To ensure proper dosage of a drug,analytical quantification of it in biofluid is necessary.Liquid chromatography mass spectrometry(LC-MS)is the conventional method of choice as it permits accurate identification and q...To ensure proper dosage of a drug,analytical quantification of it in biofluid is necessary.Liquid chromatography mass spectrometry(LC-MS)is the conventional method of choice as it permits accurate identification and quantification.However,it requires expensive instrumentation and is not appropriate for bedside use.Using soluble epoxide hydrolase(sEH)inhibitors(EC5026 and TPPU)as examples,we report development of a nanobody-based enzyme-linked immunosorbent assay(ELISA)for such small molecules and its use to accurately quantify the drug chemicals in human samples.Under optimized conditions,two nanobody-based ELISAs were successfully established for EC5026 and TPPU with low limits of detection of 0.085 ng/mL and 0.31 ng/mL,respectively,and two order of magnitude linear ranges with high precision and accuracy.The assay was designed to detect parent and two biologically active metabolites in the investigation of a new drug candidate EC5026.In addition,the ELISAs displayed excellent correlation with LC-MS analysis and evaluation of inhibitory potency.The results indicate that nanobody-based ELISA methods can efficiently analyze drug like compounds.These methods could be easily implemented by the bedside,in the field in remote areas or in veterinary practice.This work illustrates that nanobody based assays offer alternative and supplementary analytical tools to mass spectrometry for monitoring small molecule medicines during clinical development and therapy.Attributes of nanobody based pharmaceutical assays are discussed.展开更多
Soluble epoxide hydrolase(sEH) is related to arachidonic acid cascade and is over-expressed in a variety of diseases, making sEH an attractive target for the treatment of pain as well as inflammatory-related diseases....Soluble epoxide hydrolase(sEH) is related to arachidonic acid cascade and is over-expressed in a variety of diseases, making sEH an attractive target for the treatment of pain as well as inflammatory-related diseases. A new series of memantyl urea derivatives as potent sEH inhibitors was obtained using our previous reported compound 4 as lead compound. A preferential modification of piperidinyl to 3-carbamoyl piperidinyl was identified for this series via structure-based rational drug design. Compound A20 exhibited moderate percentage plasma protein binding(88.6%) and better metabolic stability in vitro. After oral administration, the bioavailability of A20 was 28.6%. Acute toxicity test showed that A20 was well tolerated and there was no adverse event encountered at dose of 6.0 g/kg. Inhibitor A20 also displayed robust analgesic effect in vivo and dose-dependently attenuated neuropathic pain in rat model induced by spared nerve injury, which was better than gabapentin and sEH inhibitor(±)-EC-5026. In one word, the oral administration of A20 significantly alleviated pain and improved the health status of the rats, demonstrating that A20 was a promising candidate to be further evaluated for the treatment of neuropathic pain.展开更多
可溶性环氧化物水解酶(Soluble epoxide hydrolases,sEH)是一种能代谢环氧脂肪酸的酶,它在哺乳动物中广泛存在,能将内源性环氧二十碳三烯酸(Epoxyeicosatrienoic acids or EETs)转化为二羟基二十碳三烯酸(Dihydroxy epoxyeicosatrienoic...可溶性环氧化物水解酶(Soluble epoxide hydrolases,sEH)是一种能代谢环氧脂肪酸的酶,它在哺乳动物中广泛存在,能将内源性环氧二十碳三烯酸(Epoxyeicosatrienoic acids or EETs)转化为二羟基二十碳三烯酸(Dihydroxy epoxyeicosatrienoic acids or DHETs)。内源性EETs是由花生四烯酸(Arachidonic acid or AA)经细胞色素P450氧化而来,它是生物体内重要的信号分子,具有调节离子转运和基因表达、血管扩张、抗炎等作用。在动物体内,有很多种途径可以降解EETs,其中sEH将EETs代谢为DHETs是最主要的代谢途径,使EETs的浓度降低,生理活性下降,从而使体内的血压升高,并进一步影响肾脏,心脏等功能。研究表明,抑制sEH的活性可治疗多种心血管疾病及炎症。因此开发新型sEH的抑制剂在治疗相关疾病中具有很好的应用价值。主要概述了sEH的抑制剂的作用机理以及抑制剂研究的最新进展,并展望了抑制剂今后的研究方向。展开更多
基金supported by NIEHS(RIVER Award,R35 ES030443)NIEHS(Superfund Award,P42 ES004699)+6 种基金NINDS(Counter ActProgram U54 NS127758)Juvenile Diabetes Research Foundation(2-SRA-2022-1210-S-B)Guangzhou Science and Technology Foundation(Grant No.:201903010034)Natural Resources Science Foundation of Guangdong Province(Grant No.:2018A030313926)Science and Technology Foundation Key R&D Program of Guangdong Province(Grant Nos.:2019B020209009 and 2019B020218009)R&D Program of Guangdong Province Drug Administration(Grant Nos.:2021TDZ09 and 2021YDZ06)supported by China Scholarship Council(CSC)(202108440382).
