Prolyl hydroxylase domain-containing protein inhibitors as stabilizers of hypoxia-inducible factor: small molecule-based therapeutics for anemia

Importance of the field: Anemia caused by chronic kidney disease and other chronic diseases or conditions can be managed by the treatment of biologic-based erythropoiesis stimulating agents (ESAs). Although these ESAs are successful in treating these anemic conditions, a small molecule-based anti-anemia medicine can potentially revolutionize the treatment of anemia by bringing convenience to patients and being cost effective. Prolyl hydroxylase domain-containing protein (PHD) inhibitors may provide an opportunity for the development of small molecule anti-anemia medicines.

Areas covered in this review: This review covers efforts to target PHD enzymes for stabilization of hypoxia-inducible factor (HIF)-α subunits under normal oxygen levels as an attractive strategy to upregulate the expression of erythropoietin and genes involved in iron metabolism for the treatment of anemia.

What the reader will gain: The reader will gain a brief summary of recent advances in HIF and PHD biology and a review of patents/patent applications on the subject of PHD inhibitors as HIF stabilizers for the treatment of anemia.

Take home message: Several classes of PHD enzyme inhibitors have been disclosed and several are currently in clinical trials for the development of small molecule-based therapeutics for the treatment of anemia.     

Differential pharmacokinetic analysis of in vivo erythropoietin receptor interaction with erythropoietin and continuous erythropoietin receptor activator in sheep

The two erythropoiesis stimulating agents (ESAs), short acting recombinant human erythropoietin (EPO) and long acting continuous erythropoietin receptor activator (CERA), have been hypothesized to share an in vivo elimination pathway that involves binding to erythropoietin receptor (EPOR) and subsequent internalization. A physiologically based recirculation model and a pharmacokinetic tracer interaction methodology (TIM) were used to compare the in vivo interaction kinetics with EPOR between the two ESAs in adult sheep. Animals treated with EPO experienced a greater EPOR up-regulation than those treated with CERA, as evidenced by an eightfold-higher initial EPOR normalized production rate constant, k(syn) /R(0) , versus a twofold-larger EPOR degradation rate constant, k(deg) . In agreement with in vitro studies, EPO had a lower in vivo equilibrium dissociation constant from EPOR than CERA (K(D) = 6 versus 88.4 pmol/l, respectively, p < 0.01). The internalization and/or degradation of the EPO-EPOR complex was faster than that of the CERA-EPOR complex (k(int) = 24 versus 2.41 h(-1) , respectively, p < 0.01). The adopted model enables a mechanism-based explanation for CERA's slower elimination and greater erythropoietic activity in vivo. As predicted by the model, the slower elimination of CERA is due to: (1) less EPOR up-regulation induced by CERA administration; (2) slower binding of CERA to EPOR; and (3) reduced internalization and/or degradation rate of surface-bound CERA. Slower CERA/EPOR complex elimination explains the greater in vivo erythropoiesis reported for CERA, despite its lower affinity to EPOR. A sensitivity analysis showed that the model parameters were reliably estimated using the TIM methodology.     

促红细胞生成素预处理对视网膜神经元缺氧性损伤的保护作用

目的研究缺氧对培养大鼠视网膜神经元的影响及促红细胞生成素(erythropoietin,EPO)预处理的保护作用。方法取体外原代培养3d的大鼠视网膜神经元,利用1mmol.L-1的连二亚硫酸钠消除培养基中的氧合并培养基质缺糖持续6h,导致神经元缺氧性损伤。采用免疫组化染色技术检测了EPO受体在原代培养视网膜神经元的表达,乳酸脱氢酶(LDH)释放率作为评价损伤的指标,TUNEL染色检测视网膜神经元的凋亡,并进行形态学观察。结果原代培养的大鼠视网膜神经元正常下可见EPOR的弱阳性表达,表达部位定位于神经元的胞体和突起,缺氧后EPOR的表达明显增加;神经元LDH释放率和凋亡百分率增加;在损伤前加入浓度2.5～40kU.L-1的rhEPO均可有效抑制LDH释放(P<0.05)。TUNEL结果显示rhEPO预处理能抑制神经元的凋亡,明显改善细胞缺氧性损伤的形态学变化。结论EPO预处理对神经元的缺氧损伤具有保护作用,且具有浓度和时间依赖性。     

