quantification-immunosuppressant-tacrolimus-on-dried-blood-spots

Quantification of the Immunosuppressant Tacrolimus on Dried Blood Spots Using LC-MS/MS

The calcineurin inhibitor tacrolimus is the cornerstone of most immunosuppressive treatment protocols after solid organ transplantation in the United ...

University of Colorado, Anschutz Medical Campus

Multinuclear NMR spectroscopy is used to investigate the effect of glutamine on neuronal glucose metabolism. Primary neurons were incubated with [1-(13C)]glucose in the absence or presence of glutamine (2 mM) and/or NH4Cl (5 mM). After ammonia-treatment, the concentrations of high-energy phosphates decreased up to 84% of control, which was aggravated in glutamine-containing medium (up to 42% of control). These effects could not be attributed to changes in mitochondrial glucose oxidation. Withdrawal of glutamine decreased amino acid concentrations, e.g. of glutamate to 53%, but also considerably lessened the 13C enrichment in [4-(13C)]glutamate to 8.3% of control, and decreased the 13C-enrichment in acetyl-CoA entering the Krebs cycle (P < 0.001). Thus, although glutamine is potent in replenishing neuronal glutamate stores, glutamate formation is mainly attributed to its de novo synthesis from glucose. Furthermore, mitochondrial glucose metabolism strongly depends on the supply of carbons from glutamine, indicating that exogenous glutamine is a well-suited substrate to replenish neuronal Krebs cycle intermediates.

Contribution of extracellular glutamine as an anaplerotic substrate to neuronal metabolism: a re-evaluation by multinuclear NMR spectroscopy in primary cultured neurons.

The bacterium Actinoplanes sp. ATCC 53771 is known to perform drug metabolism of several xenobiotics similarly to humans. We identified a cytochrome P450 enzyme from this strain, CYP107E4, and expressed it in Escherichia coli using the pET101 vector. The purified enzyme showed the characteristic reduced-CO difference spectra with a peak at 450 nm, indicating the protein is produced in the active form with proper heme incorporation. The CYP107E4 enzyme was found to bind the drug diclofenac. Using redox enzymes from spinach, the reconstituted system is able to produce hydroxylated metabolites of diclofenac. Production of the human 4'-hydroxydiclofenac metabolite by CYP107E4 was confirmed, and a second hydroxylated metabolite was also produced.

Identification and characterization of a bacterial cytochrome P450 for the metabolism of diclofenac.

Statins are cholesterol-lowering drugs with pleiotropic activities including inhibition of isoprenylation and reduction of signals driving cell proliferation and survival responses.

Effects of lovastatin on breast cancer cells: a proteo-metabonomic study.

Quantification of F(2)-isoprostanes is considered a reliable index of the oxidative stress status in vivo. Several immunoassays and chromatography/mass spectrometry-based assays are available for 15-F(2t)-isoprostane quantification. However, it remains unclear if results of immunoassays using different assays can be compared with those of liquid chromatography/mass spectrometry (LC/MS) assays. Previous studies comparing enzyme-linked immunosorbent assay (ELISA) and more specific gas chromatography/mass spectrometry assays have already indicated that ELISAs may overestimate 15-F(2t)-isoprostane concentrations in human plasma. Concentrations of 15-F(2t)-isoprostane in 25 human plasma and urine samples were measured by three commercially available ELISA assays (Assay Designs, Cayman Chemical and Oxford Biomedical Research) and compared with the concentrations measured with a validated, semi-automated high-throughput HPLC tandem mass spectrometry assay (LC/LC-MS/MS). All three ELISAs measured substantially higher 15-F(2t)-isoprostane concentrations (2.1-182.2-fold higher in plasma; 0.4-61.9-fold higher in urine) than LC/LC-MS/MS. Utilization of solid-phase extraction (SPE) columns, especially isoprostane affinity purification columns, brought ELISA isoprostane urine concentrations closer to the LC/LC-MS/MS results. However, SPE did not have much of an effect on ELISA plasma concentrations which remained significantly higher than corresponding LC/LC-MS/MS results. A poor correlation not only between LC/LC-MS/MS and immunoassay results, but also among the immunoassays was found. Especially in plasma, ELISAs grossly overestimate 15-F(2t)-isoprostane concentrations and are not comparable with each other or with LC/LC-MS/MS. It is most disturbing that a sample with relatively high concentrations measured with one ELISA may show low concentrations with another ELISA, and vice versa, potentially affecting the conclusions drawn from such data. The use of specific mass spectrometry-based assays seems advisable.