文摘To ensure proper dosage of a drug,analytical quantification of it in biofluid is necessary.Liquid chromatography mass spectrometry(LC-MS)is the conventional method of choice as it permits accurate identification and quantification.However,it requires expensive instrumentation and is not appropriate for bedside use.Using soluble epoxide hydrolase(sEH)inhibitors(EC5026 and TPPU)as examples,we report development of a nanobody-based enzyme-linked immunosorbent assay(ELISA)for such small molecules and its use to accurately quantify the drug chemicals in human samples.Under optimized conditions,two nanobody-based ELISAs were successfully established for EC5026 and TPPU with low limits of detection of 0.085 ng/mL and 0.31 ng/mL,respectively,and two order of magnitude linear ranges with high precision and accuracy.The assay was designed to detect parent and two biologically active metabolites in the investigation of a new drug candidate EC5026.In addition,the ELISAs displayed excellent correlation with LC-MS analysis and evaluation of inhibitory potency.The results indicate that nanobody-based ELISA methods can efficiently analyze drug like compounds.These methods could be easily implemented by the bedside,in the field in remote areas or in veterinary practice.This work illustrates that nanobody based assays offer alternative and supplementary analytical tools to mass spectrometry for monitoring small molecule medicines during clinical development and therapy.Attributes of nanobody based pharmaceutical assays are discussed.
基金funded by the Liaoning Revitalization Talents Program(XLYC1908031,China)Basic Research Project of Department of Education of Liaoning Province-natural sciences(2020LJC02,China)+2 种基金Major Basic Research Project of Natural Science Foundation of Shandong Province(ZR2018ZC1056,China)partial support was provided by the NIH-NIEHS RIVER Award(R35 ES030443-01,USA)the NIEHS Superfund Research Program(P42 ES004699,USA)。
文摘Soluble epoxide hydrolase(sEH) is related to arachidonic acid cascade and is over-expressed in a variety of diseases, making sEH an attractive target for the treatment of pain as well as inflammatory-related diseases. A new series of memantyl urea derivatives as potent sEH inhibitors was obtained using our previous reported compound 4 as lead compound. A preferential modification of piperidinyl to 3-carbamoyl piperidinyl was identified for this series via structure-based rational drug design. Compound A20 exhibited moderate percentage plasma protein binding(88.6%) and better metabolic stability in vitro. After oral administration, the bioavailability of A20 was 28.6%. Acute toxicity test showed that A20 was well tolerated and there was no adverse event encountered at dose of 6.0 g/kg. Inhibitor A20 also displayed robust analgesic effect in vivo and dose-dependently attenuated neuropathic pain in rat model induced by spared nerve injury, which was better than gabapentin and sEH inhibitor(±)-EC-5026. In one word, the oral administration of A20 significantly alleviated pain and improved the health status of the rats, demonstrating that A20 was a promising candidate to be further evaluated for the treatment of neuropathic pain.
文摘可溶性环氧化物水解酶(Soluble epoxide hydrolases,sEH)是一种能代谢环氧脂肪酸的酶,它在哺乳动物中广泛存在,能将内源性环氧二十碳三烯酸(Epoxyeicosatrienoic acids or EETs)转化为二羟基二十碳三烯酸(Dihydroxy epoxyeicosatrienoic acids or DHETs)。内源性EETs是由花生四烯酸(Arachidonic acid or AA)经细胞色素P450氧化而来,它是生物体内重要的信号分子,具有调节离子转运和基因表达、血管扩张、抗炎等作用。在动物体内,有很多种途径可以降解EETs,其中sEH将EETs代谢为DHETs是最主要的代谢途径,使EETs的浓度降低,生理活性下降,从而使体内的血压升高,并进一步影响肾脏,心脏等功能。研究表明,抑制sEH的活性可治疗多种心血管疾病及炎症。因此开发新型sEH的抑制剂在治疗相关疾病中具有很好的应用价值。主要概述了sEH的抑制剂的作用机理以及抑制剂研究的最新进展,并展望了抑制剂今后的研究方向。
