recovery) of BG, but no B, was detected in the intestinal lumens of germ-free rats 4 h after oral administration of B (12.1 mg/kg), in comparison with a substantial recovery (55.1%) of unabsorbed BG 4 h after its administration. During the in situ rat jejunal loop absorption experiment, B disappeared rapidly, and 8% of the lost B was excreted into the loop as BG 20 min after infusing 0.1 mM B. In an in vitro absorption experiment using everted rat jejunal sac, BG also appeared outside the sac, accompanied by the disappearance of B from the outer (mucosal) side. However, very little of B was transferred to the inner (serosal) side of the sac, and only a trace of BG was detected inside the sac. Thus, in both the loop and the everted sac systems, the efflux of BG from the mucosal surface was saturated with the concentration of B added. Moreover, the efflux rate of BG in the everted jejunal sac from Eisai hyperbilirubinemic rat (EHBR) was significantly lower by 56.4% than that from Sprague-Dawley rat. CONCLUSIONS: These results indicate that, in rat, a large proportion of any B absorbed is retained, transformed into BG within the intestinal mucosal cells, and coordinately excreted through multidrug resistance-associated protein 2 (MRP2) into the intestinal lumen.     

Optimization of a UDP-glucuronosyltransferase assay for trout liver S9 fractions: activity enhancement by alamethicin,a pore-forming peptide

1.?An existing assay for UDP-glucuronosyltransferase (UGT) activity in trout liver microsomes was optimized using trout liver S9 fractions. Individual experiments were conducted to determine the time dependence of UGT activity as well as optimal levels of S9 protein, uridine 5'-diphosphoglucuronic acid (UDPGA), substrate (p-nitrophenol) and alamethicin, a pore-forming agent added to eliminate latency.

2.?Addition of Mg^2+?(to 1?mM) or bovine serum albumin (BSA; to 2% w/v) had variable effects on activity, but these effects were minor. Eliminating alamethicin from the system resulted in very low levels of activity. A portion of this activity could be recovered by adding Triton X-100 or Brij 58; however, the optimal concentration range for either detergent was very narrow.

3.?When expressed on a pmol/min/g liver basis, UGT activities determined using this updated assay were substantially higher than those reported previously for uninduced trout.

4.?These results clearly demonstrate the advantages of using alamethicin for the removal of latency in UGT activity studies with trout and may have broad implications for the study of UGTs in other fish species.     

Urinary excretion of the main metabolites of methamphetamine,including p-hydroxymethamphetamine-sulfate and p-hydroxymethamphetamine-glucuronide,in humans and rats

The urinary concentrations of the main metabolites of methamphetamine (MA), specifically p-hydroxymethamphetamine-sulfate (?p-OHMA-Sul) and p-hydroxymethamphetamine-glucuronide (?p-OHMA-Glu), were directly measured in MA users and rats using an optimized LC-ESI MS method. The concentrations of the two conjugates in 50 MA human users’ urine ranged from 0.09 to 88.6?µM (0.02–21.7?µg?ml^?1) for p-OHMA-Sul and from <0.05 to 7.13?µM (<0.02–2.43?µg?ml^?1) for p-OHMA-Glu; the ratios of sulfate to glucuronide (S/G ratios) ranged from 2.2 to 37.1 (13.8?±?8.1). The results demonstrate that the sulfation is quantitatively more important than glucuronidation for the conjugation of p-OHMA in humans. The urinary concentration time-dependency in two MA users also revealed that the conjugates were mostly excreted in urine within 3 days post-intake. In contrast, in rat, almost all of the conjugated p-OHMA (>99%) was excreted as the glucuronide in urine. These findings confirm that a large species variation exists in the conjugation of p-OHMA between humans and rats.     

Involvement of UDP-Glucuronosyltransferases in the Extensive Liver and Intestinal First-Pass Metabolism of Flavonoid Baicalein

Purpose The present study aims to investigate the involvement of UDP-glucuronosyltranferase (UGT) in the extensive liver and intestinal first-pass glucuronidation of baicalein (B) in both rats and humans and also to study sulfation and P450 mediated hydroxylation of B.Materials and Methods B was incubated with liver and intestine microsome, cytosol, S9 fractions from human, rat and various human recombinant UGT isozymes, respectively. The generated metabolites were identified by HPLC/MS/MS and quantified by HPLC/UV.Results Three metabolites of B namely baicalein 7-O-glucuronide (BG), the isomer of baicalein 7-O-glucuronide (BG’), and baicalein sulfate were found. BG, the predominant metabolite of B, was extensively generated in liver and jejunum microsomes in both humans and rats. Its formation was mainly catalyzed by UGT 1A9 and also mediated by UGT 1A1, 1A3, 1A8, 1A7 and 2B15 with different kinetic profiles. UGT 1A8 mediated formation of BG’ was mainly found in human intestine and rat liver microsomes. Sulfation and P450 mediated hydroxylation of B were much less significant than glucuronidation.Conclusions Extensive liver and intestinal first-pass glucuronidation of B were found in both humans and rats. Under the current experimental conditions, UGT 1A9 and UGT 1A8 demonstrated the fastest formation rate of BG in human liver preparations and BG’ in human intestine preparations, respectively.     

