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Validated Simple HPLC-UV Method for Mycophenolic Acid (MPA) Monitoring in Human Plasma. Internal Standardization: Is It Necessary? Molecules 2021; 26:molecules26237252. [PMID: 34885834 PMCID: PMC8658973 DOI: 10.3390/molecules26237252] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/04/2021] [Revised: 11/23/2021] [Accepted: 11/26/2021] [Indexed: 11/16/2022] Open
Abstract
The aim of the work was to prepare a simple but reliable HPLC-UV method for the routine monitoring of mycophenolic acid (MPA). Sample preparation was based on plasma protein precipitation with acetonitrile. The isocratic separation of MPA and internal standard (IS) fenbufen was made on Supelcosil LC-CN column (150 × 4.6 mm, 5 µm) using a mobile phase: CH3CN:H2O:0.5M KH2PO4:H3PO4 (260:700:40:0.4, v/v). UV detection was set at 305 nm. The calibration covered the MPA concentration range: 0.1–40 µg/mL. The precision was satisfactory with RSD of 0.97–7.06% for intra-assay and of 1.92–5.15% for inter-assay. The inaccuracy was found between −5.72% and +2.96% (+15.40% at LLOQ) and between −8.82% and +5.31% (+19.00% at LLOQ) for intra- and inter-assay, respectively, fulfilling acceptance criteria. After a two-year period of successful application, the presented method has been retrospectively calibrated using the raw data disregarding the IS in the calculations. The validation and stability parameters were similar for both calculation methods. MPA concentrations were recalculated and compared in 1187 consecutive routine therapeutic drug monitoring (TDM) trough plasma samples from mycophenolate-treated patients. A high agreement (r2 = 0.9931, p < 0.0001) of the results was found. A Bland–Altman test revealed a mean bias of −0.011 μg/mL (95% CI: −0.017; −0.005) comprising −0.14% (95% Cl: −0.39; +0.11), whereas the Passing–Bablok regression was y = 0.986x + 0.014. The presented method can be recommended as an attractive analytical tool for medical (hospital) laboratories equipped with solely basic HPLC apparatus. The procedure can be further simplified by disapplying an internal standard while maintaining appropriate precision and accuracy of measurements.
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de Ruiter PE, Gadjradj Y, de Knegt RJ, Metselaar HJ, Ijzermans JNM, van der Laan LJW. Interaction of immunosuppressants with HCV antivirals daclatasvir and asunaprevir: combined effects with mycophenolic acid. World J Transplant 2018; 8:156-166. [PMID: 30211024 PMCID: PMC6134272 DOI: 10.5500/wjt.v8.i5.156] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/23/2018] [Revised: 06/14/2018] [Accepted: 06/27/2018] [Indexed: 02/05/2023] Open
Abstract
AIM To investigate the specific effects of immunosuppressants on the antiviral action of daclatasvir and asunaprevir.
METHODS The antiviral activity of daclatasvir (DCV) and asunaprevir (ASV) combined with immunosuppressants was tested using two in vitro models for hepatitis C virus (HCV) infection.
RESULTS Tacrolimus, rapamycin and cyclosporine did not negatively affect the antiviral action of DCV or ASV. Mycophenolic acid (MPA) showed additive antiviral effects combined with these direct acting antivirals (DAAs). MPA induces interferon-stimulated genes (ISGs) and is a potent GTP synthesis inhibitor. DCV or ASV did not induce ISGs expression nor affected ISG induction by MPA. Rather, the combined antiviral effect of MPA with DCV and ASV was partly mediated via inhibition of GTP synthesis.
CONCLUSION Immunosuppressants do not negatively affect the antiviral activity of DAAs. MPA has additive effect on the antiviral action of DCV and ASV. This combined benefit needs to be confirmed in prospective clinical trials.
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Affiliation(s)
- Petra E de Ruiter
- Department of Surgery, Erasmus MC-University Medical Center, Rotterdam CN 3015, the Netherlands
| | - Yashna Gadjradj
- Department of Surgery, Erasmus MC-University Medical Center, Rotterdam CN 3015, the Netherlands
| | - Robert J de Knegt
- Department of Gastroenterology and Hepatology, Erasmus MC-University Medical Center, Rotterdam CN 3015, the Netherlands
| | - Herold J Metselaar
- Department of Gastroenterology and Hepatology, Erasmus MC-University Medical Center, Rotterdam CN 3015, the Netherlands
| | - Jan NM Ijzermans
- Department of Surgery, Erasmus MC-University Medical Center, Rotterdam CN 3015, the Netherlands
| | - Luc JW van der Laan
- Department of Surgery, Erasmus MC-University Medical Center, Rotterdam CN 3015, the Netherlands
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3
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Dang W, Yin Y, Wang Y, Wang W, Su J, Sprengers D, van der Laan LJW, Felczak K, Pankiewicz KW, Chang KO, Koopmans MPG, Metselaar HJ, Peppelenbosch MP, Pan Q. Inhibition of Calcineurin or IMP Dehydrogenase Exerts Moderate to Potent Antiviral Activity against Norovirus Replication. Antimicrob Agents Chemother 2017; 61:e01095-17. [PMID: 28807916 PMCID: PMC5655111 DOI: 10.1128/aac.01095-17] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/26/2017] [Accepted: 08/05/2017] [Indexed: 12/14/2022] Open
Abstract
Norovirus is a major cause of acute gastroenteritis worldwide and has emerged as an important issue of chronic infection in transplantation patients. Since no approved antiviral is available, we evaluated the effects of different immunosuppressants and ribavirin on norovirus and explored their mechanisms of action by using a human norovirus (HuNV) replicon-harboring model and a surrogate murine norovirus (MNV) infectious model. The roles of the corresponding drug targets were investigated by gain- or loss-of-function approaches. We found that the calcineurin inhibitors cyclosporine (CsA) and tacrolimus (FK506) moderately inhibited HuNV replication. Gene silencing of their cellular targets, cyclophilin A, FKBP12, and calcineurin, significantly inhibited HuNV replication. A low concentration, therapeutically speaking, of mycophenolic acid (MPA), an uncompetitive IMP dehydrogenase (IMPDH) inhibitor, potently and rapidly inhibited norovirus replication and ultimately cleared HuNV replicons without inducible resistance following long-term drug exposure. Knockdown of the MPA cellular targets IMPDH1 and IMPDH2 suppressed HuNV replication. Consistent with the nucleotide-synthesizing function of IMPDH, exogenous guanosine counteracted the antinorovirus effects of MPA. Furthermore, the competitive IMPDH inhibitor ribavirin efficiently inhibited norovirus and resulted in an additive effect when combined with immunosuppressants. The results from this study demonstrate that calcineurin phosphatase activity and IMPDH guanine synthase activity are crucial in sustaining norovirus infection; thus, they can be therapeutically targeted. Our results suggest that MPA shall be preferentially considered immunosuppressive medication for transplantation patients at risk of norovirus infection, whereas ribavirin represents as a potential antiviral for both immunocompromised and immunocompetent patients with norovirus gastroenteritis.
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Affiliation(s)
- Wen Dang
- Department of Gastroenterology and Hepatology, Erasmus MC-University Medical Center, Rotterdam, The Netherlands
| | - Yuebang Yin
- Department of Gastroenterology and Hepatology, Erasmus MC-University Medical Center, Rotterdam, The Netherlands
| | - Yijin Wang
- Department of Gastroenterology and Hepatology, Erasmus MC-University Medical Center, Rotterdam, The Netherlands
| | - Wenshi Wang
- Department of Gastroenterology and Hepatology, Erasmus MC-University Medical Center, Rotterdam, The Netherlands
| | - Junhong Su
- Medical Faculty, Kunming University of Science and Technology, Kunming, People's Republic of China
| | - Dave Sprengers
- Department of Gastroenterology and Hepatology, Erasmus MC-University Medical Center, Rotterdam, The Netherlands
| | - Luc J W van der Laan
- Department of Surgery, Erasmus MC-University Medical Center, Rotterdam, The Netherlands
| | - Krzysztof Felczak
- Center for Drug Design, University of Minnesota, Minneapolis, Minnesota, USA
| | | | - Kyeong-Ok Chang
- Department of Diagnostic Medicine and Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan, Kansas, USA
| | - Marion P G Koopmans
- Department of Viroscience, Erasmus MC-University Medical Center, Rotterdam, The Netherlands
| | - Herold J Metselaar
- Department of Gastroenterology and Hepatology, Erasmus MC-University Medical Center, Rotterdam, The Netherlands
| | - Maikel P Peppelenbosch
- Department of Gastroenterology and Hepatology, Erasmus MC-University Medical Center, Rotterdam, The Netherlands
| | - Qiuwei Pan
- Department of Gastroenterology and Hepatology, Erasmus MC-University Medical Center, Rotterdam, The Netherlands
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4
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Fang S, Su J, Liang B, Li X, Li Y, Jiang J, Huang J, Zhou B, Ning C, Li J, Ho W, Li Y, Chen H, Liang H, Ye L. Suppression of autophagy by mycophenolic acid contributes to inhibition of HCV replication in human hepatoma cells. Sci Rep 2017; 7:44039. [PMID: 28276509 PMCID: PMC5343675 DOI: 10.1038/srep44039] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/21/2016] [Accepted: 02/02/2017] [Indexed: 12/20/2022] Open
Abstract
Previous studies have shown that mycophenolic acid (MPA) has an anti-HCV activity. However, the mechanism of MPA-mediated inhibition of HCV replication remains to be determined. This study investigated whether MPA has an effect on autophagy, a cellular machinery required for HCV replication, thereby, inhibits HCV replication in Huh7 cells. MPA treatment of Huh7 cells could suppress autophagy, evidenced by decreased LC3B-II level and conversion of LC3B-I to LC3B-II, decreased autophagosome formation, and increased p62 level compared to MPA-untreated cells. Tunicamycin treatment or HCV infection could induce cellular autophagy, however, MPA also exhibited its inhibitory effect on tunicamycin- or HCV infection-induced autophagy. The expression of three autophagy-related genes, Atg3, Atg5, and Atg7 were identified to be inhibited by MPA treatment. Over-expression of these genes could partly recover HCV replication inhibited by MPA; however, silencing their expression by siRNAs could enhance the inhibitory effect of MPA on HCV. Collectively, these results reveal that suppression of autophagy by MPA plays a role in its anti-HCV activity. Down-regulating the expression of three autophagy-related genes by MPA involves in its antiviral mechanism.
