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LC-MS/MS method for the quantitation of serum Tocilizumab in rheumatoid arthritis patients using rapid tryptic digestion without IgG purification

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Journal of Pharmaceutical Analysis 2022年第6期12卷 852-859页

作者： Takashi Mochizuki Kaito Shibata Takafumi Naito Kumiko Shimoyama Noriyoshi Ogawa Masato Maekawa Junichi Kawakami Department of Hospital Pharmacy Hamamatsu University School of MedicineHamamatsu431-3192Japan Department of Pharmacy Shinshu University HospitalMatsumotoNagano390-8621Japan Third Division Department of Internal MedicineHamamatsu University School of MedicineHamamatsu431-3192Japan Department of Laboratory Medicine Hamamatsu University School of MedicineHamamatsu431-3192Japan

The quantitation of serum Tocilizumab using liquid chromatography tandem-mass spectrometry(LC-MS/MS)method has not been widely applied in clinical settings because of its time-consuming and costly sample pretreatments.The present study aimed to develop a validated LC-MS/MS method for detecting serum Tocilizumab by utilizing immobilized trypsin without an immunoglobulin G purification step and evaluate its applicability in the treatment of rheumatoid arthritis(RA)patients administered intravenously or subcutaneously with Tocilizumab.The Tocilizumab-derived signature peptide was deciphered using a nano-LC system coupled to a hybrid quadrupole-orbitrap mass spectrometer.The serum Tocilizumab was rapidly digested by immobilized trypsin for 30 min.The chromatographic peak of the signature peptide and that of the internal standard were separated from the serum digests for a total run time of 15 min.The calibration curve of serum Tocilizumab concentration was linear with a range of 2-200 μg/mL.The intra-and inter-day accuracy and relative standard deviation(RSD)were 90.7%-109.4%andTocilizumab concentrations in the RA patients receiving intravenous and subcutaneous injections were 5.8-28.9 and 2.4-63.5 μg/mL,respectively.The serum Tocilizumab concentrations using the current method positively correlated with those using the enzyme-linked immunosorbent assay,although a systematic error was observed between these methods.In conclusion,a validated LC-MS/MS method with minimal sample pretreatments for monitoring serum Tocilizumab concentrations in RA patients was developed.

关键词： Tocilizumab LC-MS/MS Immobilized trypsin IgG purification Proteomics Rheumatoid arthritis

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Tocilizumab in rheumatoid arthritis-associated peripheral ulcerative keratitis:A case report

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Rheumatology & Autoimmunity 2022年第1期2卷 45-50页

作者： Jia Huang Zhen Chen Like Zhao Yongjing Cheng Ming Gao Cibo Huang Zheng Wang Fang Wang Department of Rheumatology and Immunology Beijing HospitalNational Center of GerontologyBeijingChina Ophthalmology Department First People's Hospital of YunnanYunnanChina Department of Ophthalmology Beijing HospitalNational Center of GerontologyBeijingChina

We report a case of rheumatoid arthritis-associated peripheral ulcerative keratitis(PUK)successfully treated with Tocilizumab.Despite having been treated with systemic corticosteroids and several disease-modifying antirheumatic drugs and having,received corneal transplantation,the patient lost sight in the left eye.Tocilizumab resulted in a good response,with her systemic illness controlled and the progression of corneal lesions prevented.Tocilizumab may be an additional tool for patients with rheumatoid arthritis associated PUK that is refractory to other drugs.

关键词： peripheral ulcerative keratitis rheumatoid arthritis Tocilizumab

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Successful treatment of granulomatosis with polyangiitis using Tocilizumab combined with glucocorticoids:A case report

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World Journal of Clinical Cases 2023年第5期11卷 1144-1151页

作者： Peng-Fang Tang Long-Chuan Xu Wen-Ting Hong Hong-Ying Shi Department of Rheumatology Second Affiliated Hospital of Fujian Medical UniversityQuanzhou 362000Fujian ProvinceChina Department of Pathology Second Affiliated Hospital of Fujian Medical UniversityQuanzhou 362000Fujian ProvinceChina Department of Immunology Second Affiliated Hospital of Fujian Medical UniversityQuanzhou 362000Fujian ProvinceChina