Emerging drugs for treatment of anemia of chronic kidney disease

Introduction: Erythropoiesis-stimulating agents (ESAs) prevent transfusions among anemic patients with chronic kidney disease (CKD). Clinical trials, meta-analyses, and guidelines identify arterial and venous thromboembolism as well as myocardial event risks with the traditional ESAs, erythropoietin (EPO), and darbepoietin. Side effects of anemia treatment, considering frequency and dosage of treatment as well as targeted hemoglobin levels when utilizing ESAs, greatly impact overall well-being and the quality of life. There is a need for less frequent but equally effective ESAs in this setting.

Areas covered: The three generations of ESAs used in CKD-associated anemia are described. Cost effectiveness of the utilization of these therapies, in addition to emerging therapies, is also presented. The few clinical and controlled trials only highlight the need for clarity in molecular biology surrounding the components that control EPO levels and utilization.

Expert opinion: Anemia associated with CKD is an important area for development of newer therapies which are potentially safer and more convenient to administer.     

Erythropoietic stimulating agents

Importance of the field: The synthesis of recombinant forms of erythropoietin, epoetin α, β and darbepoetin α, has represented a major achievement in anemia therapy.

Areas covered in this review: This review analyzes the therapeutic options of existing erythropoietic stimulating agents (ESAs) in patients with cancer, renal and heart failure anemia and shows the main features of new ESAs under development. Peer-reviewed scientific literature and abstracts published during the last 10 years were reviewed.

What the reader will gain: Randomized clinical trials carried out in patients with anemia associated with chronic renal disease or malignancies have consistently demonstrated that ESAs increase hemoglobin levels, reduce blood transfusion requirements and improve quality of life. However, in spite of the positive effects, treatment with ESAs, particularly in oncologic patients, is associated with an increased mortality and cardiovascular complications. These observations imply the absolute need for using ESAs in the context of currently available guidelines.

Take home message: A razionalization in the clinical use of ESAs according to current guidelines is required to improve the benefits derived from therapy with these drugs. New ESAs are under development and their introduction in therapy could provide a significant improvement in anemia management.     

rhEPO对光损伤诱导的RPE细胞凋亡的抑制

目的研究重组人促红细胞生成素(rhEPO)在人视网膜色素上皮(RPE)细胞光化学损伤中的保护作用及其作用机制,为年龄相关性黄斑变性等的药物治疗和预防提供理论依据。方法取成人ARPE-19细胞株传代培养的2~5代细胞建立光损伤模型,光照12h后再培养24h终止培养,采用AnnexinV-FITC/PI流式双染法检测不同浓度的rhEPO干预治疗前后RPE细胞凋亡的变化;采用酶联免疫吸附实验(enzyme linked immunosorbant assay,ELISA)及免疫组织化学法检测不同浓度的rhEPO干预治疗前后RPE细胞caspase-3及Bcl-2表达的变化;并添加AG490(Jak2激酶抑制剂),探讨人rhEPO对人RPE细胞光化学损伤的保护性作用机制。结果各rhEPO组均可明显减少光化学损伤诱导的人RPE细胞的凋亡,呈浓度依赖性,以40IU·ml-1EPO组结果最明显,为(4.93±1.45)·ml-1;在40IU·ml-1EPO组caspase-3浓度最低,为(0.125±0.029)μg·L-1;在40IU·ml-1EPO组Bcl-2表达最高(168.21±3.87);在加入AG490组,人RPE细胞凋亡增高为(11.29±2.11)·ml-1;caspase-3浓度增高为(0.362±0.042)μg.L-1;Bcl-2表达降低。rhEPO抑制凋亡作用均被阻止。结论rhEPO可抑制光化学损伤诱导的人RPE细胞的凋亡,抑制caspase-3的浓度,上调Bcl-2的表达,对人RPE细胞的光化学损伤有保护治疗作用;其保护作用机制主要通过EPO与受体结合后激活Jak2激酶途径完成的。     

