Study On The Mechanism Of Decreasing Portal Pressure By Octreotide And Promoting Intestinal Absorption Of It In Jejunum

Objective: Hepatitis B is an important reason to cause liver cirrhosis. There arehundreds of patients with hepatitis B or with hepatitis B virus infection in China, andmillions of them can develop to cirrhosis very year. Portal hypertension (PH) is thetypical manifestation of patients at the decompensated stage of cirrhosis, and it is themain reason to result in various complications and death. So it is important to preventand treat of patients with PH, thus improving the prognosis and decreasing the mortalityof patients.It is revealed that heme oxygenase/carbon monoxide (HO/CO) system canaggravate PH during the middle and end stage of cirrhosis, and induce progressivedamages of various organs and tissues, thus accelerating cirrhosis process. The resultswere also confirmed by our studies on the basis of cirrhotic rats with PH. But up to now,the researches focused on HO/CO system mostly are basic studies, and lack of clinicalstudies. We had found that carboxyhemoglobin, which could reflect the level of HO/COsystem was obviously increased in patients with PH. The current research was the firsttime to perform perspective study to reveal the importance of HO/CO system onpatients with PH complicated by various complications and the effects of it on organs.As a kind of Somatostatin, Octrotide (OCT) is an ideal drug for decrease portal pressure,but the detail mechanism is unclear. Due to the effects of vascular contraction, OCT canreduce portal blood volumn and decrease portal pressure. Due to the mechanism ofendogenous OCT to maintain PH is dilation of splanchnic vessels, we thought OCTmight play effects in decreasing portal pressure by influence on HO/CO system. Thisstudy was to deeply research the mechanism of OCT effects on PH by HO/CO system,and further confirm the key role of HO/CO system on cirrhosis.On the basis of the two parts of experiments and results of previous studies, we found that HO/CO system played great role during PH development and OCT coulddecrease portal pressure by inhibition of the system. Consideration of variouspharmacological advantages of OCT, it is useful to realize oral administration of OCTin order to resolve the difficulties of prevention and treatment of PH. Due to itsstabilized structure against enzymatic degradation, OCT can partially overcome theproblems of therapeutically active peptides often limited by their short biologicalhalf-lives. But OCT has high molecular weight, less fat-soluble quality, influenced byfirst-pass effect of intestines and liver, and prohibited by intestinal absorption barrier, soits oral bioavailability is low. Up to now, many researchers are focused on increasingOCT absorption by change its chemical structures or increase paracellular absorptionsthrough adding absorption enhancers, and enhancing permeability of biologicalmembrane, but the absorption effects are not ideal. In addition, the mechanisms of OCTabsorption and ways to increase absorption have not been studied underwent PH state.We thought studies on factors which directly influence on first-pass effects of OCTabsorption might be useful for finding out the absorbed strategies. Because ofportosystemic shunt formation, collateral circulation establishment and abnormal liverfunction, hepatic first-pass effects plays little influence on OCT absorption, while thefirst-pass effects of intestine may take obvious roles. The current study was to studyp-glycoprotein (P-gp) and multidrug resistance associated protein2(MRP2) which arethe transport proteins located on intestines, and intestinal metabolic enzyme cytochromeP4503A4(CYP3A4), in order to reveal the absorption mechanisms of OCT inintestines. In addition, whether OCT absorption would be affected by changes of thesetransporters and enzyme underwent cirrhosis with PH was also been considered.Moreover, the effects of PepT1changes underwent PH on OCT absorption were alsobeen studied, for finding a transporter which could transport OCT directly.The aim of this study was to reveal the pivotal effects of HO/CO system duringcirrhosis with PH by basic and clinical researches from various angles, in order to findout the mechanism of decreasing portal pressure by OCT mediated by this system,definite the reason hindered the intestinal absorption of OCT and provide the solutionsfor reducing intestinal-pass effects. It was also useful to find out the