BACKGROUND Tocilizumab is a humanized monoclonal antibody against the interleukin-6(IL-6)receptor that is commonly used to treat large vessel vasculitis and antineutrophil cytoplasmic antibody-related small vessel vasculitis.However,Tocilizumab in combination with glucocorticoids for successfully treating granulomatosis with polyangiitis(GPA)has rarely been reported.CASE SUMMARY Here,we report a 40-year-old male patient who suffered GPA for 4 years.He was treated with multiple rounds of drugs,including cyclophosphamide,Tripterygium wilfordii,mycophenolate mofetil,and belimumab,with no improvement.In addition,he exhibited persistently high IL-6 levels.After Tocilizumab treatment,his symptoms improved,and his inflammatory marker levels returned to normal.CONCLUSION Tocilizumab may be effective for treating GPA.

关键词： Tocilizumab Granulomatosis with polyangiitis Antineutrophil cytoplasmic antibody Case report

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Use of subcutaneous Tocilizumab to prepare intravenous solutions for COVID-19 emergency shortage:Comparative analytical study of physicochemical quality attributes

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Journal of Pharmaceutical Analysis 2020年第6期10卷 532-545页

作者： Natalia Navas Jesús Hermosilla Anabel Torrente-Lopez Jose Hernandez-Jimenez Jose Cabeza Raquel Perez-Roblesa Antonio Salmeron-García Department of Analytical Chemistry Science Faculty/Biomedical Research Institute ibs.GranadaUniversity of GranadaGranadaE-18071Spain UGC Farmacia Hospitalaria Biomedical Research Institute ibs.Granada.Hospital Universitario San Cecilio de GranadaGranadaE-18012Spain

COVID-19,a disease caused by the novel coronavirus SARS-Co V-2,has produced a serious emergency for global public health,placing enormous stress on national health systems in many countries.Several studies suggest that cytokine storms(interleukins)may play an important role in severe cases of COVID-19.Neutralizing key inflammatory factors in cytokine release syndrome(CRS)could therefore be of great value in reducing the mortality rate.Tocilizumab(TCZ)in its intravenous(IV)form of administration-Ro Actemra?20 mg/m L(Roche)-is indicated for treatment of severe CRS patients.Preliminary investigations have concluded that inhibition of IL-6 with TCZ appears to be efficacious and safe,with several ongoing clinical trials.This has led to a huge increase in demand for IV TCZ for treating severe COVID-19 patients in hospitals,which has resulted in drug shortages.Here,we present a comparability study assessing the main critical physicochemical attributes of TCZ solutions used for infusion,at 6 mg/m L and 4 mg/m L,prepared from Ro Actemra?20 mg/m L(IV form)and from Ro Actemra?162 mg(0.9 m L solution pre-filled syringe,subcutaneous(SC)form),to evaluate the use of the latter for preparing clinical solutions required for IV administration,so that in a situation of shortage of the IV medicine,the SC form could be used to prepare the solutions for IV delivery of TCZ.It is important to remember that during the current pandemic all the medicines are used off-label,since none of them has yet been approved for the treatment of COVID-19.

关键词： COVID-19 Tocilizumab Critical quality attributes IV and SC medicines

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Tocilizumab promotes corneal allograft survival in rats by modulating Treg-Th17 balance

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International Journal of Ophthalmology(English edition) 2019年第12期12卷 1823-1831页

作者： Xiao-Song Wu Xiao-Li Lu Jing Wu Ming Ma Jian Yu Zhen-Yu Zhang Dpartment of Ophthalmology Nanfang HospitalSouthern Medical UniversityGuangzhou 510515Guangdong ProvinceChina Department of Huiqiao Building Nanfang HospitalSouthern Medical UniversityGuangzhou 510515Guangdong ProvinceChina Guangdong Women And Children Hospital Guangzhou 511400Guangdong ProvinceChina