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Affiliation(s)
- Shoucai Fang
- Guangxi Key Laboratory of AIDS Prevention and Treatment &Guangxi Universities Key Laboratory of Prevention and Control of Highly Prevalent Disease, School of Public Health, Guangxi Medical University, Nanning 530021, Guangxi, China.,Guangxi Collaborative Innovation Center for Biomedicine, Life Sciences Institute, Guangxi Medical University, Nanning 530021, Guangxi, China
| | - Jinming Su
- Guangxi Key Laboratory of AIDS Prevention and Treatment &Guangxi Universities Key Laboratory of Prevention and Control of Highly Prevalent Disease, School of Public Health, Guangxi Medical University, Nanning 530021, Guangxi, China.,Division of HIV/AIDS Control and Prevention, Guangxi Center for Disease Control and Prevention, Nanning 530021, Guangxi, China
| | - Bingyu Liang
- Guangxi Key Laboratory of AIDS Prevention and Treatment &Guangxi Universities Key Laboratory of Prevention and Control of Highly Prevalent Disease, School of Public Health, Guangxi Medical University, Nanning 530021, Guangxi, China.,Guangxi Collaborative Innovation Center for Biomedicine, Life Sciences Institute, Guangxi Medical University, Nanning 530021, Guangxi, China
| | - Xu Li
- Guangxi Key Laboratory of AIDS Prevention and Treatment &Guangxi Universities Key Laboratory of Prevention and Control of Highly Prevalent Disease, School of Public Health, Guangxi Medical University, Nanning 530021, Guangxi, China.,Guangxi Collaborative Innovation Center for Biomedicine, Life Sciences Institute, Guangxi Medical University, Nanning 530021, Guangxi, China
| | - Yu Li
- Guangxi Key Laboratory of AIDS Prevention and Treatment &Guangxi Universities Key Laboratory of Prevention and Control of Highly Prevalent Disease, School of Public Health, Guangxi Medical University, Nanning 530021, Guangxi, China
| | - Junjun Jiang
- Guangxi Key Laboratory of AIDS Prevention and Treatment &Guangxi Universities Key Laboratory of Prevention and Control of Highly Prevalent Disease, School of Public Health, Guangxi Medical University, Nanning 530021, Guangxi, China.,Guangxi Collaborative Innovation Center for Biomedicine, Life Sciences Institute, Guangxi Medical University, Nanning 530021, Guangxi, China
| | - Jiegang Huang
- Guangxi Key Laboratory of AIDS Prevention and Treatment &Guangxi Universities Key Laboratory of Prevention and Control of Highly Prevalent Disease, School of Public Health, Guangxi Medical University, Nanning 530021, Guangxi, China.,Guangxi Collaborative Innovation Center for Biomedicine, Life Sciences Institute, Guangxi Medical University, Nanning 530021, Guangxi, China
| | - Bo Zhou
- Guangxi Key Laboratory of AIDS Prevention and Treatment &Guangxi Universities Key Laboratory of Prevention and Control of Highly Prevalent Disease, School of Public Health, Guangxi Medical University, Nanning 530021, Guangxi, China.,Guangxi Collaborative Innovation Center for Biomedicine, Life Sciences Institute, Guangxi Medical University, Nanning 530021, Guangxi, China
| | - Chuanyi Ning
- Guangxi Key Laboratory of AIDS Prevention and Treatment &Guangxi Universities Key Laboratory of Prevention and Control of Highly Prevalent Disease, School of Public Health, Guangxi Medical University, Nanning 530021, Guangxi, China.,Guangxi Collaborative Innovation Center for Biomedicine, Life Sciences Institute, Guangxi Medical University, Nanning 530021, Guangxi, China
| | - Jieliang Li
- Department of Pathology and Laboratory Medicine, Temple University School of Medicine, Philadelphia, PA 19140, USA
| | - Wenzhe Ho
- Department of Pathology and Laboratory Medicine, Temple University School of Medicine, Philadelphia, PA 19140, USA
| | - Yiping Li
- Institute of Human Virology and Key Laboratory of Tropical Disease Control of Ministry of Education, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510080, China
| | - Hui Chen
- Geriatrics Digestion Department of Internal Medicine, The First Affiliated Hospital of Guangxi Medical University, Nanning 530021, Guangxi, China
| | - Hao Liang
- Guangxi Key Laboratory of AIDS Prevention and Treatment &Guangxi Universities Key Laboratory of Prevention and Control of Highly Prevalent Disease, School of Public Health, Guangxi Medical University, Nanning 530021, Guangxi, China.,Guangxi Collaborative Innovation Center for Biomedicine, Life Sciences Institute, Guangxi Medical University, Nanning 530021, Guangxi, China
| | - Li Ye
- Guangxi Key Laboratory of AIDS Prevention and Treatment &Guangxi Universities Key Laboratory of Prevention and Control of Highly Prevalent Disease, School of Public Health, Guangxi Medical University, Nanning 530021, Guangxi, China.,Guangxi Collaborative Innovation Center for Biomedicine, Life Sciences Institute, Guangxi Medical University, Nanning 530021, Guangxi, China
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5
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Wang L, Qiang W, Li Y, Cheng Z, Xie M. A novel freeze-dried storage and preparation method for the determination of mycophenolic acid in plasma by high-performance liquid chromatography. Biomed Chromatogr 2017; 31. [PMID: 28205247 DOI: 10.1002/bmc.3958] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/27/2016] [Revised: 02/01/2017] [Accepted: 02/11/2017] [Indexed: 11/09/2022]
Abstract
Plasma samples were conventionally stored at freezing conditions until the time of detection. Such a technique, when carried out over an extended period, is energy consuming; in addition, preparation and transportation of stored samples is inconvenient. In this study, a freeze-dried storage and preparation method was proposed to determine the presence of mycophenolic acid (MPA) in plasma. Fresh plasma samples were freeze-dried using a device, and then stored at ambient temperature. After the stored samples were soaked with methanol spiked with the internal standard, high-performance liquid chromatography was conducted to detect MPA. The proposed method was demonstrated to be precise and accurate over the linear range of 0.5-50 μg mL-1 , with both intra- and inter-day precision being <7% and biases <10%. The freeze-dried samples were stable at ambient temperature for at least 40 days. This method was also successfully applied to the pharmacokinetic study of MPA in healthy volunteers. Pharmacokinetic parameters, such as maximum plasma concentration, time point of maximum plasma concentration and elimination half-life, among others, were consistent with the results in the published study. This proposed technique was proved to be simple, reproducible and energy saving. This approach could also simplify the storage and analysis of samples in clinical and scientific drug research.
