Pharmacokinetic Mechanisms Of JBP485 On Alleviating Imipenem And Aristolochic Acid Ⅰ-induced Nephrotoxicity

Both imipenem（IMP）and aristolochic acidⅠ can cause kidney injury in clinical practice.IMP,the first commercially availableβ-lactam agent of the carbapenem class,possesses an extremely wide spectrum of antibacterial activity in vitro,encompassing Gram-negative and Gram-positive aerobic and anaerobic species.Notably,IMP is rapidly catalysed to inactive and toxic metabolites by the dehydropeptidase-I（DHP-I）enzyme at the luminal face of proximal tubular cells in the kidneys,resulting in nephrotoxicity.Aristolochic acids are organic compounds that naturally exist in plants of the Aristolochiaceae family,such as Aristolochia and Asarum.Aristolochic acidⅠ,the main toxic component of aristolochic acids,causes direct injury to kidney tubular cells and alters the expression level of renal transporters.IMP and aristolochic acidⅠ are both substrates of organic anion transporter 1（OAT1）and OAT3.It has been shown that cilastatin can simultaneously inhibit OAT1/3-mediated IMP renal excretion to reduce its accumulation in the kidney and inhibit the metabolism of IMP by DHP-I to improve its stability,thus increasing its effectiveness and reducing its toxicity.Probenecid（an inhibitor of OATs）and rhein（a substrate of OATs）also alleviate the nephrotoxicity caused by aristolochic acidⅠ via inhibiting its renal uptake.JBP485（Cyclo-trans-4-L-hydroxyprolyl-L-serine）,an anti-hepatitis dipeptide,was first isolated from Laennec（the hydrolysate of the human placenta）.JBP485 has been proved to be a substrate of OAT1/3 and is able to inhibit the renal excretion of related substrates.Meanwhile,JBP485 has antioxidant,anti-inflammatory and anti-apoptosis properties,which can reverse the changes in the expression of related transporters caused by kidney injury,thus alleviating the acute kidney injury induced by cisplatin,gentamicin and vancomycin.Combined with the above findings,we hypothesized that JBP485 may cause drug-drug interactions（DDIs）when combined with IMP or aristolichic acidⅠ.Therefore,this study aims to reveal the target of DDIs between JBP485 and IMP or aristolochic acidⅠ,and explore the possibility and mechanism of JBP485 to alleviate IMP or aristolochic acidⅠ-induced nephrotoxicity,so as to provide scientific basis for the safe and reasonable clinical application of IMP or aristolochic acidⅠ.Part I Development of LC-MS/MS methods for determination of aristolochic acidⅠ in rat plasmaObjective:To develop a LC-MS/MS analysis method for determination of aristolochic acidⅠ in rat plasma.Methods:After protein precipitation using acetonitrile,the analyte was separated on a Hypersil BDS-C18 column.The mobile phase consisted of acetonitrile and water with 0.1%（v/v）formic acid（80:20,v/v）.The flow rate was 0.4 m L/min.The ionization was conducted using an ESI source in positive ion mode for aristolochic acidⅠ and metformin（IS）.Multiple reaction monitoring（MRM）mode was utilized to detect the target compound.The selected transitions were m/z359.9→298.1 for aristolochic acidⅠ and m/z 130.0→71.2 for IS.Results:The linear range of the calibration curves for aristolochic acidⅠ was1-1000 ng/m L.The intra–day and inter–day precision（RSD）of aristolochic acidⅠ were both less than 4.45%and 2.79%,respectively.Conclusions:An efficient,sensitive,rapid and exclusive LC-MS/MS analysis method was established for determination of aristolochic acidⅠ in rat plasma.Part II JBP485,a dual inhibitor of organic anion transporters（OATs）and renal dehydropeptidase-I（DHP-I）,protects against imipenem-induced nephrotoxicityObjective:IMP possesses a broad spectrum of antibacterial activity;however,nephrotoxicity limits its clinical application in patients with renal insufficiency.In our previous studies,JBP485,was found to attenuate drug-induced kidney injury.The current study aimed to explore whether JBP485 could relieve IMP-induced kidney injury and clarify the potential molecular pharmacokinetic mechanism.Methods:The effects of JBP485 on IMP nephrotoxicity were evaluated in rabbits and human kidney 2（HK-2）cells.DDIs mediated by OATs