Screening Of Oral Hypoxia Inducible Factor-Prolyl Hydroxilase Domain(HIF-PHD)Inhibitors Treating Renal Anemia And Pharmacodynamics Study Of Candidate Compound DS015

Renal anemia is a complication of chronic kidney disease(CKD),which not only affects the quality of patients' life,but also is an independent risk factor cardiovascular disease in CKD patients and can accelerate the progression of CKD,increase the rate of hospitalization and mortality.The existing standard treatment of renal anemia is the combination therapy of Erythropoiesis stimulating agents(ESAs)and iron agent.The ultra-high plasma EPO level is induced by this standard therapy,which results in cardiovascular adverse reactions such as hypertension,heart failure,stroke and myocardial infarction,leading to increased mortality.ESAs should be administered by injection and the price is high.The main treatment population of ESAs is CKD patients who are about to be undergo hemodialysis and renal failure patients who have underwent hemodialysis.Besides,patients before CKD stage 5 rarely receive the treatment of ESAs.Therefore,there is still a large clinical demand for renal anemia that has not been met.HIF-PHD inhibitors promote erythropoiesis through a variety of effects,including promoting endogenous EPO production,improving iron metabolism,increasing the responsiveness of Hematopoietic stem cells to EPO,and so on,which reduce the level of plasma EPO and finally can not only avoid cardiovascular adverse reactions caused by ultra-high EPO,but also achieve the therapy purpose.In addition,HIF-PHD inhibitors are small-molecule drugs and have lots of advantages,such as oral administration,low treatment cost,good compliance and widely usage in CKD stage 3-4 anemia patients.Existing experiment results have shown that HIF-PHD inhibitors had good therapeutic effect and safety,and are expected to overcome the shortcomings of existing treatment and replace it.Objective: In this project,though establishing mature and stable screening system,including in vitro cellular and in vivo animal experiments,we intend to study the preclinical efficacy of HIF-PHD inhibitors.Our aim is to screen out some HIF-PHD inhibitors,which have the same or better in vitro and in vivo activity than the reference compound Roxadutat(FG-4592).Then through combining pharmacokinetic and toxicological preliminary evaluation,as well as the chemical properties,we hope to determine one candidate compound.Finally,the pharmacodynamics effects of this candidate compound will be fully validated in vitro and in vivo.Content: This project mainly includes two parts: The first part is to screen the candidate compound.Firstly,we established a cellular experiment to evaluate the effect of compounds on EPO secretion in Hep 3B cell,and use it to screen the new synthesized compounds.Secondly,compounds that had good activity on EPO secretion,low cytotoxicity and good solubility were chosen to evaluated the pharmacokinetics(PK)in rats.Thirdly,compounds with good pharmacokinetic profiles were selected and investigated the pharmacological activities in ICR mice after single oral administration.Next,combining the chemical properties,we selected the compounds that had good pharmacological activities to evaluate the toxicity in rats and dogs for 14 days,and finally selected the candidate compound.The second part is to verify the pharmacodynamics effects of DS015 in vitro and in vivo.The in vitro experiments included the HIF-PHD enzyme affinity test,which evaluate the enzymatic activity of DS015,and two cellular experiments in Hep 3B cell,which evaluate the effect of DS015 on EPO transcription and secretion,and the effect of DS015 on EPO secretion after inhibited by inflammatory cytokines.The in vivo experiments were evaluated in normal cynomolgus monkey and the rat renal anemia models.In normal cynomolgus monkey,the effects of DS015 on EPO secretion and erythropoietic parameters were studied.In addition,the therapeutic effect of DS015 were investigated in several renal anemia models,including 5/6 nephrectomy,gentamycin or cisplatin induced renal anemia in rats.Results: Part 1: Screening candidate compounds by in vitro and in vivo experiment studies.After preliminary screening it was found that 4 compounds showed high activity on EPO secretion in Hep 3B cells,their activities are comparable with those of FG-4592.Of these 4 compouds,DS015 with good PK in rats.A single dose of DS015 36,120 or 360 ??/kg were administered to ICR mice respectively,tend to increase plasma EPO level in a dose dependent manner,it's activities is comparable with those of FG-4592.Preclinical safety evaluation shows that the toxicity of DS015 is similar with FG-4592 in rats and beagle dogs,and finally choose DS015 as a candidate compound for subsequent pharmacodynamic verification.Part 2: Pharmacodynamic verification of DS015 in vitro and in vivo.In vitro HIF-PHD enzyme activity test showed that DS015 is a relatively potent inhibitor of HIF-PHD1/2/3 enzymes with IC50 values of 0.11,0.42 and 0.043 ?? respectively,which is slightly better than FG-4592,and with similar selectivity.In the experiment of Hep 3B cell EPO transcription and secretion activity,DS015 can promote EPO secretion in a concentration-dependent manner with minimum effective concentration(MEC)of 0.93 ??,and the activity is slightly better than FG-4592.Inflammatory cytokines such as TNF-? and IL-1? are reported to suppress EPO secretion.However,EPO concentration increased when 0.93?25 ?? DS015 was added to Hep 3B cells in the presence of TNF-?(1 ng/m L)and IL-1?(10 ng/m L),and its activity is slightly better than FG-4592.DS015 can increase plasma EPO levels after single doing of 30?60 mg/kg in normal cynomolgus monkeys.In 4-week repeat oral dosing of DS015(20?80 mg/kg),erythropoietic parameters were increased compared with cynomolgus monkeys that received vehicle.At ?8 mg/kg,erythropoietic parameters significantly increased in the DS015 groups as compared with the vehicle group 4 weeks after start of treatment in rats with 5/6 nephrectomized-induced renal anemia.In rats with gentamicin-induced renal anemia,treatment with DS015(12 mg/kg)2-week prevented the decline in HGB level.Oral DS015 8?20 mg/kg was administered to rats with cisplatin-induced renal anemia 3 times weekly for 4 weeks,erythropoietic parameters significantly increased in the DS015 groups as compared with the vehicle group.Conclusion: In this dissertation,both in vitro and in vivo models were established for evaluation of HIF-PHD inhibitors.Candidate compound DS015 was screened out from 27 compounds through erythropoietin release in vitro and in vivo,pharmacokinetic evaluation and toxicological experiments.Then,DS015 was further tested in vitro and in vivo models for the assessment of pharmacodynamic activity.In vitro HIF-PHD enzyme activity test showed that DS015 is a relatively potent inhibitor of HIF-PHD1/2/3 enzymes,and DS015 also can promote EPO secretion in Hep 3B cells with a concentration-dependent manner,which is slightly better than that of FG-4592.In vivo experimental studies shows that DS015 can increase erythropoietic parameters with qd or tiw oral dosing in renal anemia rat models,the minimum effective dose is 8 mg/kg(qd/tiw).This profile deems DS015 as a attractive HIF-PHD inhibitor with the potential for treatment of renal anemia.