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Affiliation(s)
- Lei Wang
- School of Life Sciences, Central South University, Changsha, Hunan, China.,Research Institute of Drug Metabolism and Pharmacokinetics, School of Pharmaceutical Sciences, Central South University, Changsha, Hunan, China
| | - Wei Qiang
- Research Institute of Drug Metabolism and Pharmacokinetics, School of Pharmaceutical Sciences, Central South University, Changsha, Hunan, China
| | - Ying Li
- School of Food and Pharmaceutical Engineering, Guiyang College, Guiyang, China
| | - Zeneng Cheng
- Research Institute of Drug Metabolism and Pharmacokinetics, School of Pharmaceutical Sciences, Central South University, Changsha, Hunan, China
| | - Mengmeng Xie
- Research Institute of Drug Metabolism and Pharmacokinetics, School of Pharmaceutical Sciences, Central South University, Changsha, Hunan, China
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Chen B, Huang JJ, Chen HF, Xu BM. Clinical pharmacy service practice in a Chinese tertiary hospital. Drug Metab Pers Ther 2016; 30:215-30. [PMID: 26457791 DOI: 10.1515/dmpt-2015-0009] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/17/2015] [Accepted: 09/01/2015] [Indexed: 12/31/2022]
Abstract
Clinical pharmacy service is focused on the rationality and safety of medication therapy. Clinical pharmacists play an important role in designing therapeutic regimen, preventing medication errors, reducing the incidence of adverse drug reaction, and saving medical costs. Although clinical pharmacy service in China is in its early stage, its development is rapid. In this manuscript, the working model of clinical pharmacists in a Chinese tertiary hospital is introduced, including ward rounds, consultation, stewardship of antimicrobial therapy, drug adverse reaction monitoring, therapeutic drug monitoring, clinical pharmacokinetics and pharmacogenetics, and training system. With the efforts of clinical pharmacists, there will be a significant increase in the optimization of medication therapy and a notable reduction in preventable adverse drug events as well as health-care cost in China.
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7
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Chen K, Cao W, Li J, Sprengers D, Hernanda PY, Kong X, van der Laan LJW, Man K, Kwekkeboom J, Metselaar HJ, Peppelenbosch MP, Pan Q. Differential Sensitivities of Fast- and Slow-Cycling Cancer Cells to Inosine Monophosphate Dehydrogenase 2 Inhibition by Mycophenolic Acid. Mol Med 2015; 21:792-802. [PMID: 26467706 DOI: 10.2119/molmed.2015.00126] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/22/2015] [Accepted: 10/12/2015] [Indexed: 01/03/2023] Open
Abstract
As uncontrolled cell proliferation requires nucleotide biosynthesis, inhibiting enzymes that mediate nucleotide biosynthesis constitutes a rational approach to the management of oncological diseases. In practice, however, results of this strategy are mixed and thus elucidation of the mechanisms by which cancer cells evade the effect of nucleotide biosynthesis restriction is urgently needed. Here we explored the notion that intrinsic differences in cancer cell cycle velocity are important in the resistance toward inhibition of inosine monophosphate dehydrogenase (IMPDH) by mycophenolic acid (MPA). In short-term experiments, MPA treatment of fast-growing cancer cells effectively elicited G0/G1 arrest and provoked apoptosis, thus inhibiting cell proliferation and colony formation. Forced expression of a mutated IMPDH2, lacking a binding site for MPA but retaining enzymatic activity, resulted in complete resistance of cancer cells to MPA. In nude mice subcutaneously engrafted with HeLa cells, MPA moderately delayed tumor formation by inhibiting cell proliferation and inducing apoptosis. Importantly, we developed a lentiviral vector-based Tet-on label-retaining system that enables to identify, isolate and functionally characterize slow-cycling or so-called label-retaining cells (LRCs) in vitro and in vivo. We surprisingly found the presence of LRCs in fast-growing tumors. LRCs were superior in colony formation, tumor initiation and resistance to MPA as compared with fast-cycling cells. Thus, the slow-cycling compartment of cancer seems predominantly responsible for resistance to MPA.
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Affiliation(s)
- Kan Chen
- Department of Gastroenterology and Hepatology, Erasmus MC Cancer Institute, Erasmus University Medical Center, Rotterdam, The Netherlands.,Bio-X Center, College of Life Sciences, Zhejiang Sci-Tech University, Hangzhou, China
| | - Wanlu Cao
- Department of Gastroenterology and Hepatology, Erasmus MC Cancer Institute, Erasmus University Medical Center, Rotterdam, The Netherlands
| | - Juan Li
- Department of Gastroenterology and Hepatology, Erasmus MC Cancer Institute, Erasmus University Medical Center, Rotterdam, The Netherlands
| | - Dave Sprengers
- Department of Gastroenterology and Hepatology, Erasmus MC Cancer Institute, Erasmus University Medical Center, Rotterdam, The Netherlands
| | - Pratika Y Hernanda
- Laboratory of Medical Genetics, Biomolecular Research Center, Wijaya Kusuma University, Surabaya, Indonesia
| | - Xiangdong Kong
- Bio-X Center, College of Life Sciences, Zhejiang Sci-Tech University, Hangzhou, China
| | - Luc J W van der Laan
- Department of Surgery, Erasmus University Medical Center, Rotterdam, The Netherlands
| | - Kwan Man
- Department of Surgery, Hong Kong University, Hong Kong, China
| | - Jaap Kwekkeboom
- Department of Gastroenterology and Hepatology, Erasmus MC Cancer Institute, Erasmus University Medical Center, Rotterdam, The Netherlands
| | - Herold J Metselaar
- Department of Gastroenterology and Hepatology, Erasmus MC Cancer Institute, Erasmus University Medical Center, Rotterdam, The Netherlands
| | - Maikel P Peppelenbosch
- Department of Gastroenterology and Hepatology, Erasmus MC Cancer Institute, Erasmus University Medical Center, Rotterdam, The Netherlands
| | - Qiuwei Pan
- Department of Gastroenterology and Hepatology, Erasmus MC Cancer Institute, Erasmus University Medical Center, Rotterdam, The Netherlands
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Chen H, Chen B. Clinical mycophenolic acid monitoring in liver transplant recipients. World J Gastroenterol 2014; 20:10715-10728. [PMID: 25152575 PMCID: PMC4138452 DOI: 10.3748/wjg.v20.i31.10715] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/17/2013] [Revised: 06/03/2014] [Accepted: 06/26/2014] [Indexed: 02/06/2023] Open
Abstract
In liver transplantation, the efficacy of mycophenolate mofetil (MMF) has been confirmed in clinical trials and studies. However, therapeutic drug monitoring for mycophenolic acid (MPA) has not been fully accepted in liver transplantation as no long-term prospective study of concentration controlled vs fixed-dose prescribing of MMF has been done. This review addressed MPA measurement, pharmacokinetic variability and reasons of this variation, exposure related to acute rejection and MMF-associated side effects in liver transplant recipients. Limited sampling strategies to predict MPA area under the concentration-time curve have also been described, and the value of clinical use needs to be investigated in future. The published data suggested that a fixed-dosage MMF regimen might not be suitable and monitoring of MPA exposure seems helpful in various clinical settings of liver transplantation.
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Dasgupta A, Johnson M, Tso G. Mathematical equations to calculate true mycophenolic acid concentration in human plasma by using two immunoassays with different cross-reactivities with acyl glucuronide metabolite: comparison of calculated values with values obtained by using an HPLC-UV method. J Clin Lab Anal 2014; 27:290-3. [PMID: 23852786 DOI: 10.1002/jcla.21599] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/26/2012] [Accepted: 02/06/2013] [Indexed: 11/05/2022] Open
Abstract
BACKGROUND Both immunoassays and chromatographic methods are available for therapeutic drug monitoring of mycophenolic acid (MPA). Although chromatographic methods are more precise, immunoassays are widely used in clinical laboratories due to ease of adopting such assays on automated analyzers. We studied the possibility of using mathematical equations to calculate true MPA concentration by accounting for acyl glucuronide cross-reactivities with immunoassays by using two immunoassays with widely different cross-reactivities with the metabolite. METHODS We determined MPA concentrations in 20 specimens obtained from transplant recipients using cloned enzyme donor immunoassay (CEDIA) assay and a new particle enhanced turbidimetric inhibition immunoassay (PETINIA) assay. Then we developed mathematical equations to calculate true MPA concentration using values obtained by both immunoassays and reported cross-reactivity of acyl glucuronide with respective immunoassays. Calculated concentrations were compared with values obtained by using a high-performance liquid chromatography combined with ultraviolet detection (HPLC-UV) method. RESULTS We obtained good correlation between calculated MPA concentrations and corresponding MPA level obtained by using HPLC-UV method. Using x-axis as the MPA concentrations determined by the HPLC-UV method and y-axis as the calculated MPA level, we observed the following regression equation: y = 1.083x - 0.0995 (r = 0.99, n = 20). CONCLUSIONS Mathematical equations can be used to calculate true MPA concentrations using two immunoassays with different cross-reactivities with acyl glucuronide metabolite.
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Affiliation(s)
- Amitava Dasgupta
- Department of Pathology, University of Texas-Houston Medical School, Houston, TX 77030, USA.