Quantification of 15-F2t-isoprostane in human plasma and urine: results from enzyme-linked immunoassay and liquid chromatography/tandem mass spectrometry cannot be compared.

SAR-943 (32-deoxo rapamycin) is a proliferation signal inhibitor via interaction with the mammalian target of rapamycin (mTOR). Most importantly, SAR-943 has improved chemical stability compared to rapamycin (sirolimus) and is currently under investigation as a drug coated on coronary stents. It was the goal of this study to identify the SAR-943 metabolites generated after incubation with human liver microsomes using high-resolution mass spectrometry (MS) and MS/iontrap (MS(n)) and comparison of fragmentation patterns of the metabolites with those of SAR-943 and other known rapamycin derivatives. Our study showed that SAR-943 is mainly hydroxylated and/or demethylated by human liver microsomes. The structures of the following metabolites were identified: O-demethylated metabolites: 39-O-desmethyl, 16-O-desmethyl and 27-O-desmethyl SAR-943; hydroxylated metabolites: hydroxy piperidine SAR-943, 11-hydroxy, 12-hydroxy, 14-hydroxy, 23-hydroxy, 24-hydroxy, 25-hydroxy, 46-hydroxy and 49-hydroxy SAR-943; didemethylated metabolites: 16,39-O-didesmethyl and 27,39-O-didesmethyl SAR-943; demethylated-hydroxylated metabolites: 39-O-desmethyl, 23- or 24-hydroxy and 39-O-desmethyl, hydroxy piperidine SAR-943 and dihydroxylated metabolites: 12-,23- or 24-dihydroxy SAR-943. In addition, several other demethylated-hydroxylated and dihydroxylated metabolites were detected. However, their exact structures could not be identified.

Structural identification of SAR-943 metabolites generated by human liver microsomes in vitro using mass spectrometry in combination with analysis of fragmentation patterns.

Mycophenolic acid (MPA) is used as an immunosuppressant after organ transplantation and for the treatment of immune diseases. There is increasing evidence that therapeutic drug monitoring and plasma concentration-guided dose adjustments are beneficial for patients to maintain immunosuppressive efficacy and to avoid toxicity. The major MPA metabolite that can be found in high concentrations in plasma is MPA glucuronide (MPAG). A metabolite usually present at lower concentrations, MPA acyl-glucuronide (AcMPAG), has been implicated in some of the adverse effects of MPA. We developed and validated an automated high-throughput ultra-high performance chromatography-tandem mass spectrometry (U-HPLC-MS/MS) assay using liquid-handling robotic extraction for the quantification of MPA, MPAG, and AcMPAG in human EDTA plasma and urine. The ranges of reliable response were 0.097 (lower limit of quantitation) to 200 μg/mL for MPA and MPAG and 0.156-10 μg/mL for AcMPAG in human urine and plasma. The inter-day accuracies were 94.3-104.4%, 93.8-105.0% and 94.4-104.7% for MPA, MPAG and AcMPAG, respectively. Inter-day precisions were 0.7-7.8%, 0.9-6.9% and 1.6-8.6% for MPA, MPAG and AcMPAG. No matrix interferences, ion suppression/enhancement and carry-over were detected. The total assay run time was 2.3 min. The assay met all predefined acceptance criteria and the quantification of MPA was successfully cross-validated with an LC-MS/MS assay routinely used for clinical therapeutic drug monitoring. The assay has proven to be robust and reliable during the measurement of samples from several pharmacokinetics trials.

A high-throughput U-HPLC-MS/MS assay for the quantification of mycophenolic acid and its major metabolites mycophenolic acid glucuronide and mycophenolic acid acyl-glucuronide in human plasma and urine.