effective ways forincreasing oral bioavailability of OCT, realize portal pressure reduction by oraladministration of it, and provide theory evidence for rational use of OCT in clinic at thesame time. Part IStudy on the mechanism of decreasing portal pressure by Octreotidemediated by heme oxygenase/carbon monoxide systemQuarter IThe effects of heme oxygenase/carbon monoxide system on portal hypertensionObject: The aim of this study is to detect changes of carboxyhemoglobin (COHb)levels and the relationship between them and portal hypertension (PH) complicated byadvanced complications, and to know the correlation of COHb levels and hepaticencephalopathy (HE) degrees on patients of hepatitis B related cirrhosis with HE,hoping for revealing the effects of heme oxygenase/carbon monoxide (HO/CO) systemon patients with PH complicated by complications and on various organs.Methods: According to the diagnostic and excluding criterions,68inpatients withhepatitis B virus-related cirrhosis (HBC) complicated by HE who were admitted to theGastrointestinal Department of the First Affiliated Hospital of Dalian MedicalUniversity without treatment were enrolled (group H).22patients who were inemergency department and excluded vital organs disorders (all related clinical testswere normal) were selected as control group (group N). The information of clinical datawas collected. The patients were performed related tests, and the data ofcarboxyhemoglobin (COHb) levels, partial pressure of oxygen (PaO2), oxygensaturation (SaO2) and so on were gathered. The patients were received diagnosticanalyses of HE stages, esophagogastric varices, hepatic renal syndrome (HRS),spontaneous bacterial peritonitis (SBP) and hypoxemia. The differentiations of COHb inHE patients with PH complications and none were compared, and the relationship ofCOHb level among grades of HE, PaO2and SaO2were analyzed.Results: The level of COHb in Group H was higher than in Group N((2.102±1.021)%vs.(0.983±0.231)%, P=0.000）. The COHb standard has positivecorrelation with Grade of HE (p=0.003, r=0.358). The content of COHb was increasedin patients without HRS comparison to patients with HRS ((1.981±1.020)%vs.(2.502±1.073)%，p=0.029), with no differences between patients with SBP or not(p=0.75), and with esophagogastric varices or none (p>0.05). COHb level has anegative correlation with PaO2(p=0.006, r=-0.336) and no relative to SaO2(p=0.576,r=-0.072). It was higher when the two parameters met diagnostic criteria of hypoxemia ((2.72±0.362)%vs.(1.93±0.242)%，p=0.012).Conclusions：Heme oxygenase/carbon monoxide system played an important rolein pathophysiological process of hepatic cirrhosis with portal hypertension, and its levelhas tissue-specific. This system has great clinical meanings in cirrhotic patients with PHcomplicated by various complications. Quarter IIThe research on the mechanism of decreasing portal hypertension by Octreotidemediated by heme oxygense/carbon monoxide systemObject: The aim was to understand the mechanism of reducing portal pressure byOCT, to observe whether the process was mediated by heme oxygenase/carbonmonoxide (HO/CO) system and to confirm the pivotal effects of HO/CO on cirrhosiswith portal hypertension (PH).Methods:34Male Sprague-Dawley rats were divided randomly into a Sham group(n=10), PH group (n=12) and OCT group (n=12). The cirrhotic rats with PH wereestablished by common bile duct ligation. The group which intraperitoneallyadministrated Otreotide (100μg/kg/d) was regarded as group OCT. The rats in group PHand group Sham was treated with the same doses of saline. After drugs administrationfor3days, all rats were measured portal venous pressure and then were killed forcollection of blood and liver tissues. HE staining was performed to observe thepathomorphology of liver tissues. The expressions of HO-1protein and mRNA weretested by the ways of immunohistochemistry, western blot and reverse transcriptionpolymerase chain reaction (RT-PCR), respectively. The levels of alanineaminotransferase (ALT) and aspartate aminotransferase (AST) were assayed byautomatic blood biochemistry analyzer.Results: In group PH, the levels of ALT and AST were much higher, and wascharacterized by fibrosis proliferation, amount of inflammatory cells infiltration,pseudolobule formation, and proliferation of interlobular bile duct, moreover, the portalpressure ((18.52±1.83) mmHg) was greatly increased, all the results were exited statistical difference between group Sham (p<0.01). In