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10
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Dasgupta A, Johnson M. Positive bias in mycophenolic acid concentrations determined by the CEDIA assay compared to HPLC-UV method: is CEDIA assay suitable for therapeutic drug monitoring of mycophenolic acid? J Clin Lab Anal 2013; 27:77-80. [PMID: 23325745 DOI: 10.1002/jcla.21565] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/24/2012] [Accepted: 11/01/2012] [Indexed: 11/10/2022] Open
Abstract
BACKGROUND Both immunoassays and chromatographic methods are available for therapeutic drug monitoring of mycophenolic acid (MPA), an immunosuppressant. We studied the suitability of cloned enzyme donor immunoassay (CEDIA) assay for routine monitoring of MPA by comparing values obtained by the CEDIA assay with corresponding values obtained by using a high-performance liquid chromatography combined with ultraviolet detection (HPLC-UV) method. METHODS We compared MPA concentrations obtained by a reference HPLC-UV method and CEDIA assay on Hitachi 917 analyzer (Roche Diagnostics, Indianapolis, IN) using 60 patient specimens (18 liver transplant recipient and 42 kidney transplant recipients). RESULTS When MPA concentrations in all 60 transplant recipients obtained by the HPLC-UV (x-axis) method were compared with corresponding values obtained by the CEDIA method (y-axis), the following regression equation was obtained: y = 1.1558x + 0.2876 (r = 0.97). Interestingly, much lower bias was observed in 42 renal transplant recipients as revealed by the following regression equation; y = 1.1181x + 0.2745 (r = 0.98). However, more significant positive bias was observed in 18 liver transplant recipients as following regression equation as observed: y = 1.3337x + 0.1493 (r = 0.94). CONCLUSIONS We conclude that MPA concentrations determined by the CEDIA assay showed significant positive bias compared to HPLC-UV method. Therefore, caution must be exercised in interpreting therapeutic drug monitoring result of MPA if CEDIA assay is used.
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Affiliation(s)
- Amitava Dasgupta
- Department of Pathology, University of Texas-Houston Medical School and Laboratory Services Memorial-Hermann Hospital at Texas Medical Center, Houston, TX, USA.
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11
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Upadhyay V, Trivedi V, Shah G, Yadav M, Shrivastav PS. Determination of mycophenolic acid in human plasma by ultra performance liquid chromatography tandem mass spectrometry. J Pharm Anal 2013; 4:205-216. [PMID: 29403884 PMCID: PMC5761118 DOI: 10.1016/j.jpha.2013.06.001] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/28/2013] [Accepted: 06/03/2013] [Indexed: 11/16/2022] Open
Abstract
A simple, sensitive and high throughput ultra performance liquid chromatography tandem mass spectrometry method has been developed for the determination of mycophenolic acid in human plasma. The method involved simple protein precipitation of MPA along with its deuterated analog as an internal standard (IS) from 50 µL of human plasma. The chromatographic analysis was done on Acquity UPLC C18 (100 mm×2.1 mm, 1.7 µm) column under isocratic conditions using acetonitrile and 10 mM ammonium formate, pH 3.00 (75:25, v/v) as the mobile phase. A triple quadrupole mass spectrometer operating in the positive ionization mode was used for quantitation. In-source conversion of mycophenolic glucuronide metabolite to the parent drug was selectively controlled by suitable optimization of cone voltage, cone gas flow and desolvation temperature. The method was validated over a wide concentration range of 15-15000 ng/mL. The mean extraction recovery for the analyte and IS was >95%. Matrix effect expressed as matrix factors ranged from 0.97 to 1.02. The method was successfully applied to support a bioequivalence study of 500 mg mycophenolate mofetil tablet in 72 healthy subjects.
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Affiliation(s)
- Vivek Upadhyay
- Chemistry Department, Kadi Sarva Vishwavidyalaya, Gandhinagar 382015, India
| | - Vikas Trivedi
- Chemistry Department, Kadi Sarva Vishwavidyalaya, Gandhinagar 382015, India
| | - Gaurang Shah
- Chemistry Department, Kadi Sarva Vishwavidyalaya, Gandhinagar 382015, India
| | - Manish Yadav
- Chemistry Department, Kadi Sarva Vishwavidyalaya, Gandhinagar 382015, India
| | - Pranav S. Shrivastav
- Chemistry Department, Kadi Sarva Vishwavidyalaya, Gandhinagar 382015, India
- Department of Chemistry, School of Sciences, Gujarat University, Navrangpura, Ahmedabad 380009, India
- Corresponding author at: Department of Chemistry, School of Sciences, Gujarat University, Navrangpura, Ahmedabad 380009, India. Tel.: +91 079 2630 0969; fax: +91 079 2630 8545.
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Dostalek M, Gohh RY, Akhlaghi F. Inosine monophosphate dehydrogenase expression and activity are significantly lower in kidney transplant recipients with diabetes mellitus. Ther Drug Monit 2013; 35:374-83. [PMID: 23666569 PMCID: PMC4109137 DOI: 10.1097/ftd.0b013e3182852697] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/25/2022]
Abstract
BACKGROUND Inosine 5'-monophosphate dehydrogenase (IMPDH) is a target of the immunosuppressive drug, mycophenolic acid (MPA). A 12-hour clinical pharmacokinetic and pharmacodynamic study was conducted to compare IMPDH1 and IMPDH2 gene expression, IMPDHI and IMPDHII protein levels, and enzyme activity between kidney transplant recipients with respect to diabetes status. METHODS Nondiabetic (ND, n = 11) and diabetic (D, n = 9) kidney transplant recipients and on nontransplant nondiabetic (n = 10) and diabetic (n = 10) volunteers were included in the study. RESULTS Area under the effect curve values for gene expression: IMPDH1 [ND: 22.1 (13.8-31.3) versus D: 4.5 (2.3-6.5), P < 0.001] and IMPDH2 [ND: 15.3 (11.0-21.7) versus D: 6.1 (4.6-8.6), P < 0.001], protein level: IMPDHI [ND: 1.0 (0.5-1.3) versus 0.5 (0.4-0.7), P = 0.002] and IMPDHII [ND: 1.0 (0.6-1.6) versus D: 0.7 (0.6-0.8) P < 0.001] and enzyme activity [ND: 180 (105-245) versus D: 29.9 (15.3-35.6) µmole·s(-1)·mole(-1) adenosine monophosphate, P < 0.001] was significantly lower in transplant recipients with diabetes. Similar results were observed in nontransplanted volunteers. Kinetic studies of MPA-mediated suppression of IMPDH activity in nontransplanted individuals revealed an approximately 2.5-fold lower half-maximum effective concentration (EC50) for diabetic as compared with nondiabetic [ND: 50.2 (49.8-50.7) versus D: 15.8 (15.6-16.3) nmole/L, P = 0.004] volunteers. This difference was not related to several IMPDH gene variants. CONCLUSIONS This study indicates a significantly lower IMPDH gene expression, protein level, and enzyme activity in diabetic patients. Further clinical studies in a larger number of patients are warranted to verify whether MPA dosing must be optimized for kidney transplant recipients with diabetes mellitus.
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Affiliation(s)
- Miroslav Dostalek
- Department of Biomedical and Pharmaceutical Sciences, University of Rhode Island, Kingston, RI, USA
| | - Reginald Y. Gohh
- Division of Organ Transplantation, Rhode Island Hospital, Warren Alpert Medical School of Brown University, Providence, Rhode Island, USA
| | - Fatemeh Akhlaghi
- Department of Biomedical and Pharmaceutical Sciences, University of Rhode Island, Kingston, RI, USA
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Comparison of mycophenolic acid concentrations determined by a new PETINIA assay on the Dimension EXL analyzer and a HPLC-UV method. Clin Biochem 2013; 46:685-7. [DOI: 10.1016/j.clinbiochem.2012.11.025] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/29/2012] [Revised: 11/15/2012] [Accepted: 11/27/2012] [Indexed: 11/19/2022]
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Patel CG, Ogasawara K, Akhlaghi F. Mycophenolic acid glucuronide is transported by multidrug resistance-associated protein 2 and this transport is not inhibited by cyclosporine, tacrolimus or sirolimus. Xenobiotica 2012; 43:229-35. [PMID: 22934787 DOI: 10.3109/00498254.2012.713531] [Citation(s) in RCA: 33] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022]
Abstract
1. The purpose of this study was to investigate the contribution of MRP2 to the efflux of mycophenolic acid (MPA), and its phenyl glucuronide (MPAG) and acyl glucuronide (AcMPAG) metabolites, using Madin-Darby canine kidney II cells stably transfected with human MRP2 gene (MDCKII/MRP2 cells). 2. Compared to parental MDCKII cells, MPAG was significantly translocated from basolateral (BL) to apical (AP) side in MDCKII/MRP2 cells, indicating MPAG is a substrate for MRP2. AcMPAG is highly translocated from BL to AP side in both cells, suggesting that AcMPAG is actively secreted possibly through an efflux transporter other than MRP2. Appreciable translocation of MPA was not observed in MDCKII/MRP2 cells. 3. Furthermore, using MRP2-expressing Sf9 membrane vesicles, the Michaelis-Menten constant (Km) value for MRP2-mediated MPAG transport was calculated at 224.2 ± 42.7 µM. In the vesicle system, cyclosporine, tacrolimus and sirolimus did not inhibit the uptake of MPAG via MRP2. 4. These findings indicate that only MPAG not MPA and AcMPAG is a substrate for MRP2 and that the interaction between MPAG and concomitantly administered immunosuppressive agents does not occur at MRP2 level.