人体尿液中新型促红细胞生成受体激动剂的检测

In the present study, isoelectronic focusing with different pH gradients ( pH 3−5, 2−6) or migrating distances (8.5, 12 and 17 cm) and SDS-PAGE was used to separate continuous erythropoietin receptor activator (CERA), recombinant human erythropoietin (rhEPO), darbepoitin and endogenous EPO spiked in human urine with 37 ℃ overnight incubation.  Double blotting and chemiluminescent visualization were used to detect the IEF and SDS-PAGE profiles.  The bands of CERA profile were detected and well separated from the endogenous EPO and the other two EPO preparations with both SDS-PAGE and the IEF method using a gradient pH 3−5 and a migrating distance of 17 cm, and a significant particular band of CERA profile was found in the IEF result.  These preliminary results indicated that the methods were reliable and reproducible for detecting CERA, and could be used as a routine procedure for anti-doping analysis.     

Investigational therapies for renal disease-induced anemia

Introduction: The main pillars for the treatment of chronic kidney disease (CKD) associated anemia are peptidic erythropoiesis stimulating agents (ESAs) and iron preparations. Both approaches benefit from long-term efficacy and safety data but are surrounded by clinical and economic concerns, driving the search for novel anti-anemic drugs.

Areas covered: By answering pivotal questions, the authors describe the recent developments of next generation ESAs, introduce cutting-edge iron formulations and focus on investigational approaches that interact with pathways involved in erythropoietin (Epo) synthesis and myeloid hematopoiesis. Finally, the challenges encountered with these drug candidates are discussed.

Expert opinion: Current peptidic ESAs are effective and well-tolerated, but are costly, require parenteral application and iron supplementation. ESA resistance may develop calling for increased doses. Therefore, orally available hypoxia-inducible factor (HIF) stabilizing compounds are attractive alternatives, which may be approved in the near future. Prominent compounds are molidustat, daprodustat and roxadustat. HIF stabilizers suppress hepcidin production and improve iron balance as the present ESAs, but also raise safety concerns in association with their pleiotropic actions. Other investigational erythropoietic biologics are growth-differentiation factor-11 (GDF11) ligand traps (sotatercept, luspatercept), which are also well advanced in development. Possibly, they will provide an add-on for established therapies. However, immunogenicity of these compounds still needs to be carefully investigated.     

Pharmacodynamic analysis of stress erythropoiesis: change in erythropoietin receptor pool size following double phlebotomies in sheep

A feedback receptor regulation model was incorporated into a pharmacodynamic model to describe the stimulation of hemoglobin (Hb) production by endogenous erythropoietin (EPO). The model considers the dynamic changes that take place in the EPO receptor (EPOR) pool under phlebotomy-induced anemia. Using a (125)I-rhEPO tracer the EPO clearance changes are evaluated longitudinally prior to and following phlebotomy-induced anemia indirectly to evaluate changes in the EPOR pool size, which has been shown to be linearly related to the clearance. The proposed model simultaneously captures the general behavior of temporal changes in Hb relative to EPO plasma clearance in five lambs (r = 0.95), while accounting for the confounding variables of phlebotomy and changes in the blood volume in the growing animals. The results indicate that under anemia the EPOR pool size is up-regulated by a factor of nearly two over baseline and that the lowest and highest EPOR pool sizes differ by a factor of approximately four. The kinetic model developed and the data-driven mechanism proposed serves as a starting point for developing an optimal EPO dosing algorithm for the treatment of neonatal anemia.     

