<i>In vitro</i> immunosuppressive activity of tacrolimus dihydrodiol precursors obtained by chemical oxidation and identification of a new metabolite of SDZ‐RAD by electrospray and electrospray‐linked scan mass spectrometry

Lhoëst, Georges; Hertsens, R.; Verbeeck, Roger K.; Maton, Nicole; Wallemacq, Pierre; Dehoux, Jean‐Paul; Latinne, Dominique

doi:10.1002/jms.190

Cited by 10 publications

(6 citation statements)

References 22 publications

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“…479 [(M − 210 − 111)Na] + , based on the analysis of fragment characteristics of FK506 and structurally similar compounds as reported in Lhoest, G's work [31], we have confirmed that the impurity produced in large quantities upon adding n-propanol to S. hygroscopicus ATCC 14891 is a structural analogue of FK520, namely FK523. Therefore, while the addition of n-propanol increases the yield of FK520, the large amounts of close structural resemblance of FK523 (the peak area of FK523 reached 40% of FK520) undoubtedly complicates the later stages of FK520 isolation and extraction.…”

Section: Short-chain Alcohols Facilitate Fk520 Accumulationsupporting

confidence: 81%

Boosting ascomycin production through n-butanol addition in the fermentation process ofStreptomyces hygroscopicus var. ascomyceticusATCC 14891

Meng,

Chen,

et al. 2023

Preprint

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Ascomycin (FK520) is a macrolide antibiotic known for its immunosuppressive activities. In this study, we screened several short-chain alcohols to enhance FK520 production inStreptomycin hygroscopicus var. ascomyceticusATCC 14891, with a particular focus on n-butanol addition. After optimizing the n-butanol addition process, we achieved a FK520 yield of 569.37 mg/L with 0.8% n-butanol addition at 27 h, representing a 1.72-fold increase compared with the control group. We subsequently found that ROS levels of n-butanol addition group reached 1.49×105RFU/g biomass at 29 h, which was 3.02 times higher than the control group. The 0.8% n-butanol addition also promote the accumulation of biosynthesis precursors for FK520 production. The highest ethylmalonyl-CoA content surged to 93.1 mol/g DCW at 48 h, marking a 5.3-fold increase. Likewise, the highest methylmalonyl-CoA and malonyl-CoA levels increased remarkable 4.33-fold and 3.33-fold compared with the control group at 72 h and at 120 h, respectively. Then, we explored the effects of oxygen supply on FK520 production with n-butanol addition, and improving oxygen supply caused a significant increase of FK520. Our research has revealed that addition of short-chain alcohols can regulate carbon flux toward FK520 biosynthesis by supplementing various CoA-esters, including ethylmalonyl-COA,methylmalonyl-CoA, and malonyl-CoA.

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Section: Short-chain Alcohols Facilitate Fk520 Accumulationsupporting

confidence: 81%

Boosting ascomycin production through n-butanol addition in the fermentation process ofStreptomyces hygroscopicus var. ascomyceticusATCC 14891

Meng,

Chen,

et al. 2023

Preprint

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“…Because of the lack of detailed structural information of everolimus fragments, previous attempts to identify everolimus structures using LC/MS were not only of limited success,22 but also led to incorrect conclusions 15, 16. We would like to emphasize again that the H/D experiments are critical to prove the number of hydroxylations, and clearly excluded epoxy metabolites of sirolimus and everolimus,15, 16 which have been proposed based on nonvalidated hypothetical fragmentation patterns.…”

Section: Discussionmentioning

confidence: 90%

“…This is the only experiment that distinguished between an epoxide and a hydroxyl group as a result of two possible biotransformation reactions. Because epoxy metabolites of sirolimus and everolimus have been proposed,15, 16 this experiment was important for determining the correct structures of all metabolites.…”