Enhancement of calcineurin inhibitor nephrotoxicity by sirolimus (SRL) is limiting the clinical use of this drug combination. We compared the dose-dependent effects of the structurally related everolimus (EVL) and sirolimus (SRL) alone, and in combination with cyclosporine (CsA), on the rat kidney. Lewis rats were treated by oral gavage for 28 days using a checkerboard dosing format (0, 3.0, 6.0 and 10.0 CsA and 0, 0.5, 1.5 and 3.0 mg/kg/day SRL or EVL, n = 4/dose combination). After 28 days, oxidative stress, energy charge, kidney histologies, glomerular filtration rates, and concentrations of the immunosuppressants were measured along with (1)H-magnetic resonance spectroscopy (MRS) and gas chromatography- mass spectrometry profiles of cellular metabolites in urine. The combination of CsA with SRL led to higher urinary glucose concentrations and decreased levels of urinary Krebs cycle metabolites when compared to controls, suggesting that CsA+SRL negatively impacted proximal tubule metabolism. Unsupervised principal component analysis of MRS spectra distinguished unique urine metabolite patterns of rats treated with CsA+SRL from those treated with CsA+EVL and the controls. SRL, but not EVL blood concentrations were inversely correlated with urine Krebs cycle metabolite concentrations. Interestingly, the higher the EVL concentration, the closer urine metabolite patterns resembled those of controls, while in contrast, the combination of the highest doses of CsA+SRL showed the most significant differences in metabolite patterns. Surprisingly in this rat model, EVL and SRL in combination with CsA had different effects on kidney biochemistry, suggesting that further exploration of EVL in combination with low dose calcineurin inhibitors may be of potential benefit.

Everolimus and sirolimus in combination with cyclosporine have different effects on renal metabolism in the rat.

Endothelial lipase (EL) plays a pivotal role in HDL metabolism. We sought to characterize EL and its interaction with HDL as well as its natural variants genetically, functionally and structurally. We screened our biethnic population sample (n = 802) for selected missense mutations (n = 5) and identified T111I as the only common variant. Multiple linear regression analyses in Hispanic subjects revealed an unexpected association between T111I and elevated LDL-C (p-value = 0.012) and total cholesterol (p-value = 0.004). We examined lipase activity of selected missense mutants (n = 10) and found different impacts on EL function, ranging from normal to complete loss of activity. EL-HDL lipidomic analyses indicated that EL has a defined remodeling of HDL without exhaustion of the substrate and a distinct and preference for several fatty acids that are lipid mediators and known for their potent pro- and anti-inflammatory properties. Structural studies using homology modeling revealed a novel α/β motif in the C-domain, unique to EL. The EL dimer was found to have the flexibility to expand and to bind various sizes of HDL particles. The likely impact of the all known missense mutations (n = 18) on the structure of EL was examined using molecular modeling and the impact they may have on EL lipase activity using a novel structure-function slope based on their structural free energy differences. The results of this multidisciplinary approach delineated the impact of EL and its variants on HDL. Moreover, the results suggested EL to have the capacity to modulate vascular health through its role in fatty acid-based signaling pathways.

Genetic and structure-function studies of missense mutations in human endothelial lipase.

Biomarkers, or more specifically molecular markers, can detect biochemical changes associated with disease processes and drug effects before histopathological and pathophysiological changes occur. Multiplexing technologies such as high-performance liquid chromatography/mass spectrometry (LC-MS) allow for the measurement of molecular marker patterns that confer significantly more information than the measurement of a single parameter alone. The use of multiplexing assays for drug development, and as diagnostic tools, is attractive but will require regulatory review and approval and thus requires validation following regulatory guidances. Multiplexing assays always constitute a compromise. The number of analytes that can reasonably be included in a mass spectrometry-based multiplexing assay depend on the physico-chemical properties of the analytes and their integration into a single assay in terms of extraction, HPLC separation, ionization conditions and mass spectrometry detection. Another aspect includes biomedical considerations such as the differences in physiological concentrations of analytes, the required concentration range, and how much variability is acceptable before the clinical utility of a marker is negatively affected. Regulatory considerations include validation and quality control during sample analysis. Current bioanalytical regulatory guidelines have mostly been developed for single drug compounds and are not always adequate for multiplexing molecular marker assays that often quantify endogenous compounds. Specific guidances for multiplexing assays should be developed. Even if it is possible to integrate a wide variety and large number of analytes into a multiplexing assay, it should always be taken into consideration that a set of shorter, more specialized assays, may offer a more manageable and efficient alternative.

Mass Spectrometry-Based Multiplexing for the Analysis of Biomarkers in Drug Development and Clinical Diagnostics- How Much is too Much?