前列腺癌患者血清EPO浓度和癌组织EPOR的表达及其临床意义

目的探讨前列腺癌患者血清促红细胞生成素(EPO)浓度和癌组织中促红细胞生成素受体(EPOR)的表达及临床意义。方法采用ELISA测定35例初诊前列腺癌(A组)、12例激素非依赖性前列腺癌(B组)、20例前列腺增生(C组)及15例健康人(D组)血清EPO水平;免疫组化方法检测A、C组列腺组织EPOR表达。分析A组血清EPO水平、癌组织EPOR的表达与Gleason评分、TNM分期的关系。结果 A组血清EPO水平为(32.71±26.13)mIu/ml,明显高于C组的(18.47±12.97)mIu/ml和D组的(16.41±9.37)mIu/ml(P<0.01),明显低于B组的(59.91±33.34)mIu/ml(P<0.05)。A组EPOR的表达明显高于C组(P<0.01)。A组血清EPO水平和癌组织中EPOR的表达与临床分期呈正相关(P<0.05)。结论 EPO和EPOR在前列腺癌的进展过程中可能起着重要作用。检测血清EPO水平和癌组织EPOR的表达有助于前列腺癌的诊断。     

Structure-based drug design for hypoxia-inducible factor prolyl-hydroxylase inhibitors and its therapeutic potential for the treatment of erythropoiesis-stimulating agent-resistant anemia: raising expectations for exploratory clinical trials

Introduction: Anemia occurs in various chronic diseases and its treatment is dramatically improved after the appearance of erythropoiesis-stimulating agents (ESA). However, there are several problems regarding the use of ESA including: i) invasiveness, ii) high cost and iii) ESA resistance. Therefore, there is a need to develop small molecule drugs which can improve these problems. Hypoxia-inducible factor (HIF) plays a key role in regulating erythropoietin production. HIF stabilizers, particularly, prolyl hydroxylase domain-containing protein (PHD) inhibitors, have emerged as small molecule-based anti-anemia medicine.

Areas covered: This article discusses the current status of PHD inhibitors and the pros and cons of currently tested methods. Specifically, the article reviews the advantages of structure-based drug design in the development of PHD inhibitors and looks at future perspectives within the field.

Expert opinion: Despite the fact that structure-based drug design has dramatically improved drug discovery, testing on humans is still one of the most time-consuming parts of drug discovery and one that is not accelerated by structural approaches. Exploratory clinical trials, first-in-man studies have emerged as a new strategy for preclinical and clinical development of drugs. Exploratory clinical trials will not only reduce the time and cost in preclinical trials but also provide important information on candidate drug's pharmacological effects in humans. Exploratory clinical trials may be a potential alternative strategy for the drug discovery in the future.     

Data from a microdosed recombinant human erythropoietin administration study applying the new biotinylated clone AE7A5 antibody and a further optimized sarcosyl polyacrylamide gel electrophoresis protocol

Erythropoietin (EPO) is a hormone, which stimulates the production of red blood cells. Due to its performance-enhancing effect, it is prohibited by the World Anti-Doping Agency (WADA). In order to reduce the detection window of EPO doping, athletes have been applying low doses of recombinant EPO (e.g., <10 IU/kg body weight, daily or every second day) instead of larger doses twice or more per week (e.g., 30 IU/kg). Microdoses of Retacrit (epoetin zeta), an EPO biosimilar, were administered intravenously and subcutaneously to human males and females. Urine and serum samples were collected and analysed applying the new biotinylated clone AE7A5 EPO antibody and a further optimized sarcosyl polyacrylamide gel electrophoresis (SAR-PAGE) protocol. With the improved protocol, microdosed Retacrit (7.5 IU/kg body weight [BW]) was detectable for at least 52 h after intravenous administration. Detection windows were approximately the same for serum and urine and doubled after subcutaneous administration (~104 h). Previous studies applying different electrophoretic techniques and the not further optimized SAR-PAGE protocol revealed considerably shorter detection windows for recombinant human erythropoietin (rhEPO) microdoses. Because the new biotinylated antibody performed significantly more sensitive than the nonbiotinylated version, the new protocol will improve the sensitivity and hence detectability of recombinant EPO in doping control.     