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Affiliation(s)
- Chirag G Patel
- Clinical Pharmacokinetics Research Laboratory, Department of Biomedical and Pharmaceutical Sciences, College of Pharmacy, University of Rhode Island, Kingston, RI 02881, USA
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15
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Mycophenolate mofetil inhibits hepatitis C virus replication in human hepatic cells. Virus Res 2012; 168:33-40. [PMID: 22728816 DOI: 10.1016/j.virusres.2012.06.009] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/15/2012] [Revised: 06/06/2012] [Accepted: 06/08/2012] [Indexed: 12/13/2022]
Abstract
Hepatitis C virus (HCV) infection is the most common indication for liver transplantation and the major cause of graft failure. A widely used immunosuppressant, cyclosporine A (CsA), for people who receive organ transplantation, has been recognized to have the ability to inhibit HCV replication both in vivo and in vitro. In this study, we investigated the effects of several other immunosuppressants, including mycophenolate mofetil (MMF), rapamycin and FK506, on HCV replication in human hepatic cells. MMF treatment of hepatic cells before or during HCV infection significantly suppressed full cycle viral replication, as evidenced by decreased expression of HCV RNA, protein and production of infectious virus. In contrast, rapamycin and FK506 had little effect on HCV replication. Investigation of the mechanism(s) disclosed that the inhibition of HCV replication by MMF was mainly due to its depletion of guanosine, a purine nucleoside crucial for synthesis of guanosine triphosphate, which is required for HCV RNA replication. The supplement of exogenous guanosine could reverse most of anti-HCV effect of mycophenolate mofetil. These data indicate that MMF, through the depletion of guanosine, inhibits full cycle HCV JFH-1 replication in human hepatic cells. It is of interest to further determine whether MMF is indeed beneficial for HCV-infected transplant recipients in future clinical studies.
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Pan Q, de Ruiter PE, Metselaar HJ, Kwekkeboom J, de Jonge J, Tilanus HW, Janssen HLA, van der Laan LJW. Mycophenolic acid augments interferon-stimulated gene expression and inhibits hepatitis C Virus infection in vitro and in vivo. Hepatology 2012; 55:1673-83. [PMID: 22213147 DOI: 10.1002/hep.25562] [Citation(s) in RCA: 86] [Impact Index Per Article: 6.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/11/2011] [Accepted: 12/16/2011] [Indexed: 12/13/2022]
Abstract
UNLABELLED Mycophenolic acid (MPA) is a highly effective immunosuppressant that has broad antiviral activity against different viruses and can act in synergy with interferon-α (IFN-α) on hepatitis C virus (HCV) replication. MPA is a potent inosine monophosphate dehydrogenase (IMPDH) inhibitor but the antiviral mechanisms are less understood. The aim of this study was to investigate the inhibition of HCV infection by MPA and the molecular basis for its synergy with IFN-α. The role of IMPDH and interferon-stimulated genes (ISGs) was investigated in two HCV models using gain- or loss-of-function approaches. The in vivo effect of MPA treatment was studied in NOD/SCID mice engrafted with HCV replicon cells. Potent antiviral effects of MPA at clinically relevant concentrations were observed with both the subgenomic and JFH1-derived infectious HCV models. MPA treatment in mice resulted in a specific and robust inhibition of HCV replication. Ectopic expression of an MPA-resistant IMPDH2 mutant in HCV host cells completely reversed the antiproliferative effect of MPA but only partially affected the antiviral potency. However, similar to ribavirin, MPA induced expression of multiple antiviral ISGs, including interferon regulatory factor 1 (IRF1). Cotreatment of MPA with IFN-α resulted in additive effects on ISG expression and enhanced IFN-induced luciferase reporter activity. Knockdown of IRF1, but not IFITM3, significantly attenuated the inhibition of HCV replication by MPA. CONCLUSION MPA exerts a potent anti-HCV effect in vitro and in mice and acts in synergy with IFN-α. MPA's antiviral activity partially depends on IMPDH but also involves stimulation of ISGs, providing a molecular basis for its synergy with IFN-α.
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Affiliation(s)
- Qiuwei Pan
- Department of Gastroenterology & Hepatology, Erasmus MC-University Medical Center, Rotterdam, Netherlands
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Laverdière I, Caron P, Couture F, Lévesque E, Guillemette C. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for monitoring drug exposure in hematopoietic stem cell transplant recipients. J Chromatogr B Analyt Technol Biomed Life Sci 2012; 885-886:131-7. [PMID: 22265668 DOI: 10.1016/j.jchromb.2011.12.029] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/05/2011] [Revised: 12/23/2011] [Accepted: 12/30/2011] [Indexed: 11/30/2022]
Abstract
A liquid chromatography-tandem mass spectrometry method was developed for the quantification of circulating levels of multiple immunosuppressant drugs including cyclosporine (CsA), tacrolimus, methotrexate (Mtx), prednisone, prednisolone, methylprednisone, total and free mycophenolic acid (MPA), as well as MPA phenolic (MPAG) and acyl (AcMPAG) glucuronide metabolites. Linearity, precision and accuracy were validated within the typical therapeutic range of concentrations for each compound. The assay was linear over 0.125-25ng/mL for tacrolimus, 1-500ng/mL for prednisone/methylprednisone, 2-400ng/mL for Mtx, 2-1000ng/mL for prednisolone and from 7.5 to 1500ng/mL for CsA with the lowest limit of quantification (LLOQ) being 0.125, 1.00, 2.00, 2.00 and 7.5ng/mL, respectively. The calibration curve concentrations for MPA and MPAG ranged from 50 to 50,000ng/mL (LLOQ: 50ng/mL) and 10 to 10,000ng/mL (LLOQ: 10ng/mL) for AcMPAG. Mean recoveries in blood and plasma were 84%±5.7%. The method could measure individual drugs with high sensitivity, accuracy (bias≤14%), and reproducibility (CV≤12.8%). Its clinical application was validated by measuring levels of these drugs in samples obtained from hematopoietic stem cell transplant recipients treated with combined immunosuppressive drug therapy. Our results indicate that this approach is suitable for simultaneous determination of in vivo levels of immunosuppressive drugs commonly used in combined therapies.
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Affiliation(s)
- Isabelle Laverdière
- Pharmacogenomics Laboratory, Centre Hospitalier Universitaire de Québec (CHUQ) Research Center, Canada
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Vinay KB, Revanasiddappa HD, Raghu MS, Abdulrahman SAM, Rajendraprasad N. Spectrophotometric Determination of Mycophenolate Mofetil as Its Charge-Transfer Complexes with Two π-Acceptors. JOURNAL OF ANALYTICAL METHODS IN CHEMISTRY 2012; 2012:875942. [PMID: 22567572 PMCID: PMC3336187 DOI: 10.1155/2012/875942] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 11/10/2011] [Revised: 01/15/2012] [Accepted: 01/20/2012] [Indexed: 05/03/2023]
Abstract
Two simple, selective, and rapid spectrophotometric methods are described for the determination of mycophenolate mofetil (MPM) in pure form and in tablets. Both methods are based on charge-transfer complexation reaction of MPM with p-chloranilic acid (p-CA) or 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) in dioxane-acetonitrile medium resulting in coloured product measurable at 520 nm (p-CA) or 580 nm (DDQ). Beer's law is obeyed over the concentration ranges of 40-400 and 12-120 μg mL(-1) MPM for p-CA and DDQ, respectively, with correlation coefficients (r) of 0.9995 and 0.9947. The apparent molar absorptivity values are calculated to be 1.06 × 10(3) and 3.87 × 10(3) L mol(-1) cm(-1), respectively, and the corresponding Sandell's sensitivities are 0.4106 and 0.1119 μg cm(-1). The limits of detection (LOD) and quantification (LOQ) are also reported for both methods. The described methods were successfully applied to the determination of MPM in tablets. Statistical comparison of the results with those of the reference method showed excellent agreement. No interference was observed from the common excipients present in tablets. Both methods were validated statistically for accuracy and precision. The accuracy and reliability of the methods were further ascertained by recovery studies via standard addition procedure.