group OCT, the contents of ALTand AST, the lesions degree of liver tissues by HE staining and portal pressure((13.17±1.12mmHg) were greatly lightened (p<0.05), but worse than in group Sham(p<0.05). The methods of immunohistochemistry and western blot showed that theprotein expressions of HO-1in group OCT was evidently lower than in group PH(P<0.01), but higher than in group Sham (p<0.05), moreover, the way of RT-PCR wasconfirmed the results form mRNA levels.Conclusions: The expressions of heme-1in liver can be inhibited by OCT at thestage of cirrhosis with portal hypertension. The regulation of OCT to heme oxygense/carbon monoxide system may be regarded as a mechanism of it effects on portalpressure. The heme oxygense/carbon monoxide system plays an important role on theformation of portal hypertension. Part IIThe effects of transports and metabolic enzymes onOctreotide absorption in intestinesObject: The transports, such as P-glycoprotein (P-gp) and multidrug resistanceassociated protein2(MRP2), and metabolic enzyme cytochrome P4503A4(CYP3A4)located on intestines are regared as key factors for affecting on drugs transport andmetabolism. The aim of this study was to know the relationship of them and Octreotide(OCT), and to understand whether their changes underwent cirrhosis with portalhypertension (PH) can influence on OCT absorption.Methods: The effects of P-gp or Mrp2inhibitors rhodamine123(1mM) andprobenecid (1mM) on the uptake and transepithelial transport of OCT (10μM) wereobserved by permeability direction rate (PDR) and from apparent permeabilitycoefficient (Papp) received by OCT bidirectional transportation from apical side (AP) orbasolateral side (BL). The effects of OCT concentrations (1μM and50μM) ontransportation were also considered. The cirrhotic rats with PH were established bycommon bile duct ligation. The effects of P-gp or MRP2inhibitors verapamil (1mM)and probenecid (1mM) on transportation of OCT (10μM) were observed by the model of everted intestinal sacs. The normal rats were divided into group A, B, C and D (n=4).The PH rats were did the same experiments and in accordance divided into group A1,B1, C1and D1(n=4). In situ jejunal perfusions of rats were also performed, and thedoses and grouping were the same as in the experiments of everted intestinal sacs (n=4).The effect of CYP3A inhibitor ketoconazole on intestinal metabolism of OCT wasdetermined by intestinal microsomes obtained from both kinds of rats. Each group wasadded OCT (100μM). According to whether ketoconazole (10μM) was added, thenormal rats were separated to group N+OCT and group N+OCT+K. The same drugs’doses and grouping were performed to PH rats, and in accordance divided into groupPH+OCT and group PH+OCT+K (n=6). The group without microsomes was regardedas control (group OCT).The experiment of human recombinant CYP3A4was applied for determinewhether OCT was one of substrates of CYP3A4. Firstly, the best incubation time wasdetermined by changes of reaction time by limitation of protein content of recombinantCYP3A4(2.5mg/mL) and the OCT concentration (100mM). Secondly, the bestincubated protein concentration of recombinant CYP3A4was observed by limitation ofother two factors. According to the optimized protein contents of CYP3A4and reactiontime, different OCT concentrations (20,50,100,200,400mM) were put into thereaction mixture and found out the metabolic productsResults1. It was no concentration dependence of OCT transportation across Caco-2cells.The permeability direction rate PDR>1.5when P-gp or/and MRP2were inhibited at theconcentrtation of OCT10μM. The Pappof transportation for10μM OCT from side B toA mediated by P-gp and MRP2was significantly increased than the Pappoftransportation from side A to B, and Papp (A-B)s were much increased when inhibitedP-gp or MRP2, especially for inhibition P-gp and MRP2at the same time, on thecontrary, Papp (B-A)s were decreased obviously.2. In the model of everted intestinal sacs, the concentration of OCT in group D wasincreased comparison to other three groups (p<0.05). Moreover, compared with group C,the OCT content in group B was much higher (p<0.05). The same phenomenon couldbe seen in PH rats. Moreover, the OCT contents in PH rats were decreased obviouslycompared with those of the corresponding normal group (p<0.05). The AUC values ingroup B, C and D were176.22%,151.39%, and220.63%of that of the group A,respectively. The AUC values of the group B1, C1and D1were151.82%,128.23%, and165.33%of