| Drug disposition involving distribution, metabolism, and excretion, has great relation with drug’s effects. The rate and amount of drug reaches the target site may influence the initial time and the degree of drug affecting. Moreover, the duration time of drug affecting last is determined primarily by the metabolism and excretion.The amount of drug distribution differs in various organs, and distribution characterization varied for diverse drugs. Similarly, metabolism and excretion characterization varied for diverse drugs. Many drugs proceed enteric and enterhepatic recycling in body by the means of phase II metabolism, such as glucuronidation and sulfation, which play a central role in prolonging the effecting time of drugs.Enterhepatic recycling scheme was recognized more than half a century ago. It involves the action of liver to excrete the conjugated phase II metabolites and action of the microflora or bacterialβ-glucuronidase or sulfatases to release the aglycones (from the conjugated phase II metabolites). Released aglycones can enter the body again and form glucuronides or sulfates again, thereby completing the recycling loop.Enterhepatic recycling scheme may help body utilize endogenous necessary material effectively. For example, during the bile digesting, cholic acid would experience enterhepatic recycling two or three times after administration once. Other similar necessary materials are vit D3, vit B12, folic acid etc. Many drugs like digoxin, cardigin, diethylstilbestrol, ampicillin etc, would also experience recycling scheme of this kind. Therefore, enterhepatic recycling is of great importance to the utilization of endogenous necessary material and exerting of drug effects.Enteric recycling has only been termed more recently. In this recycling scheme, the phase II conjugates are excreted by the enterocytes, and once again, the action of bacterialβ-glucuronidase or sulfatases is required to release aglycone for reabsorption.In our study, we have identified the third and perhaps the most important recycling scheme for compounds that are extensively glucuronidated in the gut:the local recycling. In the local recycling scheme, the phase II conjugates are excreted by the enterocytes, and also once again, the action of bacterial P-glucuronidase or sulfatases is required to release aglycone for reabsorption. After reabsorption, drug was again glucuronidated by UDP-glucuronosyltranferase (UGT), and one part of the glucuronides was excreted by the enterocytes. In this way, drug and its glucuronide could form a complete recycling loop at one enterocyte and gut outside it.Wogonin, the aglycone of wogonoside(wogonin-7-O-glucuronide), was a kind of dihydroxylflavone. It has attracted a lot of attention for its anti-tumor and anti-inflammatory activity in the gut and elsewhere and was used here as the model compound to prove the presence of this recycling scheme. Wogonin was selected because it is known to be extensively glucuronidated. It was also chosen because of its important pharmacological activities, including its ability to restore the sensitivity of tumor necrosis factor receptor apoptosis-inducing ligand (TRAIL) in TRAIL-resistant cancer cells. In human colon cancer HCT116 cells, it was shown that wogonin-induced apoptosis was via p53-dependent PUMA induction. Wogonin was also selected because it has been used widely in humans, mostly in the form of herbal formulation. For example, BZL 101, an aqueous herbal extract active against breast cancer cell lines, was shown to have a favorable toxicity profile in a phase I clinical trial and demonstrated encouraging clinical activity. Hange-shashin-to (HST), a combination of seven herbs including Scutellaria baicalensis, was found to suppress inflammatory bowel disease. Another reason to study wogonin was several flavonoids were shown to be active in management of the gastrointestinal diseases. For example, silibinin was shown to inhibit hepatitis C virus, whereas green tea flavonoids were shown to be active against colon cancer. Lastly, wogonin was also selected because large quantities of wogonoside were available commercially at a reasonable cost.Methods and Results.1. The conversion study of wogonin and wogonoside.A "four site perfusion model" was used to investigate the transport and metabolism of wogonin or wogonoside. Male Sprague-Dawley rats (80-110 days old) weighing from 280 to 350 g were obtained to operate the experiments, and four segments of the intestine (duodenum, upper jejunum, terminal ileum and colon) were perfused simultaneously with perfusate containing the compound of interest. The result showed that wogonin and wogonoside could convert to each other in the rat intestine individually. When wogonin of 5μM and 20μM concentrations was perfused, wogonoside was respectively found in all four segments of intestine. On the contrary, when wogonoside of 5μM and 20μM concentrations was perfused, wogonin was respectively found in all four segments of intestine. To our surprise, wogonin was also found in the upper segments of intestine (duodenum, jejunum). According to classical view,β-glucuronidase that hydrolyse glucuronides comes from bacteria. However, bacteria count in duodenum, jejunum was much lower than that of ileum and colon.In addition, wogonoside couldn’t be converted into wogonin in duodenum and jejunum, for lack of bacterialβ-glucuronidase.Compared with wogonoside, wogonin was smaller and less polar, and therefore it penetrated the intestinal membrane more rapidly. Moreover, P*eff of wogonin is 10 times higher than that of wogonoside. As expected, when wogonin or aglycone was perfused, it was well absorbed in all four segments of intestine. When wogonin of 5μM concentration was perfused, its absorption percentage was 66.87%,66.73%, 67.00%,74.62% in order at