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Gao JW, Peng ZH, Li XY, Sun B, Guo YK, Liu GL. simultaneous determination of mycophenolic acid and its metabolites by HPLC and pharmacokinetic studies in rat plasma and bile. Arch Pharm Res 2011; 34:59-69. [DOI: 10.1007/s12272-011-0107-2] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/08/2010] [Revised: 07/02/2010] [Accepted: 07/05/2010] [Indexed: 11/29/2022]
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Dostalek M, Court MH, Hazarika S, Akhlaghi F. Diabetes mellitus reduces activity of human UDP-glucuronosyltransferase 2B7 in liver and kidney leading to decreased formation of mycophenolic acid acyl-glucuronide metabolite. Drug Metab Dispos 2011; 39:448-55. [PMID: 21123165 PMCID: PMC3061563 DOI: 10.1124/dmd.110.036608] [Citation(s) in RCA: 31] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/02/2010] [Accepted: 12/01/2010] [Indexed: 01/28/2023] Open
Abstract
Mycophenolic acid (MPA) is an immunosuppressive agent commonly used after organ transplantation. Altered concentrations of MPA metabolites have been reported in diabetic kidney transplant recipients, although the reason for this difference is unknown. We aimed to compare MPA biotransformation and UDP-glucuronosyltransferase (UGT) expression and activity between liver (n = 16) and kidney (n = 8) from diabetic and nondiabetic donors. Glucuronidation of MPA, as well as the expression and probe substrate activity of UGTs primarily responsible for MPA phenol glucuronide (MPAG) formation (UGT1A1 and UGT1A9), and MPA acyl glucuronide (AcMPAG) formation (UGT2B7), was characterized. We have found that both diabetic and nondiabetic human liver microsomes and kidney microsomes formed MPAG with similar efficiency; however, AcMPAG formation was significantly lower in diabetic samples. This finding is supported by markedly lower glucuronidation of the UGT2B7 probe zidovudine, UGT2B7 protein, and UGT2B7 mRNA in diabetic tissues. UGT genetic polymorphism did not explain this difference because UGT2B7*2 or *1c genotype were not associated with altered microsomal UGT2B7 protein levels or AcMPAG formation. Furthermore, mRNA expression and probe activities for UGT1A1 or UGT1A9, both forming MPAG but not AcMPAG, were comparable between diabetic and nondiabetic tissues, suggesting the effect may be specific to UGT2B7-mediated AcMPAG formation. These findings suggest that diabetes mellitus is associated with significantly reduced UGT2B7 mRNA expression, protein level, and enzymatic activity of human liver and kidney, explaining in part the relatively low circulating concentrations of AcMPAG in diabetic patients.
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Affiliation(s)
- Miroslav Dostalek
- Department of Biomedical and Pharmaceutical Sciences, College of Pharmacy, University of Rhode Island, Kingston, Rhode Island 02881, USA
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Establishment of high-performance liquid chromatography and enzyme multiplied immunoassay technology methods for determination of free mycophenolic acid and its application in Chinese liver transplant recipients. Ther Drug Monit 2011; 32:653-60. [PMID: 20814351 DOI: 10.1097/ftd.0b013e3181f01397] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022]
Abstract
The objective of this study is to investigate the correlation between methods of high-performance liquid chromatography (HPLC) and enzyme multiplied immunoassay technology (EMIT) for determination of total mycophenolic acid (tMPA) and free (fMPA) concentration and to study pharmacokinetics of fMPA in Chinese liver transplant recipients. An HPLC method with fluorometric detection and an EMIT assay were established to determine fMPA in plasma ultrafiltrates. Pharmacokinetic parameters of tMPA and fMPA in 51 patients were estimated. The calibration range of fMPA was 0.0025 to 1.0 μg/mL for the HPLC method and 0.0050 to 0.50 μg/mL for the EMIT method. Mean recovery of the two methods was 98.0% and 97.1%, respectively. The intraday and interday coefficient of variations were 0.93% to 3.1% and 1.6% to 2.9% for HPLC and 4.51% to 15.8% and 5.83% to 19.5% for EMIT, respectively. The relationship of the two methods was EMIT = 1.074 × HPLC + 0.582 (r2 = 0.918, n = 470, P < 0.05) for tMPA and EMIT = 1.068 × HPLC + 0.004 (r2 = 0.945, n = 297, P < 0.05) for fMPA. There was a positive mean bias of EMIT for tMPA (27.0%) and fMPA (23.3%). The AUC0-12 of tMPA and fMPA obtained by HPLC in 51 patients was 34.7 ± 11.1 and 0.72 ± 0.38 μg·h/mL, respectively. The free fraction of MPA was 1.60 ± 1.21% (Median:1.36%, interquartile: 0.72, 2.22), [corrected] which was significantly correlated with 7-O-glucuronide conjugate of MPA AUC0-12 (r2 = 0.705, P < 0.001), albumin (r2 = -0.529, P < 0.001), and the clearance of creatinine (r2 = -0.417, r2 = 0.005). Both HPLC and EMIT assay are suitable for the determination of fMPA. A considerable interindividual variability exists in pharmacokinetics of fMPA among Chinese liver transplant recipients. 7-O-Glucuronide conjugate of MPA and albumin concentrations are two factors correlated to fMPA variance.
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Guo D, Xu LY, Pang LF, Tan ZR, Han Y, Yang H, Zhou G, Chen Y, Ouyang DS, Zhou HH. UPLC Analysis of Mycophenolic Acid and Its Phenol and Acyl Glucuronide Metabolites in Human Plasma. Chromatographia 2010. [DOI: 10.1365/s10337-010-1715-6] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/05/2022]
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The level of ATP production in mitogen-stimulated CD4+ lymphocytes is independent of the time of ingestion of immunosuppressive agents. Ther Drug Monit 2010; 32:116-7. [PMID: 20104140 DOI: 10.1097/ftd.0b013e3181c324df] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022]
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Kuypers DR, Meur YL, Cantarovich M, Tredger MJ, Tett SE, Cattaneo D, Tönshoff B, Holt DW, Chapman J, Gelder TV. Consensus Report on Therapeutic Drug Monitoring of Mycophenolic Acid in Solid Organ Transplantation. Clin J Am Soc Nephrol 2010; 5:341-58. [DOI: 10.2215/cjn.07111009] [Citation(s) in RCA: 240] [Impact Index Per Article: 16.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/23/2022]
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Sam WJ, Akhlaghi F, Rosenbaum SE. Population pharmacokinetics of mycophenolic acid and its 2 glucuronidated metabolites in kidney transplant recipients. J Clin Pharmacol 2009; 49:185-95. [PMID: 19179297 DOI: 10.1177/0091270008329558] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/17/2022]
Abstract
The population pharmacokinetics of mycophenolic acid (MPA) and its phenolic (MPAG) and acyl (AcMPAG) glucuronide metabolites were studied in patients taking enteric-coated mycophenolate sodium. Plasma samples (n = 232), obtained from 18 renal transplant recipients, were analyzed for MPA, MPAG, and AcMPAG using a validated high-performance liquid chromatography/ultraviolet assay. Population pharmacokinetic analysis was performed using NONMEM. The pharmacokinetics of MPA were best described by a 2-compartment model, with MPAG and AcMPAG produced from the central compartment and with enterohepatic recirculation of MPA via these 2 metabolites. Population mean estimates for MPA were apparent clearance (CL/F) of 10.6 L/h (interindividual variability [IIV] = 21.4%) and apparent volume of distribution of the central compartment (V(1)/F) of 25.9 L (IIV = 87.8%). Mean elimination rate constants of MPAG and AcMPAG were 0.323 h(-1) (IIV = 29.1%) and 0.206 h(-1) (IIV = 48.8%), respectively. The mean fraction of MPA converted to MPAG and AcMPAG, normalized by their volumes of distribution (FM(AG) and FM(AC), respectively), was also estimated. The elimination rate constant for MPAG and FM(AC) was influenced by glomerular filtration rate in patients with renal impairment. The visual predictive check, based on 100 simulated data sets each for MPA, MPAG, and AcMPAG, found that the final pharmacokinetic model adequately predicts the observed concentrations of all 3 species.
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Affiliation(s)
- Wai-Johnn Sam
- Department of Biomedical and Pharmaceutical Sciences, University of Rhode Island, 41 Lower College Road, Kingston, RI 02881, USA
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A fast ultra-performance liquid chromatography method for simultaneous quantification of mycophenolic acid and its phenol- and acyl-glucuronides in human plasma. Ther Drug Monit 2009; 31:110-5. [PMID: 19057465 DOI: 10.1097/ftd.0b013e318191897d] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/02/2023]
Abstract
Several studies have demonstrated a close relationship between mycophenolic acid (MPA) exposure and the risk for graft rejection or side effects. Measurements of MPA and its metabolites plasma levels are therefore recommended. A new chromatographic method has been developed using ultra-performance liquid chromatography (UPLC) to improve both analytical throughput and sensitivity. MPA and its phenol-glucuronide and acyl-glucuronide were extracted from plasma using Isolute C2 solid phase extraction (SPE) cartridges (100 mg, 3 mL). UPLC separations were performed with a Waters BEH C18 column (50 x 2.1 mm, 1.7 microm) maintained at 65 degrees C on a Waters Acquity instrument equipped with a photodiode array detector. The total UPLC run time was 3.5 minutes. The method was linear in the range of 0.1-40 microg/mL for MPA and acyl-glucuronide, and 1-400 microg/mL for phenol-glucuronide. Relative standard error and mean relative prediction error were <15% for all tested quality controls (in-house and external proficiency panels). UPLC performances are characterized by a dramatic reduction in retention times together with an improvement of the sensitivity without affecting peak resolution. Further validations have been obtained by analyzing routine and clinical trial patients' samples. Significant improvement of the analytical throughput (reduction of run time from >10 to 3.5 minutes) was obtained using UPLC for MPA analyses. This retention time reduction was accompanied by an improvement of other analytical performances such as sensitivity.