影响葛根总黄酮生物粘附片体外释放度的因素研究

目的 :探讨影响葛根总黄酮生物粘附片释放度的主要因素。方法 :以羟丙基甲基纤维素 (HPMC)与卡波姆 (CP)为生物粘附材料和骨架材料、乳糖为致孔剂制备葛根总黄酮生物粘附片 ;采用转篮法测定释放度 ,以0 1mol/LHCl为溶出介质 ,转速为100r/min ,测定累积释放度 ,同时考察HPMC用量、CP用量、致孔剂种类、乳糖用量、压片颗粒大小、介质 pH等因素对释放度的影响。结果与结论 :HPMC用量、CP用量、致孔剂种类、乳糖用量、压片时颗粒大小、介质 pH等因素对生物粘附片溶出均有明显影响     

Both phenolic and acyl glucuronidation pathways of diflunisal are impaired in liver cirrhosis

Summary The pharmacokinetics of diflunisal, a salicylate derivative that undergoes phenolic and acyl glucuronidation as well as sulphate conjugation, has been studied after a single oral dose (250 mg) in patients with cirrhosis (n=5) and in healthy controls (n=5).The plasma clearance of total (bound + unbound) diflunisal was 10.2 ml · min^–1 in the control subjects and it was not affected by cirrhosis (10.9 ml · min^–1). The plasma protein binding of diflunisal was significantly reduced in cirrhosis; the percentage of unbound diflunisal in plasma was 0.089 in the controls and 0.147 in the patients with cirrhosis. Plasma clearance of unbound diflunisal was significantly impaired in cirrhosis: 11.51 · min^–1 in control subjects vs 7.41 · min^–1 in cirrhotics.In cirrhotic patients, the unbound partial clearances to the phenolic and acyl glucuronides were both significantly reduced, by approximately 38%. The unbound partial clearance to the sulphate conjugate was not significantly affected by cirrhosis.The results show that both the phenolic and acyl glucuronidation pathways of diflunisal are equally susceptible to the effects of liver cirrhosis.     

山楂叶总黄酮磷脂复合物大鼠在体肠吸收动力学研究

目的研究山楂叶总黄酮磷脂复合物的大鼠在体小肠吸收动力学特征。方法采用大鼠在体肠单向灌流模型,以山楂叶总黄酮原料药为对照,采用分光光度法测定灌流液中总黄酮含量,计算肠吸收速率常数（Ka值）与单位时间吸收转化率（A值）,考察药物浓度与pH值对山楂叶总黄酮磷脂复合物肠吸收的影响。结果浓度为0．1mg·mL^-1的山楂叶总黄酮和山楂叶总黄酮磷脂复合物,Ka值与A值分别为0．0109h^-1,1．20％和0．0391h^-1,3．73％;药物浓度为0．1mg．mL^-1,pH为6．0,7．4,8．0的肠循环液,山楂叶总黄酮和山楂叶总黄酮磷脂复合物的Ka值分别为0．0076．0．0109．0．0056h^-1和0．0376,0．0391,0．0305h^-1,A值分别为0．69％,1．20％,0．42％和3．19％,3．73％,2．81％;表明在总黄酮浓度和溶液pH相同条件下,山楂叶总黄酮磷脂复合物的大鼠在体肠吸收明显优于山楂叶总黄酮。结论磷脂固体分散技术可改善山楂叶总黄酮的小肠吸收。     

Time course of acetaminophen-protein adducts and acetaminophen metabolites in circulation of overdose patients and in HepaRG cells

Abstract

1.?It has been suggested that acetaminophen (APAP)-protein adducts can be measured in circulation to diagnose APAP-induced liver injury. However, the full-time course of plasma adducts has not been studied specifically in early-presenting overdose patients. In fact, surprisingly little work has been done on the metabolism of APAP after overdose in general.

2.?We measured APAP, five APAP metabolites and APAP-protein adducts in plasma samples from early- and late-presenting overdose patients, and APAP-protein adducts in culture medium from HepaRG cells.

3.?In contrast to earlier rodents studies, we found that APAP-protein adducts were lower at early time points and peaked around the time of peak liver injury, suggesting that these adduct levels may take longer to become elevated or remain elevated than previously thought.

