Study On The Mechanism Of Morphine Antinociception Based On Drug Metabolism And Transport

Pain prevention and treatment are the international front-burner issues in the clinic medical domain.When the drastic pains like trauma,surgery or burns as well as the intense pains like radio or chemotherapy generate,analgesic opioid drug,morphine which is the longest and most widely used in clinic will be performed its strong antinociception.However,side-effects such as tolerance,dependent or addiction can significantly weaken its antinociception,which means regulating its side-effects would be necessary for the change of its pharmacological effect.Recent research demonstrates morphine is mainly metabolized in human liver and colon by the glucuronidation of drug enzyme,uridine diphosphate-glucuronosyltransferase 2B7(UGT2B7).In this process,two main glucuronides including morphine-3-glucuronide(M3G)and morphine-6-glucuronide(M6G)were produced at the meantime.The antinociception of M6G is much stronger than M3G,but after administrating same doses of M6G and morphine to mice,the former one need more to make the tolerant ones recover initial response.Meanwhile,M3G can antagonize this tolerance produced by M6G and it would be responsible to the tolerance reverse.That reflects M6G may perform different roles in morphine antinociception and tolerance reverse.Besides that,morphine and its metabolites can cross the brain blood barrier(BBB)to the central nervous system then come into play.Since there are few reports to illustrate whether some uptake transporters mediate this process,so novel mechanisms explosion for morphine brain transport will be indispensable.On the account of these two aspects,our study was from the perspective of morphine's metabolism and transport to explore the relationship between morphine antinociception enhancement and tolerance reverse.Several regulations of main enzymes and transporters in the maladies such as single nucleotide polymorphisms(SNPs),dimerization,epigenetic modifications even endogenous chemicals would affect their expressions then alter their funtions.To eliminate these mechanisms,we divided the dissertation into quartered section to make a further investigation.Sectionl:Establishment of HPLC-MS/MS method for simultaneous analysis of morphine and its two glucuronides.To determine the concentrations of morphine and its two glucuronides from different biological matrixes,we estiblished and validated the HPLC-MS/MS method.The deuterated internal standard,M6G-d3,was mixed with prepared samples at 1 Ong/mL final concentration.The sample was injected into the Aglient HILIC PLUS SB-C18(2.1 mm×50mm,3.5?m)column with gradient elution for separation.The mobile phase consisted of purified water solution containing 0.5‰ formic acid(pH3.2)and methanol.Multiple reaction monitoring,MRM,has been applied to analyze three compounds in the positive ion mode.MRM ion pairs are morphine(m/z 286.0-200.9),M3G and M6G(m/z 462.1-286.1),M6G-d3(m/z 465.1-289.1).The results of method validation illustrate the specificity was good,and it has a linear calibration range from 0.05 to 10ng for all the three analytes with calibration coefficients(r2)>0.9930.The lower limit of quantitation(LLOQ)for each analyte was 0.05ng/mL,accuarcy and precision were good at low,medium and high concentrations for all of them.No remarkable matrix effect was observed for each biological sample,the stability for each of the analytes was also reasonable.In conclusion,this method was successful established,and can be used to measure morphine and its two glucuronides in biological samples.Section2:The Regioselective Glucuronidation of Morphine by Dimerized Human UGTs and Their Allelic Variants.We primary researched the genetic effects of UGT2B7 induced its enzymatic activity change to regioseletive alter morphine metabolism.UGT2B7 and its allelic variants can dimerize with the homologous enzymes UGT1A1 and UGT1A9,as well as their allelic variants,and then change their enzymatic activities in the process of substrate catalysis.The current study was designed to identify this mechanism when induced by morphine,which is a substrate of UGT2B7.The production ratio of its aberrant metabolism and subsequent metabolites,M3G and M6G,were found to change regioselectively.Single-recombinant allozymes,including UGT2B7*1(wild type),UGT2B7*71S(A71S,211G>T),UGT2B7*2(H268Y,802C>T),UGT2B7*5(D398N,1192G>A),and double-recombinant allozymes formed by the dimerization of UGT1A9*1(wild type),UGT1A9*2(C3Y,8G>A),UGT1A9*3(M33T,98T>C),UGT1A9*5(D256N,766G>A),UGT1A1(wild type)with its splicing variant UGT1 Alb were established and incubated with morphine in vitro.Each sample was analyzed by HPLC-MS/MS.All enzyme kinetic parameters were then measured and analyzed.Double-recombinant allozymes exhibit stronger enzymatic activity catalyzing morphine than the single-recombinant allozymes.Compared to UGT2B7*11 UGT2B7*2 singles or doubles have lower Km values for M3G and