A simple method to immunopurify erythropoiesis stimulating agents from urine,aiming to optimize erythropoietin screening by SAR‐PAGE

The efficiency of the immunopurification step of urinary erythropoietin (EPO) and recombinant forms is important for their optimal detection in antidoping screening. Previous investigations of immunopurification techniques have been done for immunomagnetic beads, EPO Purification Kit (EPK) columns (MAIIA Diagnostics), and enzyme‐linked immunosorbent assay (ELISA) microplates (Stemcell Technologies) conjugated/coated with anti‐EPO antibodies. In this study, a new immunopurification technique using anti‐EPO sepharose gel beads, developed by MAIIA Diagnostics, to simplify and minimize sample handling was evaluated. This EPO Purification Gel Kit (EPGK) was compared with our current routine EPK for limit of detection (LOD). Linearity, recovery, repeatability, sample incubation time, and sample volume were also evaluated for EPGK. The LODs and linearity for EPK and EPGK were comparable to each other and the recovery for BRP, NESP, CERA, and EPO‐Fc were within the range of other studies, and concentration of the sample eluate improved the recovery results. Little variation was seen within days, between days, and between operators. A 90 minute incubation of the sample with the sepharose gel beads is sufficient for most of the erythropoiesis stimulating agents (ESAs) tested, with 10 mL being an optimal sample volume for EPGK. The improved sample handling, higher sample throughput and the reduced working time demonstrate that the EPGK is a better alternative to the current MAIIA EPK immunopurification method for urine. The EPO Purification Gel Kit (from MAIIA Diagnostics) was evaluated and validated for immunopurification of endogenous erythropoietin and exogenous erythropoiesis stimulating agents from urine samples. The kit was a better alternative to that currently used (EPO Purification Kit) in many antidoping laboratories because it improves sample handling and increases sample throughput.     

A novel synthetic derivative of human erythropoietin designed to bind to glycosaminoglycans

To synthesize long-acting and antiangiogenic erythropoietin to be clinically applied for treatment of patients with solid tumors, we synthesized a hybrid molecule of human erythropoietin added onto the C-terminus with a heparin-binding motif of human PLGF-2 to develop a novel derivative of long-acting and antiangiogenic erythropoietin: heparin-binding erythropoietin (HEPO), and studied the characteristics of this novel erythropoietin derivative. HEPO cDNA was synthesized, expressed in insect cells, and the protein was purified using a heparin-sepharose affinity column. The erythropoietic and angiogenic effects of the partially purified protein were analyzed in vitro and in vivo. The erythropoietic activity of the protein was equivalent to natural EPO in vitro. In vivo administration of the protein to mice revealed its long-acting erythropoietic activity as expected. Administration of the protein inhibited angiogenesis in a mouse limb ischemia model. In conclusion, the heparin-binding motif of PLGF-2 may act as, so to speak, a superendostatin. This novel long-acting erythropoietin derivative may have an advantage to inhibit tumor growth while preserving hematopoietic and tissue-protective effects.     

Discovery of c.577del in EPO: Investigations into endogenous EPO double-band detected in blood with SAR-PAGE

Recently, some athletes were repetitively found to have rEPO positive results, including a characterized double-band pattern in blood samples, in routine doping analysis. In contrast to previous findings from excretion studies, this double-band pattern showed the same relative intensity even when the samples were collected weeks (/months) apart. We therefore suspected that these “positive” doping control samples were related with a novel pathway of endogenous EPO production. Thus, follow-up investigations were warranted to characterize the origin of such analytical test results and to avoid the issuing of adverse analytical findings in the absence of rEPO by identifying the root cause of these “constantly positives.” In this study, we designed and conducted a series of causal studies, including population screening of EPO profiles, exploration of EPO de-N-glycosylation, single nucleotide polymorphism (SNP) browsing in EPO, sequencing of EPO exons, genealogical analysis of the c.577del EPO variant, and finally expression and investigation of mutant EPO. In summary, we found that these “constantly positives” were related to endogenous EPO production associated with the c.577del EPO variant. The frequency of this variant was 0.39% in our Chinese population pool. The mutant EPO encoded by this variant is 27 amino acids longer than the wild-type. The molecular weight of this mutant EPO is approximately the same as that of rEPO, exhibiting a similar electrophoretic behavior. To prevent charges against carriers of the c.577del variant, a revised rEPO testing strategy has been implemented in the new version of TD EPO.     