Mycophenolate mofetil (MMF) per se is not known to have negative effects on the kidney. MMF alone or in combination with sirolimus, can be the basis of calcineurin inhibitor (CNI)-free, kidney sparing drug protocols. However, long-term outcomes in patients on MMF/SRL seem to be inferior to those treated with regimens that include the CNI tacrolimus (TAC) due to an increased risk of allo-immune reactions. Interestingly, potential enhancement of the negative effects of SRL and TAC on the kidney by MMF has never been considered.

Mycophenolate mofetil enhances the negative effects of sirolimus and tacrolimus on rat kidney cell metabolism.

Stearoyl-CoA desaturase 1 (SCD1) plays a role in the development of obesity and related conditions, such as insulin resistance, and potentially also in neurological and heart diseases. The activity of SCD1 can be monitored using the desaturation index (DI), the ratio of product (16:1n-7 and 18:1n-9) to precursor (16:0 and 18:0) fatty acids. Here, different analytical strategies were applied to identify the method which best supports SCD1 biology. A novel effective approach was the use of the SCD1-independent fatty acid (16:1n-10) as a negative control. The first approach was based on a simple extraction followed by neutral loss triglyceride fatty acid analysis. The second approach was based on the saponification of triglycerides followed by fatty acid analysis (specific for the position of the double bond within monounsaturated fatty acids (MUFAs)). In addition to the analytical LC-MS assays, different matrices (plasma total triglyceride fraction and the very low-density lipoprotein (VLDL) fraction) were investigated to identify the best for studying changes in SCD1 activity. Samples from volunteers on a high-carbohydrate diet were analyzed. Both ultra HPLC (UHPLC)-MS-based assays showed acceptable accuracies (75-125% of nominal) and precisions (<20%) for the analysis of DI-specific fatty acids in VLDL and plasma. The most specific assay for the analysis of the liver SCD activity was then validated for specificity and selectivity, intra- and interday accuracy and precision, matrix effects, dilution effects, and analyte stability. After 3 days of high-carbohydrate diet, only the specific fatty acids in human plasma VLDL showed a significant increase in DI and associated SCD1 activity.

Article	Year
Contribution of extracellular glutamine as an anaplerotic substrate to neuronal metabolism: a re-evaluation by multinuclear NMR spectroscopy in primary cultured neurons. Neurochemical research\| PubMed ID: 16341588	2005
Identification and characterization of a bacterial cytochrome P450 for the metabolism of diclofenac. Applied microbiology and biotechnology\| PubMed ID: 19636551	2010
Effects of lovastatin on breast cancer cells: a proteo-metabonomic study. Breast cancer research : BCR\| PubMed ID: 20205716	2010
Quantification of 15-F2t-isoprostane in human plasma and urine: results from enzyme-linked immunoassay and liquid chromatography/tandem mass spectrometry cannot be compared. Rapid communications in mass spectrometry : RCM\| PubMed ID: 21259353	2011
Structural identification of SAR-943 metabolites generated by human liver microsomes in vitro using mass spectrometry in combination with analysis of fragmentation patterns. Journal of mass spectrometry : JMS\| PubMed ID: 21671437	2011
A high-throughput U-HPLC-MS/MS assay for the quantification of mycophenolic acid and its major metabolites mycophenolic acid glucuronide and mycophenolic acid acyl-glucuronide in human plasm... Journal of chromatography. B, Analytical technologies in the biomedical and life sciences\| PubMed ID: 21839692	2012
Everolimus and sirolimus in combination with cyclosporine have different effects on renal metabolism in the rat. PloS one\| PubMed ID: 23118926	2012
Genetic and structure-function studies of missense mutations in human endothelial lipase. PloS one\| PubMed ID: 23536757	2013
Mass Spectrometry-Based Multiplexing for the Analysis of Biomarkers in Drug Development and Clinical Diagnostics- How Much is too Much? Microchemical journal, devoted to the application of microtechniques in all branches of science\| PubMed ID: 23645936	2012
Mycophenolate mofetil enhances the negative effects of sirolimus and tacrolimus on rat kidney cell metabolism. PloS one\| PubMed ID: 24497939	2014
Fatty acid desaturation index in human plasma: comparison of different analytical methodologies for the evaluation of diet effects. Analytical and bioanalytical chemistry\| PubMed ID: 25116600	2014

Touraj Shokati

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