the four segments of intestine. When wogonoside was perfused, its absorption percentage was lower than that of wogonin at respective region of intestine.After blank intestinal perfusate of four segments were collected, wogonin and wogonoside was put in it individually. It was found that wogonoside was unstable in blank intestinal perfusate, wogonoside of 20μM concentration was almost hydrolysis into its aglycone within 24 hours. It proved that enzymes hydrolyzing wogonoside did exist in the blank intestinal perfusate, and further research should be carried on to determine the kinds and sources of the hydrolysis enzymes. Wogonin was found stable in blank intestinal perfusate, and wouldn’t convert into its glucuronide. It showed that no UGT exists in the blank intestinal perfusate, and the glucuronide found in the perfusate when wogonin was perfused, should be released from the enterocytes.2. The dissolubility, stability, adsorbability study of wogonin and wogonoside.The dissolubility, stability, adsorbability were investigated individually during experiment to assure the accuracy of all data. It was found that two compounds dissolve completely in HBSS buffer in the concentration range of 0.3125-160μM, and stability of the two chemicals could meet the requirement of absorption and metabolism study, all silica tube and EP tube used in the experiment didn’t show adsorbability to the two chemicals. All above results indicate that the wogonoside hydrolysis in gut and perfusate has no relation with their chemical stability, or dissolubility and adsorbability.3. The kinds study of the hydrolysis enzymes of wogonoside.Blank intestinal perfusate was collected to investigate the kinds of wogonoside hydrolysis enzymes. A stop solution (consisting of 94% acetonitrile and 6% glacial acetic acid) was first developed to ensure the enzyme hydrolysis could be ended at any time. It was demonstrated that wogonoside is stable in blank intestinal perfusate during 24 hours after the addition of the stop solution. Hence, the stop solution was added into all samples before their collections to stabilize the drug in samples.The influence of bacteria was first considered, and experiments were performed to determine the effects of bacteria presence. The results indicated that removal of bacteria via centrifugation (13,000 rpm,30 min) and aseptic filtration did not improve stability of wogonoside. Therefore, free hydrolysis enzymes might be the main reason for the wogonoside hydrolysis.Lactasc phlorizin hydrolase (LPH) is a major contributor to the glucoside hydrolysis in the intestine. After addition of gluconolactone, a specific inhibitor of the LPH, it was found that the hydrolysis of wogonoside was not inhibited.When 0.1 mM saccharolactone, the specific inhibitor of the glucuronidase was investigated, it was found that the hydrolysis of wogonoside was inhibited significantly. When the concentration rose from 0.1 mM to 4.4 mM, the hydrolysis of wogonoside was found inhibited thoroughly.The later perfusion experiment showed that the wognoside absorption and its aglycone conversion rate on the upper intestine would be inhibited by the specific inhibitor of the glucuronidase,0.1 mM saccharolactone, not by the specific inhibitor of LPH, gluconolactone. All the results showed thatβ-glucuronidase is exactly the enzyme responsible for the wogonoside hydrolysis.4. The source study of the hydrolysis enzymes of wogonoside.The P-glucuronidase in intestinal gut may come from three sources. The first source may be the liver, and the enzymes excreted there may enter gut with bile. The second source may be the intestinal microflora, this is the most obvious source and has been widely accepted. The third source may be the enterocytes.After adding wogonin and wogonoside into bile, both of the compounds were found stable. Moreover, a bile duct cannulation was made in perfusion experiments to avoid the bile entering the intestinal gut. Therefore, it’s impossible for theβ-glucuronidase to come from liver in this study.Sequential perfusate samples were collected from all four regions, and amount of bacteria in every sample was determined and then plotted against hydrolysis reaction rates measured using 20μM wogonoside. The results indicated that there was no correlation between the numbers of bacteria present in the perfusate and rate of wogonoside hydrolysis.To make acute antibiotic treatment, an antibiotic mixture of penicillin and gentamicin was added into the HBSS buffer containing 20μM wogonoside before perfusion. The results indicated that absorption or metabolism of wogonoside was not impacted. All these results didn’t accordance with the opinion that the P-glucuronidase in intestinal gut supplied by the intestinal microflora, and the intestinal bacteria couldn’t be the sole source for the intestinal gut. Based on above results, we conclude that enterocytes in rat intestine gut might be the main source ofβ-glucuronidase in intestinal gut.5. The kinetic validation of the source of GUS enzymeBecause of the lack of commercial monoclonal antibody to distinguish bacteriaβ-glucuronidase from mammalian one, kinetic parameters were used to distinguish them. To do so, rat intestine S9 fractions were prepared for S9 contains the smooth endoplasmic reticulum with most enterocyte enzymes in it, and the P-glucuronidase in S9 fractions could be referred, when the source of intestinal gut was considered. In addition, rat feces extract was also prepared, and the P-glucuronidase in feces extract could be referred, when the source of bateria was considered.We also selected four perfusate samples with drastically different amounts of bacteria (difference of greater than 2,700-fold), and found that 90-120 min jejunal perfusate sample had the highest Vmax value even though it has the lowest