慢性丙型肝炎感染患者内源性循环红细胞自体抗体与贫血的相关性研究

目的研究内源性循环红细胞(EPO)自体抗体与慢性丙型肝炎感染相关的贫血之间的关联性。方法收集孝昌县第一人民医院2014年1月—2016年8月被诊断出丙型肝炎病毒感染的抗病毒治疗患者的人口学和临床检查信息,共有121名患者纳入本研究。采用酶联免疫吸附试验测定患者血清抗EPO抗体水平,采用商用试剂盒测定血清中EPO水平,RT-PCR法测定丙型肝炎病毒RNA水平。多变量logistic回归分析血清抗EPO抗体与慢性丙型肝炎病毒感染相关的贫血之间的关联性。结果慢性丙型肝炎病毒感染患者中共有53名贫血和67名非贫血患者,贫血患者年龄、网织红细胞比例、血清EPO水平和抗EPO抗体阳性率均高于非贫血组。但血红蛋白、白蛋白和丙型肝炎病毒RNA负荷水平均低于非贫血组。慢性丙型肝炎病毒感染伴随抗EPO抗体阳性患者的血清EPO水平较低(P0.001),但丙型肝炎病毒RNA负载却呈现出较高的水平(P0.001)。慢性丙型肝炎病毒感染伴随抗EPO抗体阴性患者中血清EPO水平与血红蛋白水平呈现出负向相关性(P0.001),并呈现出明显的反向剂量反应关系,但该相关性并未在抗EPO阳性患者中发现。多变量logistic回归分析发现网织红细胞比例每增加1%,血清EPO水平每增加1 mU/mL,贫血发生风险分别增加32.2%、8.9%,抗EPO抗体阳性患者贫血发生风险增高5倍。结论丙型肝炎病毒相关性贫血的发生与自身免疫有关。慢性丙型肝炎病毒感染伴随抗EPO抗体阳性患者有较高的贫血发生风险。     

Clinical effectiveness and safety evaluation of long-term pioglitazone treatment for erythropoietin responsiveness and insulin resistance in type 2 diabetic patients on hemodialysis

Background: We aimed to assess the effect of long-term pioglitazone treatment on erythropoietin responsiveness and insulin resistance in type 2 diabetic patients on hemodialysis.

Methods: We conducted a prospective, open-label, parallel-group, controlled study of 63 type 2 diabetic hemodialysis patients who were randomly assigned to two groups: pioglitazone group (P-group; 15 – 30 mg/day pioglitazone plus conventional oral hypoglycemic agents) and control group (C-group; conventional oral hypoglycemic agents alone). We determined the efficacy of pioglitazone by monitoring anemia, glycemic control, insulin resistance, and levels of inflammatory cytokines and high-molecular-weight (HMW) adiponectin for 96 weeks.

Results: Pioglitazone effectively reduced erythropoietin dose and maintained the target hemoglobin levels by improving insulin resistance up to the end of the study. In the P-group, hemoglobin A_1c, glycated albumin, and triglycerides significantly decreased compared with the C-group. There was a significant reduction in homeostasis model assessment for insulin resistance and the level of high-sensitivity C-reactive protein, and a significant increase in HMW adiponectin level in the P-group; these changes were significantly different compared with values for the C-group. No serious adverse effects such as hypoglycemia, liver impairment, or heart failure were observed in any of the patients.

Conclusion: Pioglitazone treatment resulted in better glycemic control, improved lipid levels, an increase in insulin sensitivity and adiponectin levels, and a decrease in inflammatory markers, thus improving the risk factors of cardiovascular disease. Erythropoietin responsiveness improved with a reduction in erythropoietin dose and may be associated with the improvement in insulin resistance due to long-term pioglitazone treatment.