M6G,whereas UGT2B7*5 allozymes show opposite effects.The double allozymes of UGT1A9*2 or UGT1A9*5 with UGT2B7 tend to produce M6G.Interestingly,the majority of single or double allozymes significantly reduce the ratio of M3G to M6G.The UGT1A9*2-UGT2B7*1 double enzyme has the lowest M3G:M6G ratio,which reflects that more M6G would form in morphine glucuronide metabolism.Single-or double-recombinant allozymes of UGT2B7,UGT1A1 and UGT1A9 as well as their allelic variants can influence the regioselectivity of morphine glucuronidation and alter enzyme-substrate affinities.Section3:Mechanism Research of the Repression of UGT2B7 Mediated Morphine Glucuronidation Alteration in Colorectal Carcinoma.UGT2B7,as one of significant drug enzymes,is responsible for the glucuronidation of abundant endobiotics or xenobiotics.We here report that it is markedly repressed in the tumor tissues of colorectal carcinoma(CRC)patients.Accordingly,morphine in CRC cells will stimulate the expression of its main metabolic enzyme,UGT2B7 during tolerance generation by activating the positive signals in histone 3,especially for trimethylated lysine 27(H3K4Me3)and acetylated lysine 4(H3K27Ac).Further study reveals that brain-derived neutrophilic factor(BDNF),a secretory neurotrophin,enriched in CRC can interact and inhibit UGT2B7 by primarily blocking the positive signals of H3K4Me3 as well as activating H3K27Ac on the promoter region of UGT2B7.Meanwhile,BDNF repression attributes to the sensitizations of main core factors in poly-comb repressive complex(PRC)1 rather than PRC2 as the reason of the depression of SUZ12 in the later complex.Besides that,the productions of two main morphine glucuronides are both increased in the BDNF deficient or TSA and BIX-01294 treated morphine tolerance-like HCT-116 cells.On the same condition,active metabolite,M6G was accumulated more than inactive M3G.Our findings imply that enzymatic activity enhancement and substrate regioselective catalysis alteration of UGT2B7 may release morphine tolerance under the cure of tumor-induced pain.Section4:Reverse of Acute and Chronic Morphine Tolerance by Lithocholic Acid via Down-regulating UGT2B7.We further researched some endogenous chemicals induced UGT2B7 enzymatic activity alteration to regio-selective impact morphine glucuronidation.Interestingly,lithocholic acid(LCA)deposited in human livers always induces drastic pains which need analgesic drug,like morphine to release.Our research showed that LCA can effectively inhibit UGT2B7 in morphine tolerance-like human normal liver cells,HL-7702,then increase ?-opioid receptor(MOR)and calcium-calmodulin dependent protein kinase ?a(CaMKIIa)expression.In vivo assay,UGT2B7 was significantly inhibited in the livers of acute or chronic morphine tolerance mice pretreated with LCA(10,50 and 100 mg/kg,p.o.).To investigate the connections between LCA and enzymatic activity changes of UGT2B7 in mice livers,two morphine metabolites,M3G and M6G were quantified by solid phase extraction(SPE)connected with HPLC-MS/MS.The result indicated no matter in acute or chronic tolerance,the concentrations of M3G and M6G were all decreased.In addition,50mg/kg of LCA administration can prevent auto-phosphorylation of CaMK?a at Thr286 in acute or chronic morphine tolerance mice prefrontal cortexes(mPFCs)due to synthesis increase of cyclic adenosine monophosphate(cAMP).Therefore,UGT2B7 depression mediated by LCA can affect its catalysis to morphine,then that may be responsible to acute or chronic morphine tolerance alleviation.These findings might assist to modify antinociception of morphine in clinic.Section5:Mechanism Research of Acute Morphine Tolerance Reverse via OATP2B1.Regulating main brain-uptake transporter of morphine may restrict its tolerance generation,then modify its antinociception.In this study,more than 2-fold higher intracellular uptake concentrations for morphine and M6G were observed in stable expression cells,HEK293-hOATP2B1 than HEK293-MOCK.Specifically,the Km value of morphine to OATP2B1(57.581±8.90?M)is 1.4-time more than that of M6G(80.31±21.75?M);Cyclosporine A(CsA),an inhibitor of OATP2B1,can inhibit their intracellular accumulations with IC50=3.90±10.50?M for morphine and IC50=6.04±0.86?M for M6G,respectively.To further investigate the role of OATP2B1 in morphine brain transport and tolerance,the novel nanoparticles of DGL-PEG/dermorphin capsulated siRNA(OATP2B1)were applied to deliver siRNA into mouse bra:in.Along with OATP2B1 depressed,a main reduction was found for each of morphine or M6G in cerebrums or epencephalons of acute morrphine tolerance mice.Furthermore,calcium/calmodulin-dependent protein kinase ?a(CaMK?a)in mouse prefrontal cortex(mPFC)underwent dephosphorylation at Thr286.In conclusion,OATP2B1 downregulation in mouse brain can suppress tolerance via blocking morphine and M6G brain transport.These findings based on the metabolism and transport might help to improve the pharmacological effects of morphine.