bacteria count. In contrast,60-90 min colon perfusate sample with the highest bacteria count had the lowest Vmax value.The above results showed that samples with higher bacteria count did not have faster hydrolysis rates. To confirm this finding, we also determine the metabolism of wogonoside in different perfusate collected at the same time, and the results again indicated that samples with the highest bacteria count (colon) did not have faster hydrolysis rates. However, the amounts of enzyme (as represented by the Vmax values) present in the perfusate appeared to decrease with time, indicating amount of enzymes present in the perfusate was less after continuous single-pass perfusion.To determine if protein concentration in the perfusate could correlate with the hydrolysis activities, the amounts of protein in the perfusate were plotted against hydrolysis rates, and surprisingly, there was no correlation either. In addition, we found that protein concentration did not decrease with perfusion time, indicating that protein in the perfusate may be due to sloughing off from the enterocytes or mucus or both. Lastly, there was no correlation between bacteria counts and protein concentration in the perfusate. The kinetic parameters were determined and Eadie-Hofstee plots were generated forβ-glucuronidase derived from intestinal S9 fractions, microbial enzymatic extract, and perfusate samples. The results indicated that enzyme-catalyzed hydrolysis of wogonoside in different sample matrix all followed a classic Michaelis-Menten kinetic profile. The Km values of intestinal S9 fractions from all three small intestinal segment were similar (25-30μM), but they were significantly higher than those derived from microbial enzymatic extract (7.7μM). When comparing to the Km values derived from the intestinal perfusate samples (15.8-25.6μM), they were all close to the Km values derived from the intestinal S9 fractions, except for two terminal ileal perfusate samples, which displayed Km values (6.3-6.9μM) closely resembled that of the microbial enzymatic extract. All the results showed that (3-glucuronidase in blank perfusion should mainly come from intestinal gut.6. The pharmacokinetic study of wogonin and wognosideTo study the pharmacokinetic characterization of wogonin and wognoside, blood samples were collected from fossa orbitalis to determine drug concentrations in blood after rat intragastric administration. Pharmacokinetic parameters such as AUCo-24h were obtained using 3P97 statistical moment to calculate. It was found that the blood drug concentration curves of the two compounds both displayed double peaks, and their curve profiles were similar. Most chemicals in blood were wogonoside, only little amount of wogonin exist, this indicated that wognonin was glucuronidated extensively in vivo, and exist in the form of wogonoside in blood. For pharmacokinetic parameters, the MRTo-T values of both wogonin and wogonoside were 11.53±1.27 and 9.36±0.95hour respectively, this might have relation with the local recycling in the gut.Conclusions.1. Wogonin and wogonoside were found to be converted to each other in the rat intestine, wogonoside could be hydrolyzed into its aglycone, which was then absorped rapidly.2. Wogonoside is mainly hydrolyzed byβ-glucuronidase. The result comes from the following facts. The wogonoside hydrolysis could be inhibited by the specific inhibitor of glucuronidase, saccharolactone. Moreover, the wogonoside absorption was found decrease significantly when saccharolactone was used to co-perfuse with wogonoside, and the absorption didn’t decrease when the specific inhibitor of LPH or antibiotics was used to co-perfuse with wogonoside.3. The activities ofβ-glucuronidase in the gut were mediated by enteric. The result comes from the fact that the enzyme hydrolysis activity in upper intestinal segments (ie.jejunum) was higher than that in colon, while the bacteria counts result showed that colon perfusate samples with the highest bacteria count than other three segments of the intestine.4. Theβ-glucuronidase activities were higher in upper small intestinal blank perfusate and that the Km values of hydrolysis reaction in duodenal and jejunal blank perfusate were nearly identical to those of their intestinal S9 fraction, respectively. Their kinetic profiles were also very similar as shown by both regular concentration versus rate plots and Eadie-Hofstee plots.5. After rat intragastric administration, it was found that the MRTo-T values of both wogonin and wognoside were rarely high, their blood drug concentration curves displayed double peaks, and curve profiles were similar.6. No free glucuronidation enzyme exist in the gut, and they only exists in the enteroytes. Wogonin was very stable in blank intestinal perfusate, the glucuronide found in the perfusate when wogonin was perfused, should be released from the enterocytes.Our conclusion that flavonoid glucuronides can be recycled locally was well-supported by the above results. In conclusion, we have discovered a novel local recycling mechanism that will significantly enhance the local bioavailability and residence time of flavonoids such as wogonin. This local recycling mechanism has the potential to significantly impact the bioavailabilities of other drugs and dietary chemicals that also undergo extensive glucuronidation in the gut. Because a large numbers of phenolics are glucuronidated significantly in gut, this newly discover recycling mechanism should have a significant physiological role in governing the local bioavailability and residence time of phenolics, which will ultimate impact the pharmacodynamic and toxicological effects of this class of important compounds, many of which are showing promise as chemopreventive agents. |
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