4.?APAP and its major metabolites were elevated in plasma at early time points and rapidly decreased.

5.?Although clinical measurement of APAP-protein adducts holds promise as a diagnostic tool, we suggest caution in its interpretation in very early-presenting patients. Our data also support the idea that sulfation is saturated even at low doses but glucuronidation has a much higher capacity, highlighting the importance of glucuronidation in APAP metabolism.     

紫外分光光度法测定毛鸡骨草提取物中总黄酮含量

建立用紫外分光光度法测定毛鸡骨草提取物中总黄酮含量的方法。以其主成分芹菜素-6-C-阿拉伯糖-8-C-葡萄糖苷为对照品,三氯化铝显色,测定波长382 nm,线性范围为4.1~24.6μg.mL-1(r2=0.9999)。     

In Vivo Absorption of Theophylline and Salicylic Acid from Rat Small Intestine

Abstract: In vivo absorption of theophylline and salicylic acid from the rat small intestine was studied by a closed segment technique. The drugs were administered at 1 and 4 mM in dissolved form, both in the presence and absence of phlorizin (0.01 mM). Drug concentrations were measured by high-pressure liquid chromatography. Phlorizin inhibited the absorption of theophylline (pK_a = 8.6) at 4 mM but not at 1 mM. In contrast, the absorption of salicylic acid (pK_a = 3) both at I and 4 mM was unaffected by phlorizin. This suggests that an active transport system, sensitive to phlorizin, is involved in the rat intestinal absorption of theophylline, but not in that of salicylic acid. Apart from differences in drug structure, differences in the degree of ionization may influence the access of acidic drugs to this transport system.     

In vitro/in vivo investigations to examine the gender differences in the pharmacokinetics of novel oral Janus kinase (JAK) inhibitor ASP015K and sulfate metabolite M2 in rats

Abstract

1. Although marked gender differences have been reported for the exposure level of the sulfate metabolite M2 of ASP015K in rats, no such differences have been reported for the unchanged drug. To clarify the cause of these pharmacokinetic gender differences, we investigated the in vitro hepatic sulfation, glucuronidation, and cytochrome P450 (CYP) metabolism of ASP015K in rat liver cytosols or rat liver microsomes. Further, in vivo excretion and metabolic profiles were investigated using rat urine, bile, and feces post-ASP015K administration.

2. In vitro metabolism study using liver cytosols clearly suggested that the gender differences in the M2 exposure were mainly attributed to the female-predominant ASP015K metabolism mediated by sulfotransferase (SULT). Metabolic profiles in urine and bile from male rats suggested that the major elimination pathway of ASP015K is glucuronidation in rats. No remarkable gender differences in the in vitro glucuronidation were observed.

3. The contribution of the sulfation pathway to the clearance of ASP015K was markedly lower than that of the glucuronidation pathway in both male and female rats. These results might explain why gender differences were not marked for ASP015K exposure but were for M2.     

Effect of Colchicine on Drug Absorption from the Rat Small Intestine in Situ and in Vitro

Abstract The effect of colchicine (1 mg/kg intraperitoneally on two successive days) on the absorption of isoniazid, quinidine and sulphafurazole (sulfisoxazole) from the rat small intestine was studied in situ and in vitro. Colchicine produced two different types of histological damage in the small intestine, one with degenerative and the other with regenerative changes predominating. The small intestinal surface area was variably reduced. The colchicine-treated rats were lethargic and hypothermic as compared to controls. Colchicine retarded the disappearance of fluid and all three drugs from the small intestinal lumen in situ 2 days after the first colchicine injection. In vitro the total amounts of fluid and drugs passed through the intestinal wall were not significantly changed by colchicine, although there was a slight tendency towards an increased absorption of quinidine. Hence, colchicine as an antimitotic drug decreases drug absorption from the rat small intestine in situ, apparently due to the decreased surface area of the small intestine, the decreased water flux through the intestinal wall, the retarded intestinal motility and hypothermia of the rats. In vitro the changes are small, which makes the in vitro tests less suitable for studying the effect of colchicine on absorption.     