that in the group A1. While the AUC of the group A1was21.54%ofthat in the group A.3. In the model of in situ jejunal perfusions of rats, the levels of OCT in Group Dwere increased significantly (p<0.05). The OCT content in group C was much higherthan in group C (p<0.05). The same phenomenon could be seen in PH rats. In addition,the OCT contents in PH rats were decreased compared with those of the correspondingnormal group (p<0.05). The AUCs of the group B, C and D were521.08%,418.95%,618.40%of that of the respective control group (group A). The AUCs of the Group B1,C1and D1were384.61%,236.28%,461.78%of that in the group A1. While the AUCof the group A1was96%of that of the group A.4. In the experiments of intestinal microsomes, the levels of OCT were eminentlylowered in group PH+OCT than in group N+OCT (p<0.05), were obviously increasedwhen usage of ketoconazole (group N+OCT<group N+OCT+K, group PH+OCT<group PH+OCT+K, p<0.01). The contents of OCT in group OCT were much higherthan in Group N+OCT and in group PH+OCT (p<0.01). There was no difference ofOCT contents between group N+OCT+K or PH+OCT+K with group OCT (p>0.05).5. In the experiments of human recombinant CYP3A4, the best incubation timewas10min and the best protein was2.5mg/mL. Under the optimized incubation timeand protein content of CYP3A4, the residual OCT contents were decreased along withreduced initial concentration of OCT (p<0.05), and also many micromolecularmetabolites were found by test of Liquid chromatography-tandem mass spectrometry.Conclusions: P-glycoprotein, multidrug resistance associated protein2, andmetabolic enzyme cytochrome P4503A4were played inhibitory effects on Octreotideintestinal absorption, and Octreotide is the substrate of them. Up-regulated expressionsor activities of them were found under cirrhosis with portal hypertension, and enhancedblocking effects of OCT in intestines. Part IIIThe research on pormoting intestinal absorption ofOctreotide and decreasing portal pressuer under cirrhosiswith portal hypertensionObject: The aim was to know the influence of inhibitors of P-glycoprotein (P-gp),multidrug resistance associated protein2(MRP2) and cytochrome P4503A4(CYP3A4)on intestinal absorption of Octreotide (OCT) and the effects of them on decreasingportal pressuer. In addition, the impact of oligopeptide transporter PepT1on OCTabsorption was also studied, in order to find an effective way for realizaion of OCT byoral administration.Methods:1. The in vivo absorption experiment in rats was performed. The Normal rats weredivided randomly into two groups:1) intra-jejunal (IJ) injection of OCT (1mg/kg),2) i.v.infusion of OCT (0.1mg/kg)(n=4). PH rats were divided randomly into three groups:1)IJ injection of OCT (1mg/kg),2) i.v. infusion of OCT (0.1mg/kg),3) IJ injection ofOCT (1mg/kg) and mixed inhibitors (verapamil hydrochloride4.9mg/kg, probenecid300mg/kg and ketoconazole5.3mg/kg)(n=4, separately). The pharmacokineticparameters were caculated.2. The normal rats were divided into three groups according to if administratedOCT or not (group N and group N+OCT, respectively). PH rats were divided inaccordance to group PH and group PH+OCT, and the rats with OCT and mixedinhibitors (verapamil hydrochloride4.9mg/kg, probenecid300mg/kg and ketoconazole5.3mg/kg, once a day) were regared as group PH+OCT+I (n=4, separately). The dose ofOCT was1mg/kg, once a day. After3days, all rats were killed and the ways of westernblot, immunohistochemistry and reverse transcription polymerase chain reaction(RT-PCR) were performed to test the protein and mRAN expressions of P-gp, MRP2and CYP3A4on jejumns of each group.3. The PH rats were divided into three groups according to whether oraladministration of OCT (1mg/kg, once a day), and if orally administrated mixedinhibitors or not (group PH, group OCT, and group PH+OCT+I). The normal rats weregiven routine food and water without any drug. The doses of inhibitors were the same asabove (n=4, separately). After3days, the portal pressure of each rat was measured.4. The normal rats were divided into two groups according to whether oral administration of OCT (1mg/kg, once a day) or not (group N and group N+OCT). ThePH rats were did the same experiments and were grouping in accordance to group PHand group PH+OCT. After3days, the rats were killed and the ways of western blot,immunohistochemistry and RT-PCR were performed to test the protein and mRANexpressions of PepT1on jejumns of each group. The effects of PepT1inhibitorglycylsarcosine (Gly-Sar)(1mM) on the uptake and transepithelial transport of OCT(10μM) in Caco-2cells were observed by permeability direction rate (PDR) and fromapparent permeability coefficient (Papp) received by OCT bidirectional transportationfrom apical side (AP) or basolateral side (BL)(n=4, respectively). The models ofeverted intestinal sacs were established, and according to whether Gly-Sar wasadministrated, normal rats were divided into group N and group N+G. The PH rats weredid the same experiments, and in accordance to divided as group PH and group PH+G(n=4). In situ jejunal perfusions of rats were also performed, the doses and groupingwere the same as in the experiments of everted intestinal sacs (n=4).Results1. In vivo experiments, OCT was rapidly dispelled from plasma after i.v. infusion,and it underwent slower elimination or easier absorption in normal rats than in PH rats.Increased expression or activities of transporters and metabolic enzymes in liver mayoccur, leading to quicker elimination in PH state. By IJ administration of OCT, longerTmax, lower Cmaxand AUC0-12hwere found in PH rats than in normal rats, indicating thatincreased expressions or activities of transporters and metabolic enzymes might exist inintestines of PH rats to inhibit OCT absorption. Shorter Tmax, higher Cmaxand AUC0-12hwere observed in the group of OCT with P-gp/MRP2/CYP3A mixed inhibitorscomparing to normal rats and PH rats without inhibitors. In addition, F and ER wereobviously increased about4times for PH rats when usage of mixed inhibitors.2. The results from western blot and immunohistochemistry found that proteinexpressions of P-gp/MRP2/CYP3A4were significantly increased in Group PH than inGroup N (p<0.05), were obviously increased when usage of OCT (GroupN+OCT>Group N, Group PH+OCT> Group PH, p<0.05), and were the least in GroupPH+OCT+I among all the groups because of inhibitors usage (p<0.01), whereas noeminent difference between Group N+OCT and Group PH (p>0.05). The results ofRT-PCR were the same as western blot showed from mRNA levels.3. The average PVP level of Group PH (15.56±2.36mmHg) was increased thannormal rats (9.24±0.76mmHg, p<0.01) and was higher than that of Group PH+OCT (12.51±1.50mmHg, p<0.05). The PVP level of Group PH+OCT+I (6.95±1.12mmHg)was much lower than that of Group PH and Group PH+OCT (p<0.01). It was indicatedthat OCT with mixed inhibitors by oral via could decrease PVP effectively.4. RT-PCR showed that the expressive levels of PepT1mRNA were significantlyhigher in PH group than in group N (p<0.05). Compared the groups without OCTtreatment, the mRNA expression levels of PepT1in groups with OCT were no obviousdifference (group N+OCT vs. group N, group PH+OCT vs. group PH, p>0.05), whilein Group PH+OCT was higher than in group N+OCT (p>0.05). The results of westernblot and immunohistochemistry were the same as RT-PCR showed from protein levels.Caco-2cell model was used to examine the effects of PepT1inhibitor on OCT uptake.The Papp of transportation for OCT from side A to B mediated by influx pumptransporter PepT1was significantly decreased than the Papp of transportation from sideB to A. The PDR<1.5revealed that PepT1could not mediate OCT transport in Caco-2cells. On the basis of everted intestinal sacs, compared with Group N, the serosal sideconcentration of OCT in Group N+G was no obvious difference (p>0.05). The samephenomenon could be seen in PH rats. These results suggested PepT1could not mediateOCT transport, not only in normal rats, but also for rats with PH. While the AUC of thegroup PH was52.4%of that in the group N. In the model of in situ perfusions of rats,there was no obvious difference of OCT concentration between Group N and GroupN+G (p>0.05). The same phenomenon could be seen in PH rats. The results revealedthe similar conclusions of experiments of everted small intestinal sacs that PepT1couldnot transport the OCT uptake in situ, either for normal rats or PH rats. While the AUCof the group PH was67.2%of that in the group N.Conclusions: P-glycoprotein, multidrug resistance associated protein2andcytochrome P4503A4play great important role in intestinal absorption of Octreotide.Inhibition of them can induce OCT absorption of rats with portal hypertension, andrealize ideal effects of decreasing portal pressure. The expressions of oligopeptidetransporter PepT1were enhanced on the stage of cirrhosis with portal hypertension, butit has no effect on transportation of OCT in intestines of rats.