and DHP-I were explored through pharmacokinetic studies in rats,metabolism assays in the kidney,and uptake studies in kidney slices and OAT-over-expressing cells.Results:The results revealed that JBP485 significantly ameliorated IMP-induced nephrotoxicity in rabbits.Further,incubation of HK-2 cells with JBP485 or cilastatin markedly improved the cell survival rate,inhibited apoptosis and attenuated mitochondrial damage by improving the stability of IMP and reducing its intracellular accumulation.This suggests that DHP-I and OATs might be involved in the protective effect of JBP485.Furthermore,coadministration with JBP485 significantly increased the plasma concentration and the area under the plasma concentration-time curve（AUC）of IMP,while decreasing the renal clearance and cumulative urinary excretion of IMP.Moreover,JBP485 reduced IMP uptake in kidney slices and OAT1/3-human embryonic kidney 293（HEK293）cells.Meanwhile,the metabolism of IMP by DHP-I was inhibited by JBP485 with an IC₅₀value of 12.15±1.22μM.Finally,the molecular docking assay revealed a direct interaction between JBP485 and OAT1/3 or DHP-I.Conclusions:JBP485 protected against IMP nephrotoxicity in rabbits and HK-2cells by improving IMP stability and reducing its intracellular accumulation via simultaneous inhibition of renal OATs and DHP-I.JBP485 is a promising renoprotective agent and could serve as an effective supplement to reduce IMP-induced adverse renal reactions in the clinical setting.Part III Molecular pharmacokinetic mechanism of JBP485 againstAristolochic acidⅠ（AAI）-induced nephrotoxicityObjective:The aim of this study was to determine the protective effect and molecular pharmacokinetic mechanism of JBP485 against AAI-induced nephrotoxicity.Methods:The effects of JBP485 on AAI-induced nephrotoxicity were evaluated in vitro and in vivo.The levels of creatinine（CRE）,urea nitrogen（BUN）,indoxol sulfate in blood and urine,ratio of kidney weight to body weight and HE staining were measured to determine the renal protective effect of JBP485 against AAI nephrotoxicity.Drug-drug interactions（DDIs）mediated by Organic anion transporters（OATs）were explored through pharmacokinetic studies in rats,and uptake studies in kidney slices and OAT-over-expressing cells.Cell viability assay,western blotting,malondialdehyde（MDA）,superoxide dismutase（SOD）detection and intracellular reactive oxygen species（ROS）level were carried out to explore the protective mechanisms of JBP485 on AAI-induced nephrotoxicity.Results:JBP485 attenuated the pathological injures of rat kidney and decreased the levels of CRE,BUN and plasma indoxyl sulfate as well as increased the levels of urine indoxyl sulfate compared to the AAI group.Co-administration with AAI and JBP485 significantly increased the plasma concentration and the AUC of AAI,while decreasing AAI renal clearance and cumulative urinary excretion.Moreover,JBP485reduced AAI uptake in kidney slices and OAT1/3-HEK293 cells,indicating that JBP485 might ameliorate AAI-induced nephrotoxicity through reducing renal exposure of AAI via inhibiting Oats.At the same time,JBP485 regulated the abnormal expressions of Oat1,Oat3,organic cation transporter 2（Oct2）,multidrug resistance-associated protein 2（Mrp2）,mammal multidrug and toxin extrusion proteins 1（Mate 1）and P-glycoprotein（P-gp）in rat kidney,suggesting that JBP485improved tubular secretion in AAI-treated rats.Moreover,JBP485 reversed the decreased expression of heme oxygenase 1（HO-1）and NAD（P）H:quinone oxidoreductase-1（NQO1）,the decreased expression of B-cell lymphoma-2（Bcl-2）and the increased expressions of Bcl-2-like protein 4（Bax）induced by AAI in rat kidney.In addition,JBP485 induced the increase of cell viability and decreased in the intracellular ROS levels in NRK-52E cells.These results suggested JBP485 prevented renal oxidative stress which induced by AAI.Conclusions:JBP485 protected against AAI-induced nephrotoxicity through reducing renal exposure of AAI,restoring renal secretion function and alleviating oxidative stress.Our findings suggested JBP485 was a promising renoprotection agent for the prevention of AAI-induced nephrotoxicity.