Effect of Methotrexate on Drug Absorption from the Rat Small Intestine in Situ and in Vitro

Abstract The effect of methotrexate (20 mg/kg intramuscularly) on the absorption of phenobarbitone, sulphafurazole, mecamylamine, quinidine and isoniazid from the rat small intestine was studied in situ and in vitro. The disappearance of all drugs studied from the intestinal fluid in situ was retarded on the third day after methotrexate administration. The fluid transfer and the amount of drugs passed through the intestinal wall in vitro were also decreased. The absorption of phenobarbitone was reversible within six days, whereas the absorption of quinidine was still retarded on the sixth day after methotrexate administration. Methotrexate did not modify the amount of quinidine excreted into the intestinal lumen after intravenous administration. The levels of other drugs except isoniazid in the blood at the end of the experiment showed changes corresponding to their disappearance from the intestinal lumen. In situ the drug levels in the intestinal wall were much lower than in vitro. Their levels in the intestinal wall reflected drug absorption in vitro but not in situ. The methotrexate-induced reversible decrease in absorption seems to be attributable at least partly to diminished water flux through the intestinal wall, although other mechanisms may also exist.     

A Comparative Study on Drug Hydroxylation and Glucuronidation in Liver Microsomes of Phenobarbital and 3-Methylcholanthrene Treated Rats

The treatment of hepatic microsomes with phospholipase A or phospholipase C decreased the drug hydroxylase activity. The exception was the aryl hydrocarbon hydroxylase in hepatic microsomes from 3-methylcholanthrene pre-treated rats. Its specific activity remained unchanged after phospholipase A digestion. In hepatic microsomes from phenobarbital pretreated rats the aryl hydrocarbon hydroxylase was not significantly increased and after phospholipase A or phospholipase C digestion, it decreased in both treated and control rats. The phenobarbital or 3-methylcholanthrene induced p-nitroanisole 0-demethylase activity in liver microsomes decreased in a manner corresponding to the decrease in cytochrome P-450 (P-448) concentrations. The treatment of hepatic microsomes with phospholipase A or phospholipase C activated the membrane-bound UDP glucuronosyltransferase both in control and drug-treated rats. Phospholipase A increased the measurable UDP glucuronosyltransferase activity particularly in hepatic microsomes from 3-methylcholanthrene pretreated rats. However, digestion with trypsin was necessary in order to detect the induction in UDP glucuronosyltransferase after phenobarbital pretreatment of rats. Other agents studied, such as phospholipase A or C, digitonin, cetylpyridinium chloride, or NaSCN, were unable to do this. Digestion with trypsin released considerable amounts of UDP glucuronosyltransferase activity into 27,000 g supernatant, especially from phenobarbital microsomes, whereas phospholipase A was more active in releasing UDP glucuronosyltransferase into 27,000 g supernatant from hepatic microsomes of 3-methylcholanthrene pretreated rats. Membrane perturbating agents seem to differ in their action towards the hepatic microsomal membranes obtained from rats treated with phenobarbital or 3-methylcholanthrene.     

In Vivo Absorption of Phenytoin from Rat Small Intestine and its Inhibition by Phlorizin

Abstract In vivo absorption of phenytoin from the small intestine was studied by an in vivo closed segment technique. Phenytoin in concentrations of 1000, 2000, and 4000 μmol/1 was administered in dissolved form. Polythylene glycol 4000 was used as a non-absorbable marker. The concentrations of phenytoin in the intestinal lumen. in the mucosa, and in cardiac blood were measured both by spectrophotometry and by gas chromatography. Phenytoin was absorbed very rapidly, and the proportion absorbed increased with increasing dose. Thus, during the first 10 min. about 85 per cent of the largest dose but only 25 percent of the smallest dose had been absorbed. The phenytoin concentration in mucosa and serum increased in an analogous way; maximum values were observed within the first ten minutes. The concentrations in mucosa and serum were dose dependent during the first ten minutes. 0.01 mmol/1 and 1 mmol/1 phlorizin significantly reduced the transfer of phenytoin (4000 and 2000 μmol/1) from the gut lumen to the mucosa. No inhibition was observed when the initial phenytoin dose was 1000 μmol/1. The results suggest that an active transport mechanism, sensitive to phlorizin, is involved in the intestinal absorption of phenytoin in the rat.     

柱前衍生化HPLC法测定大鼠肝S9组分中川丁特罗对映体

目的:建立柱前衍生化HPLC法研究大鼠肝S9组分中川丁特罗对映体体外代谢的立体选择性。方法:以二-O-乙酰基-L-酒石酸酐（DATAAN）为衍生化试剂,采用反相高效液相色谱法拆分川丁特罗对映体,测定大鼠肝S9组分中川丁特罗对映体的浓度。结果:川丁特罗对映体达到基线分离,分离度为2.24,线性范围2.50～200μmol·L^-1,平均回收率（S）-川丁特罗为89.3%,（R）-川丁特罗为91.3%。平均日内、日间RSD均小于15%。在大鼠肝S9组分中川丁特罗的代谢呈现立体选择性。结论:此法专属性强、准确可靠,可用于大鼠肝S9组分中川丁特罗代谢的立体选择性研究。