AIM: To examine the therapeutic effects of Tocilizumab on experimental corneal transplantation and its effect on Treg/Th17 balance. METHODS: Allograft corneal graft was performed between host Sprague Dawley and Wistar donor rats.The rats were randomly divided into four groups: normal,autograft, allograft, and allograft treated with Tocilizumab.Kaplan-Meier was performed to draw the survival curve.The protein levels of interleukin-17A(IL-17A), vascular endothelial growth factor(VEGF), and forkhead box protein3(Foxp3) were measured by immunohistochemistry.The mRNA levels of IL-17A, VEGF, retinoid-related orphan receptor gammat(RORγt), interleukin-6(IL-6) and Foxp3 were detected by reverse transcription real-time polymerase chain reaction(RT-PCR). The Treg and Th17 cells were investigated by flow cytometry. RESULTS: The survival time of Tocilizumab group was(24±1.27 d) longer than that of allograft group(10±0.55 d).Moreover, immunohistochemical examination revealed that IL-17A and VEGF protein levels in the allograft group were significantly higher than that of Tocilizumab group(PTocilizumab treated group(PTocilizumab group(PTocilizumab group(PTocilizumab treated group was significantly higher than other groups(PTocilizumab may promote corneal allograft survival, possibly by modulating Treg-Th17 balance.

关键词： Tocilizumab corneal transplantation Th17/Treg rats

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Protective function of Tocilizumab in human cardiac myocytes ischemia reperfusion injury

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Asian Pacific Journal of Tropical Medicine 2015年第1期8卷 48-52页

作者： Hai-Feng Cheng Yan Feng Da-Ming Jiang Kai-Yu Tao Min-Jian Kong Department of Cardiovascular Surgery The Second Affiliated Hospital of Medical College of Zhejiang University Department of Interventional cardiovascular center The Second Affiliated Hospital of Medical College of Zhejiang University

Objective:To investigate the protective function of Tocilizumab in human cardiac myocytes ischemia-reperfusion injury.Methods:The human cardiac myocytes were treated by Tocilizumab with different concentrations(1.0 mg/mL,3.0 mg/mL,5.0 mg/mL) for 24 h.then cells were cultured in ischemia environment for 24 h and reperfusion environment for 1 h.The MTT and flow cytometry were used to detect the proliferation and apoptosis of human cardiac myocytes,respectively.The mRNA and protein expressions of Bcl-2 and Bax were measured by qRT-PCR and western blot,respectively.Results:Compared to the negative group,pretreated by Tocilizumab could significantly enhance the proliferation viability and suppress apoptosis of human cardiac myocytes after suffering ischemia reperfusion injury(PTocilizumab treated group were higher than NC group(PTocilizumab could significantly inhibit apoptosis and keep the proliferation viability of human cardiac myocytes after suffering ischemia reperfusion injury.Tocilizumab may obtain a widely application in the protection of ischemia reperfusion injury.

关键词： Tocilizumab Human cardiac myocytes Ischemia-reperfusion injury Protection

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Successful treatment of a case of COVID-19 pneumonia following kidney transplantation using paxlovid and Tocilizumab

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世界临床病例杂志 2023年第25期11卷 6012-6018页

作者： Qian Chen Yu-Lin Niu Department of Organ Transplantation Affiliated Hospital of Guizhou Medical UniversityGuiyang 550000Guizhou ProvinceChina

BACKGROUND Since its initial detection in 2019,coronavirus disease 2019(COVID-19)pneumonia has rapidly spread throughout the world in a global pandemic.However,reports of COVID-19 pneumonia among patients following kidney transplantation have been limited and no uniform treatment guidelines for these patients have yet to be established.CASE SUMMARY Here,we report the case of a 39-year-old patient recovering from kidney transplantation who contracted perioperative COVID-19 pneumonia that was successfully controlled with oral paxlovid and a single intravenous drip infusion of Tocilizumab following the discontinuation of immunosuppressive drugs.CONCLUSION Given the rapid spread of severe acute respiratory syndrome coronavirus 2 infections,clinicians should be aware of the potential for more cases of COVID-19 among patients following kidney transplantation and be familiar with appropriate treatment options and likely clinical outcomes.

关键词： Clinical research Kidney transplantation Coronavirus disease 2019 pneumonia Paxlovid Tocilizumab Case report

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Tocilizumab in patients with moderate or severe COVID-19: a randomized, controlled, open-label, multicenter trial

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Frontiers of Medicine 2021年第3期15卷 486-494页