Section: Resultsmentioning

confidence: 99%

Assessment and validation of the MS/MS fragmentation patterns of the macrolide immunosuppressant everolimus

et al. 2007

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Everolimus (40-O-(2-hydroxyethyl)rapamycin, Certican) is a 31-membered macrolide lactone. In lymphocytes, it inhibits the mammalian target of rapamycin (mTOR) and is used as an immunosuppressant after organ transplantation. Due to its instability in pure organic solvents and insufficient HPLC separation, NMR spectroscopy analysis of its metabolite structures is nearly impossible. Therefore, structural identification based on tandem mass spectrometry (MS/MS) and MS(n) fragmentation patterns is critical. Here, we have systematically assessed the fragmentation pattern of everolimus during liquid chromatography (LC)-electrospray ionization (ESI)-MS/MS and validated the fragment structures by (1) comparison with structurally identified derivatives (sirolimus), (2) high-resolution mass spectrometry, (3) elucidation of fragmentation pathways using ion trap mass spectrometry (up to MS(5)) and (4) H/D exchange. In comparison with the structurally related immunosuppressants tacrolimus and sirolimus, our study was complicated by the low ionization efficiency of everolimus. Detection of positive ions gave the best sensitivity, and everolimus and its fragments were mainly detected as sodium adducts. LC-ESI-MS/MS of everolimus in combination with collision-induced dissociation (CID) resulted in a complex fragmentation pattern and the structures of 53 fragments were identified. These detailed fragmentation pathways of everolimus provided the basis for structural elucidation of all everolimus metabolites generated in vivo und in vitro.

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“…2). The identity of each product was assigned based on observed mass spectral fragmentation patterns (Table 1), in comparison to that of previously published tacrolimus and metabolite spectra (Iwasaki et al, 1993;Lhoest et al, 2001). Representative metabolite spectra and formation of diagnostic ions are depicted in Fig.…”

Section: Isolation and Identification Of Tacrolimus Metabolitesmentioning

confidence: 99%

Effect of Cyp3a5 Polymorphism on Tacrolimus Metabolic Clearance in Vitro

Dai

Hébert

Isoherranen

et al. 2006

Drug Metabolism and Disposition

256

197

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ABSTRACT:Previous investigations of solid organ transplant patients treated with tacrolimus showed that individuals carrying a CYP3A5*1 allele have lower dose-adjusted trough blood concentrations compared with homozygous CYP3A5*3 individuals. The objective of this investigation was to quantify the contribution of CYP3A5 to the hepatic and renal metabolic clearance of tacrolimus. Four primary tacrolimus metabolites, 13-O-desmethyl tacrolimus (13-DMT) (major), 15-O-desmethyl tacrolimus, 31-O-desmethyl tacrolimus (31-DMT), and 12-hydroxy tacrolimus (12-HT), were generated by human liver microsomes and heterologously expressed CYP3A4 and CYP3A5. The unbound tacrolimus concentration was low (4-15%) under all incubation conditions. For CYP3A4 and CYP3A5, V max was 8.0 and 17.0 nmol/min/nmol enzyme and K m,u was 0.21 and 0.21 M, respectively. The intrinsic clearance of CYP3A5 was twice that of CYP3A4. The formation rates of 13-DMT, 31-DMT, and 12-HT were >1.7-fold higher, on average, in human liver microsomes with a CYP3A5*1/*3 genotype compared with those with a homozygous CYP3A5*3/*3 genotype. Tacrolimus disappearance clearances were 15.9 ؎ 9.8 ml/min/mg protein and 6.1 ؎ 3.6 ml/ min/mg protein, respectively, for the two genotypes. In vitro to in vivo scaling using both liver microsomes and recombinant enzymes yielded higher predicted in vivo tacrolimus clearances for patients with a CYP3A5*1/*3 genotype compared with those with a CYP3A5*3/*3 genotype. In addition, formation of 13-DMT was 13.5-fold higher in human kidney microsomes with a CYP3A5*1/*3 genotype compared with those with a CYP3A5*3/*3 genotype. These data suggest that CYP3A5 contributes significantly to the metabolic clearance of tacrolimus in the liver and kidney.

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In vitro immunosuppressive activity of tacrolimus dihydrodiol precursors obtained by chemical oxidation and identification of a new metabolite of SDZ‐RAD by electrospray and electrospray‐linked scan mass spectrometry

Cited by 10 publications

References 22 publications

Boosting ascomycin production through n-butanol addition in the fermentation process ofStreptomyces hygroscopicus var. ascomyceticusATCC 14891

Boosting ascomycin production through n-butanol addition in the fermentation process ofStreptomyces hygroscopicus var. ascomyceticusATCC 14891

Assessment and validation of the MS/MS fragmentation patterns of the macrolide immunosuppressant everolimus

Effect of Cyp3a5 Polymorphism on Tacrolimus Metabolic Clearance in Vitro

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