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27
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Zeng L, Nath CE, Shaw PJ, Earl JW, McLachlan AJ. HPLC-UV assay for monitoring total and unbound mycophenolic acid concentrations in children. Biomed Chromatogr 2009; 23:92-100. [DOI: 10.1002/bmc.1088] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022]
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Mohamed MEF, Harvey SS, Frye RF. Determination of mycophenolic acid glucuronide in microsomal incubations using high performance liquid chromatography–tandem mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci 2008; 870:251-4. [DOI: 10.1016/j.jchromb.2008.06.020] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/29/2008] [Revised: 06/11/2008] [Accepted: 06/13/2008] [Indexed: 11/15/2022]
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Simultaneous determination of mycophenolic acid and its acyl and phenol glucuronide metabolites in human serum by capillary zone electrophoresis. J Pharm Biomed Anal 2008; 47:201-6. [DOI: 10.1016/j.jpba.2007.12.028] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/12/2007] [Revised: 12/05/2007] [Accepted: 12/13/2007] [Indexed: 11/21/2022]
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Effect of diabetes mellitus on mycophenolate sodium pharmacokinetics and inosine monophosphate dehydrogenase activity in stable kidney transplant recipients. Ther Drug Monit 2008; 29:735-42. [PMID: 18043470 DOI: 10.1097/ftd.0b013e31815d8ace] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/15/2023]
Abstract
Effect of diabetes mellitus on mycophenolic acid (MPA) pharmacokinetics and catalytic activity of inosine monophosphate dehydrogenase (IMPDH) was investigated in maintenance kidney transplant recipients. Demographically matched diabetic (n=9) and nondiabetic (n=9) patients were included in a 12-hour open-label, steady-state study after oral administration of enteric-coated mycophenolate sodium. Concentrations of total MPA and free MPA, MPA-glucuronide, and acyl-MPA-glucuronide were measured and oral acetaminophen absorption was used as a marker for gastric-emptying rate. Median (range) of MPA area under the curve(0-12) was 36.7 (range, 16.4-116.4) mg*h/L in diabetic and 48.2 (range, 34.9-80.1) mg*h/L in nondiabetic patients (P=0.49). All other primary pharmacokinetic parameters, including time to maximum concentration, for total or unbound MPA as well as MPA metabolites were comparable. In contrast, IMPDH activity was 17.5+/-2.8 versus 46.6+/-2.5 nmol XMP/h/microg protein in diabetics and nondiabetics, respectively (P<0.0001) and was significantly lower in the diabetics irrespective of concomitant therapy with cyclosporine or tacrolimus. This study demonstrated that diabetes does not alter MPA pharmacokinetics when administered as enteric-coated mycophenolate sodium; however, IMPDH activity appeared to be significantly lower in patients with diabetes independent of the unbound or total concentrations of MPA. Further investigations are warranted to investigate the regulation of IMPDH enzyme in patients with diabetes.
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Mino Y, Naito T, Matsushita T, Kagawa Y, Kawakami J. Simultaneous determination of mycophenolic acid and its glucuronides in human plasma using isocratic ion pair high-performance liquid chromatography. J Pharm Biomed Anal 2008; 46:603-8. [DOI: 10.1016/j.jpba.2007.11.018] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/19/2007] [Revised: 11/14/2007] [Accepted: 11/16/2007] [Indexed: 10/22/2022]
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Elbarbry FA, Shoker AS. Liquid chromatographic determination of mycophenolic acid and its metabolites in human kidney transplant plasma: pharmacokinetic application. J Chromatogr B Analyt Technol Biomed Life Sci 2007; 859:276-81. [PMID: 17964230 DOI: 10.1016/j.jchromb.2007.09.036] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/19/2007] [Revised: 09/01/2007] [Accepted: 09/29/2007] [Indexed: 12/26/2022]
Abstract
BACKGROUND AND OBJECTIVE Difference in the hydrophilic properties of mycophenolic acid metabolites makes it technically difficult to simultaneously determine their plasma levels in one analytical run. Therapeutic drug monitoring (TDM) for MPA ensures adequate MPA exposure levels to both prevent rejection and avoid related toxicity. One measure limitation for TDM for MPA is the availability of simple, rapid and reproducible method for determination of MPA derivatives. METHOD Herein we report a single method to measure MPA and its metabolites using a gradient elution system in less than 10 min. We further tested applicability of our method in both stable and unstable renal transplant recipients with a wide range of levels. RESULTS Intra- and inter-day imprecision were less than 8% and 10%, respectively. Accuracy of the estimated concentrations ranges from 90% to 108%. CONCLUSION Collectively these data show that the new method is reasonably accurate and precise for the simultaneous determination of MPA and its metabolites in human plasma.
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Affiliation(s)
- Fawzy A Elbarbry
- Department of Medicine, Royal University Hospital, University of Saskatchewan, 103 Hospital Drive, Saskatoon S7N 0W8, Canada
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Chen B, Zhang W, Yu Z, Cai W. Determination of Mycophenolic Acid (MPA) and Its Acyl and Phenol Glucuronide Metabolits Simultaneously in Human Plasma by a Simplified HPLC Method. ANAL LETT 2007. [DOI: 10.1080/00032710701583466] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/02/2023]
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Benoit-Biancamano MO, Caron P, Lévesque E, Delage R, Couture F, Guillemette C. Sensitive high-performance liquid chromatography–tandem mass spectrometry method for quantitative analysis of mycophenolic acid and its glucuronide metabolites in human plasma and urine. J Chromatogr B Analyt Technol Biomed Life Sci 2007; 858:159-67. [PMID: 17827076 DOI: 10.1016/j.jchromb.2007.08.023] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/23/2007] [Revised: 08/14/2007] [Accepted: 08/16/2007] [Indexed: 10/22/2022]
Abstract
A method to determine total and free mycophenolic acid (MPA) and its metabolites, the phenolic (MPAG) and acyl (AcMPAG) glucuronides, using HPLC and mass spectrometry was developed. Mean recoveries in plasma and urine samples were >85%, and the lower limits of quantification for MPA, MPAG and AcMPAG were 0.05, 0.05 and 0.01 mg/L, respectively. For plasma, the assay was linear over 0.05-50 mg/L for MPA and MPAG, and from 0.01 to 10mg/L for AcMPAG. A validation study demonstrated good inter- and intra-day precision (CV<or=11%) and accuracy (bias<or=16%) and satisfactory specificity and stability. Pharmacokinetic parameters were assessed in plasma and urine from healthy volunteers after an oral dose of mycophenolate mofetil.
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Affiliation(s)
- Marie-Odile Benoit-Biancamano
- Canada Research Chair in Pharmacogenomics, Laboratory of Pharmacogenomics, Oncology and Molecular Endocrinology Research Center, CHUL Research Center and Faculty of Pharmacy, Laval University, G1V 4G2 Québec, Canada
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Elbarbry FA, Shoker AS. Therapeutic drug measurement of mycophenolic acid derivatives in transplant patients. Clin Biochem 2007; 40:752-64. [PMID: 17482154 DOI: 10.1016/j.clinbiochem.2007.03.006] [Citation(s) in RCA: 29] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/20/2006] [Revised: 03/03/2007] [Accepted: 03/07/2007] [Indexed: 02/03/2023]
Abstract
INTRODUCTION Mycophenolic acid, the active metabolite of the prodrug mycophenolate mofetil, is widely used as an immunosuppressive agent in transplant patients for the prophylaxis of acute rejection. Recent prospective trials suggested the need for therapeutic drug monitoring, which raises the necessity to acquire accurate methods to measure MPA and its metabolites. OBJECTIVE Present an overview of the reasons to monitor MPA and its metabolites as well as a review of the currently available methods for their determination. METHODS Articles published from January 1992 to December 2006 were reviewed. RESULTS Most of the cited references use either chromatographic or immunoassay techniques. Basic information about biological samples used for the analysis, sample preparation, stationary phase, mobile phase, detection mode and validation data are discussed. Current information suggests the feasibility to set up method(s) to monitor MPA and its metabolites in most centers.
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Affiliation(s)
- Fawzy A Elbarbry
- Department of Medicine, Royal University Hospital, University of Saskatchewan, Saskatoon, SK, Canada
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Saint-Marcoux F, Sauvage FL, Marquet P. Current role of LC-MS in therapeutic drug monitoring. Anal Bioanal Chem 2007; 388:1327-49. [PMID: 17520242 DOI: 10.1007/s00216-007-1320-1] [Citation(s) in RCA: 83] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/12/2007] [Revised: 03/22/2007] [Accepted: 04/24/2007] [Indexed: 10/23/2022]
Abstract
The role of liquid chromatography coupled with mass spectrometry (LC-MS) techniques in routine therapeutic drug monitoring activity is becoming increasingly important. This paper reviews LC-MS methods published in the last few years for certain classes of drugs subject to therapeutic drug monitoring: immunosuppressants, antifungal drugs, antiretroviral drugs, antidepressants and antipsychotics. For each class of compounds, we focussed on the most interesting methods and evaluated the current role of LC-MS in therapeutic drug monitoring.