作者： Dongsheng Wang Binqing Fu Zhen Peng Dongliang Yang Mingfeng Han Min Li Yun Yang Tianjun Yang Liangye Sun Wei Li Wei Shi Xin Yao Yan Ma Fei Xu Xiaojing Wang Jun Chen Daqing Xia Yubei Sun Lin Dong Jumei Wang Xiaoyu Zhu Min Zhang Yonggang Zhou Aijun Pan Xiaowen Hu Xiaodong Mei Haiming Wei Xiaoling Xu Department of Pulmonary and Critical Care Medicine the First Affiliated Hospital of University of Science and Technology of China(Anhui Provincial Hospital)Hefei 230001China Division of Life Sciences and Medicine University of Science and Technology of ChinaHefei 230027China Intensive Care Unit the First Affiliated Hospital of University of Science and Technology of China(Anhui Provincial Hospital)Hefei 230001China Drug Clinical Trail Institution the First Affiliated Hospital of University of Science and Technology of China(Anhui Provincial Hospital)Division of Life Sciences and MedicineUniversity of Science and Technology of ChinaHefei 230001China Department of Infectious Diseases Union HospitalTongji Medical CollegeHuazhong University of Science and TechnologyWuhan 430000China Department of Respiratory Medicine the Second People’s Hospital of FuyangFuyang 236000China Department of Respiratory Medicine National Key Clinical SpecialtyBranch of National Clinical Research Center for Respiratory DiseaseXiangya HospitalCentral South UniversityXiangya Lung Cancer CenterXiangya HospitalCentral South UniversityHunan Provincial Clinical Research Center for Respiratory DiseasesChangsha 410000China Lu’an People’s Hospital Affiliated to Anhui Medical University Lu’an 237005China Department of Pulmonary and Critical Care Medicine the First Affiliated Hospital of Bengbu Medical CollegeClinical Research Center for Respiratory Disease(tumor)in Anhui ProvinceBengbu 233004China Department of Respiratory Medicine Anqing Hospital Affiliated to Anhui Medical UniversityAnqing 246000China The First Affiliated Hospital of Nanjing Medical University Nanjing 210000China Department of Rheumatology and Immunology the First Affiliated Hospital of University of Science and Technology of China(Anhui Provincial Hospital)Hefei 230001China Department of Oncology the First Affiliated Hospital of University of Science and Technology of China(Anhui Provincial Hospital)Hefei 230001China Department of Endocrinology

Tocilizumab has been reported to attenuate the“cytokine storm”in COVID-19 patients.We attempted to verify the effectiveness and safety of Tocilizumab therapy in COVID-19 and identify patients most likely to benefit from this treatment.We conducted a randomized,controlled,open-label multicenter trial among COVID-19 patients.The patients were randomly assigned in a 1:1 ratio to receive either Tocilizumab in addition to standard care or standard care alone.The cure rate,changes of oxygen saturation and interference,and inflammation biomarkers were observed.Thirty-three patients were randomized to the Tocilizumab group,and 32 patients to the control group.The cure rate in the Tocilizumab group was higher than that in the control group,but the difference was not statistically significant(94.12%vs.87.10%,rate difference 95%CI−7.19%–21.23%,P=0.4133).The improvement in hypoxia for the Tocilizumab group was higher from day 4 onward and statistically significant from day 12(P=0.0359).In moderate disease patients with bilateral pulmonary lesions,the hypoxia ameliorated earlier after Tocilizumab treatment,and less patients(1/12,8.33%)needed an increase of inhaled oxygen concentration compared with the controls(4/6,66.67%;rate difference 95%CI−99.17%to−17.50%,P=0.0217).No severe adverse events occurred.More mild temporary adverse events were recorded in Tocilizumab recipients(20/34,58.82%)than the controls(4/31,12.90%).Tocilizumab can improve hypoxia without unacceptable side effect profile and significant influences on the time virus load becomes negative.For patients with bilateral pulmonary lesions and elevated IL-6 levels,Tocilizumab could be recommended to improve outcome.

关键词： Tocilizumab coronavirus disease 2019(COVID-19) cytokine storm

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Tocilizumab下调NF-κB/JNK减轻急性肺、肾损伤的效果与对脓毒症小鼠模型存活率的影响

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中文科技期刊数据库（全文版）医药卫生 2023年第9期 0093-0096页

作者：朱景法黄天宝颜才荣杨少东陈金图福建医科大学附属泉州第一医院急诊科福建泉州362000

研究分析Tocilizumab通过下调NF-κB/JNK减轻急性肺和肾损伤并提高脓毒症小鼠模型的存活率。方法本次研究选择同一实验室的48只雄性C57BL/6小鼠作为此次研究对象,随机分成两组,并运用盲肠扎穿孔法(CLP)建立脓毒血症动物模型,视为CLP组... 详细信息