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Affiliation(s)
- Franck Saint-Marcoux
- Department of Pharmacology-Toxicology, Limoges University Hospital, Unité INSERM U850, 87042 Limoges cedex, France
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Patel CG, Harmon M, Gohh RY, Akhlaghi F. Concentrations of Mycophenolic Acid and Glucuronide Metabolites Under Concomitant Therapy With Cyclosporine or Tacrolimus. Ther Drug Monit 2007; 29:87-95. [PMID: 17304155 DOI: 10.1097/ftd.0b013e3180318c35] [Citation(s) in RCA: 17] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/07/2023]
Abstract
Mycophenolate mofetil [MMF, the prodrug of mycophenolic acid (MPA)] is usually administered at double doses with cyclosporine than with tacrolimus because it is believed that MPA exposure is lower during cyclosporine therapy. This study aimed to compare 12 hour, steady-state concentration-time profiles of MPA and its phenol- and acyl-glucuronide metabolites (MPAG and AcMPAG, respectively) in stable kidney transplant recipients maintained either on cyclosporine (n = 12) or tacrolimus (n = 12). During the absorption phase in the cyclosporine group, dose-normalized concentrations of total and free MPA were significantly higher but the overall area under the concentration-time curve (AUC0-12) was not significantly different. Additionally, exposure to AcMPAG was higher in the cyclosporine group (P < 0.05). Ten of 12 patients in the cyclosporine group were on ketoconazole therapy; however, the exposure to MPA or MPAG was not different when MMF was given orally to Sprague-Dawley rats with or without ketoconazole. In conclusion, cyclosporine modulates the disposition of MPA and metabolites differently from tacrolimus; however, patients on cyclosporine may not require double doses of MMF to achieve the same exposure.
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Affiliation(s)
- Chirag G Patel
- Department of Biomedical and Pharmaceutical Sciences, College of Pharmacy, University of Rhode Island, Kingston, RI 02881, USA
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Henry SD, Metselaar HJ, Lonsdale RCB, Kok A, Haagmans BL, Tilanus HW, van der Laan LJW. Mycophenolic acid inhibits hepatitis C virus replication and acts in synergy with cyclosporin A and interferon-alpha. Gastroenterology 2006; 131:1452-62. [PMID: 17101321 DOI: 10.1053/j.gastro.2006.08.027] [Citation(s) in RCA: 92] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/02/2005] [Accepted: 07/28/2006] [Indexed: 12/15/2022]
Abstract
BACKGROUND & AIMS Chronic hepatitis C virus (HCV) infection is the leading indication for liver transplantation. Clinical evidence suggests that particular immunosuppressive agents can have an influence on HCV recurrence. Cyclosporine A (CsA) specifically inhibits HCV replication through blocking the viral RNA polymerase enzyme NS5B. In this study, we investigated the effect of mycophenolic acid (MPA) and other immunosuppressants on HCV replication. METHODS MPA and other compounds were tested in vitro using an HCV-replication model containing a luciferase reporter gene. RESULTS At clinically relevant concentrations (1.0-6.0 microg/mL), MPA inhibited HCV replication to approximately 75%. CsA and interferon (IFN)-alpha also showed inhibition in a dose-dependent manner. In these short-term (18 hours) experiments, MPA did not inhibit cell proliferation or induce cell death, which could have accounted for the antiviral effect. In contrast to the antiviral activity of MPA against West Nile virus, the effect of MPA on HCV replication was guanosine independent. When combined, MPA and CsA showed significant synergistic inhibition of replication, reaching maximum inhibition of approximately 90% at the highest doses. Synergistic effects were observed with suboptimal concentrations of IFN-alpha with MPA or CsA. The kinetics of HCV inhibition by MPA, CsA, and IFN-alpha were clearly distinct, with earliest effects seen with IFN-alpha. No specific inhibitory effects were observed with tacrolimus or rapamycin. CONCLUSIONS The immunosuppressive drug MPA is as potent as CsA as an inhibitor of HCV replication. MPA was shown to have a distinct anti-HCV mechanism of action, independent of cell proliferation and guanosine depletion.
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Affiliation(s)
- Scot D Henry
- Department of Surgery, Erasmus MC-University Medical Center, Rotterdam, The Netherlands
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Ünsalan S, Hempel G, Fobker M, Würthwein G, Boos J. Monitoring of Mycophenolic Acid in the Plasma of Transplant Patients by Capillary Electrophoresis. Chromatographia 2006. [DOI: 10.1365/s10337-006-0046-0] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/05/2022]
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Brandhorst G, Streit F, Goetze S, Oellerich M, Armstrong VW. Quantification by liquid chromatography tandem mass spectrometry of mycophenolic acid and its phenol and acyl glucuronide metabolites. Clin Chem 2006; 52:1962-4. [PMID: 16931568 DOI: 10.1373/clinchem.2006.074336] [Citation(s) in RCA: 58] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/01/2023]
Abstract
BACKGROUND We developed and validated a rapid and reliable liquid chromatography-tandem mass spectrometry (LC-MS/MS) procedure for the quantification of mycophenolic acid (MPA) and its phenol glucuronide (MPAG) and acyl glucuronide (AcMPAG) metabolites. METHODS We performed protein precipitation on all samples (calibrators, quality controls, and patient samples) and then subjected them to online solid-phase extraction followed by reversed-phase liquid chromatography for 4.0 min. The carboxybutoxy ether of MPA (MPAC) was used as the internal calibrator. The separated compounds (MPA, MPAG, AcMPAG, and MPAC) were detected by electrospray ionization-coupled MS/MS. We compared LC-MS/MS results with results for the same samples obtained with a validated HPLC procedure with an ultraviolet detector. RESULTS Comparison with the validated HPLC-ultraviolet procedure demonstrated good agreement. The Passing-Bablok regression was y = 0.968x - 0.058 for MPA, y = 1.08x - 1.697 for MPAG, and y = 0.952x + 0.076 for AcMPAG. Assay imprecision showed a CV <10% at 3 concentrations for each compound. The lower limit of quantification was 0.1 mg/L for MPA, 1.0 mg/L for MPAG, and 0.05 mg/L for AcMPAG. The mean analytical recovery was 90%-110%. The assay was linear from 0.1 to 50 mg/L for MPA (r = 0.9987), from 1 to 500 mg/L for MPAG (r = 0.9999), and from 0.05 to 10 mg/L for AcMPAG (r = 0.9988). Quantification of the compounds was not affected by in-source fragmentation or ion suppression. CONCLUSION The LC-MS/MS assay described here is valid and reliable for the quantification of total MPA, MPAG, and AcMPAG in serum.
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Affiliation(s)
- Gunnar Brandhorst
- Universitätsklinikum Göttingen, Abteilung Klinische Chemie/Zentrallabor, 37075 Göttingen, Germany.
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Mendonza AE, Gohh RY, Akhlaghi F. Analysis of Mycophenolic Acid in Saliva Using Liquid Chromatography Tandem Mass Spectrometry. Ther Drug Monit 2006; 28:402-6. [PMID: 16778726 DOI: 10.1097/01.ftd.0000211826.65607.05] [Citation(s) in RCA: 35] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/25/2022]
Abstract
Salivary levels of the immunosuppressive agent, mycophenolic acid (MPA), may provide a convenient and noninvasive method for drug monitoring. An analytical method was developed and validated for quantification of salivary MPA using liquid chromatography tandem mass spectrometry. Sample preparation included addition of 50 microL internal standard solution [500 microg/L indomethacin in methanol] to 100 microL saliva sample, followed by protein precipitation with 200 microL acetonitrile. Supernatants were dried and reconstituted in 100 microL of 85:15% (vol/vol) mixture of methanol and water containing 0.05% formic acid and 20 microL was injected onto the analytical column. The mobile phase comprised a gradient mixture of methanol and 0.05% formic acid, giving a total run time of 7.5 minutes. Chromatograms were obtained using mass transitions of m/z 319.0-->190.8 for MPA and m/z 355.9-->312.2 for indomethacin. The calibration curve was linear over a concentration range of 2.5 to 800 microg/L (r=0.9999) and the recovery of MPA from saliva was >90%. The inaccuracy was <10% and intra- and interday coefficient of variation ranged from 2.8% to 5.2%. Mean+/-SD of MPA concentrations in saliva (n=100) obtained from 11 kidney transplant recipients was 31.4+/-32.3 microg/L (range: 2.6 to 220.4 microg/L) and correlated well with total (r=0.909) and unbound (r=0.910) MPA concentrations in plasma. In conclusion, a simple, sensitive, and specific method was developed and validated for quantification of MPA in saliva. Additional clinical studies are required to establish the usefulness of this specimen in the clinical management of organ transplant recipients.
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Affiliation(s)
- Anisha E Mendonza
- College of Pharmacy, University of Rhode Island, 41 Lower College Road, Kingston, RI, USA
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