研究分析Tocilizumab通过下调NF-κB/JNK减轻急性肺和肾损伤并提高脓毒症小鼠模型的存活率。方法本次研究选择同一实验室的48只雄性C57BL/6小鼠作为此次研究对象,随机分成两组,并运用盲肠扎穿孔法(CLP)建立脓毒血症动物模型,视为CLP组,另一只作为假手术(SMAM)组,组间平均分配,每组各24只,根据给药方式不同分为SHAM组和两SHAM+TCZ组、CLP组和两CLP+TCZ,共6小组,每组8只。CLP组小鼠采取结扎盲肠全长,使用无菌针头穿刺3处,SHAM组采取剖腹术。脓毒血症动物模型分为CLP组、CLP+TCZ (Tocilizumab注射4 mg/kg)、CLP+TCZ (Tocilizumab注射8mg/kg) 。假手术组:SHAM组、SHAM+TCZ (Tocilizumab注射4 mg/kg) 组、CLP+TCZ (Tocilizumab注射8mg/kg)。观察6小组急性肺和肾损伤情况以及小鼠模型的存活率。结果 CLP+TCZ (8mg/kg)W/D、肺损伤评分均优于其他组别,(PTocilizumab治疗可明显提高脓毒症小鼠的存活率,(PTocilizumab通过下调NF-κB/JNK可减轻急性肺和神损伤并且可提高脓毒血症小鼠模型的存活率,为脓毒血症的治疗以及药物研发提供研究基础。

关键词： Tocilizumab NF-κB/JNK 急性肺和肾损伤脓毒症小鼠模型的存活率

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Clinical efficacy of Tocilizumab treatment in severe and critical COVID-19 patients

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World Journal of Clinical Cases 2020年第17期8卷 3763-3773页

作者： Jia Zeng Ming-Hui Xie Jing Yang Sheng-Wu Chao Er-Li Xu Department of Aviation Disease Naval Medical Center of PLAShanghai 200052China No.1 Department of Infection Optical Valley Campus of Hubei Maternal and Child Health Care HospitalWuhan 430073Hubei ProvinceChina Department of Interventional Therapy First Affiliated Hospitalthe Second Military Medical UniversityShanghai 200433China Department of Nephrology Naval Medical Center of PLAShanghai 200052China Department of Cardiovascular Medicine Naval Medical Center of PLAShanghai 200052China

BACKGROUND The main pathophysiological basis of coronavirus disease 2019(COVID-19)causing respiratory failure is a cytokine storm and interleukin-6(IL-6)is an important component of the COVID-19 cytokine storm.As a specific antagonist of IL-6,Tocilizumab may block the cytokine storm of COVID-19.The Diagnosis and Treatment Guidelines of New Coronavirus Pneumonia(7 th Edition)includes Tocilizumab as a recommended drug for immunotherapy in severe and critical COVID-19 patients.However,the specific clinical efficacy of Tocilizumab in the treatment of COVID-19 patients is worth studying.AIM To determine the clinical efficacy of Tocilizumab in inhibiting the cytokine storm in COVID-19.METHODS In total,19 severe and critical COVID-19 patients were enrolled in this study,and were treated with Tocilizumab in Optical Valley Campus of Hubei Maternal and Child Health Care Hospital from February 20 to March 31,2020.The imaging manifestations and clinical data before and after treatment were analyzed retrospectively,including routine peripheral venous blood tests,routine blood biochemical tests,coagulation test,C-reactive protein(CRP),IL-6,and arterial blood gas analysis.RESULTS Of the 19 patients in this group,13(68.4%)had significantly improved symptoms of COVID-19(5 patients were discharged directly and 8 patients were transferred after improvement)following treatment.One case was invalid,1 case was exacerbated,and 4 deaths(21.1%)were observed(all critical cases).The lymphocyte count,CRP,lactic acid,oxygenation index,fibrinogen(FIB)and IL-6 levels were significantly different in the improved group.CONCLUSION Tocilizumab treatment is effective against IL-6 in COVID-19 patients,but it does not completely inhibit the inflammation and cytokine storm in all patients with COVID-19.In the clinical treatment of COVID-19 patients,attention should be paid to the timing of drug administration and other adjuvant treatments.

关键词： COVID-19 Cytokine storm Tocilizumab Interleukin-6

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