Identification of a new metabolite of macrolide immunosuppressant, like rapamycin and SDZ RAD, using high performance liquid chromatography and electrospray tandem mass spectrometry

Hallensleben, Katrin; Raida, Manfred; Habermehl, Gerhard

doi:10.1016/s1044-0305(00)00123-9

Cited by 32 publications

(22 citation statements)

References 14 publications

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“…8). A ring-opened sirolimus metabolite, possibly identical to M2 characterized in this study, was recently reported as a minor metabolite in incubations of sirolimus with human liver microsomes (Hallensleben et al, 2000). We also found M2 to be a minor metabolite in human liver (and intestinal) microsomes, but it was the dominant metabolite produced by human liver and intestinal mucosal homogenates and in Caco-2 cell homogenates (Fig.…”

Section: Novel Route Of Intestinal Extraction For Sirolimus 181supporting

confidence: 84%

Identification of a Novel Route of Extraction of Sirolimus in Human Small Intestine: Roles of Metabolism and Secretion

Paine

Leung

Lim

et al. 2002

The Journal of Pharmacology and Experimental Therapeutics

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Using Caco-2 cell monolayers expressing CYP3A4, we investigated the interplay between metabolism and transport on the first-pass intestinal extraction of the immunosuppressant sirolimus, a CYP3A4/P-glycoprotein (P-gp) substrate. Modified Caco-2 cells metabolized [14 C]sirolimus to the predicted amounts of CYP3A4-mediated products based on CYP3A4 content, which was ϳ20% of that measured in human small intestinal mucosal homogenate. [14 C]Sirolimus also degraded to the known ring-opened product, seco-rapamycin. Unexpectedly, a ring-opened dihydro metabolite (M2) was the major product detected in cells at all sirolimus concentrations examined (2-100 M). Greater M2 formation after apical versus basolateral dosing (1.6-fold) was explained by higher intracellular content of sirolimus after apical dosing. M2 was not detected in incubations with human liver and intestinal microsomes but was readily detected with corresponding homogenates. M2 formation was NADPH-dependent but unaffected by the CYP3A4 inhibitors ketoconazole and troleandomycin. Although M2 was formed from purified seco-rapamycin (20 M) in the homogenates, it was not detected in cells when seco-rapamycin was added to the apical compartment, because seco-rapamycin was essentially impermeable to the apical membrane. Sirolimus, seco-rapamycin (basolaterally dosed), and M2 were all actively secreted across the apical membrane, and secretion of each was inhibited by the P-gp inhibitor LY335979 [(2R)-anti-5-{3-[4-(10,11-difluoromethanodibenzo-suber-5-yl)piperazin-1-yl]-2-hydroxypropoxy}quinoline trihydrochloride]. Along with CYP3A4-mediated metabolism and Pgp-mediated secretion, we conclude that the following novel pathway, which occurs at least in the intestine, may contribute significantly to the first-pass extraction of sirolimus in humans: intracellular degradation of sirolimus to seco-rapamycin, metabolism of seco-rapamycin to M2 by an unidentified nonmicrosomal enzyme, and P-gp-mediated secretion of M2 and secorapamycin.

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Section: Novel Route Of Intestinal Extraction For Sirolimus 181supporting

confidence: 84%

Identification of a Novel Route of Extraction of Sirolimus in Human Small Intestine: Roles of Metabolism and Secretion

Paine

Leung

Lim

et al. 2002

The Journal of Pharmacology and Experimental Therapeutics

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“…Hallensleben et al (2000) identified a novel metabolite of sirolimus (dihydrosirolimus) formed from the degradation of sirolimus to its ring opened isomer (secosirolimus) that then undergoes hydrogenation to form this new metabolite. The proposed metabolic pathway for the formation of dihydrosirolimus is shown in Fig.…”

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confidence: 99%

CYP3A4-Transfected Caco-2 Cells as a Tool for Understanding Biochemical Absorption Barriers: Studies with Sirolimus and Midazolam

Cummins¹,

Jacobsen²,

Christians³

et al. 2004

The Journal of Pharmacology and Experimental Therapeutics

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CYP3A4-transfected Caco-2 cells were used as an in vitro system to predict the importance of drug metabolism and transport on overall drug absorption. We examined the transport and metabolism of two drugs; midazolam, an anesthetic agent and CYP3A4 substrate, and sirolimus, an immunosuppressant and a dual CYP3A4/P-glycoprotein (P-gp) substrate, in the presence of cyclosporine (CsA, a CYP3A4/P-gp inhibitor) or N-{4- [2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]-phenyl}-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamine (GG918) (an inhibitor of P-gp and not CYP3A4). All major CYP3A4 metabolites were formed in the cells (1-OH Ͼ 4-OH midazolam and 39-O-desmethyl Ͼ 12-OH Ͼ 11-OH sirolimus), consistent with results from human liver microsomes. There was no bidirectional transport of midazolam across CYP3A4-transfected Caco-2 cells, whereas there was a 2.5-fold net efflux of sirolimus (1 M) that disappeared in the presence of CsA or GG918. No change in the absorption rate or extraction ratio (ER) for midazolam was observed when P-gp was inhibited with GG918. Addition of GG918 had a modest impact on the absorption rate and ER for sirolimus (increased 58% and decreased 25%, respectively), whereas a 6.1-fold increase in the absorption rate and a 75% decrease in the ER were found when sirolimus was combined with CsA. Although both midazolam and sirolimus metabolites were preferentially excreted to the apical compartment, only sirolimus metabolites were transported by P-gp as determined from inhibition studies with GG918. Using CYP3A4-transfected Caco-2 cells we determined that, in contrast to P-gp, CYP3A4 is the major factor limiting sirolimus absorption. The integration of CYP3A4 and P-gp into a combined in vitro system was critical to unveil the relative importance of each biochemical barrier.Oral bioavailability and intestinal drug absorption can be significantly limited by metabolizing enzymes and efflux transporters in the gut (Benet et al., 1996b). The most prevalent oxidative drug-metabolizing enzyme present in the intestine is cytochrome P450 3A4 (CYP3A4). Currently, more than 50% of the drugs on the market metabolized by P450 enzymes are metabolized by CYP3A4 (Benet et al., 1996a). Oral absorption of CYP3A4 substrates can also be limited by the multidrug resistance transporter P-glycoprotein (P-gp), because there is extensive substrate overlap between these two proteins (Wacher et al., 1995). P-gp is an ATP-dependent transporter on the apical plasma membrane of enterocytes that functions to limit the entry of drugs into the cell (Ambudkar et al., 1999). We have previously hypothesized that the interplay between CYP3A4 and P-gp in the intestine can serve to enhance drug metabolism and significantly decrease intestinal drug absorption (Cummins et al., 2002).

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“…It is often assumed that compound 3 is formed through the dehydration of 2. [17][18][19] Although possible, this mechanism appears to be not supported by any experimental data.…”

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confidence: 83%

Degradation of rapamycin and its ring-opened isomer: Role of base catalysis

Il’ichev¹,

Alquier²,

Maryanoff³

2007

Arkivoc

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Rapamycin is a natural macrolide immunosuppressant with a distinct mechanism of action. Quantitative analysis of rapamycin poses many challenges associated with facile degradation and the multitude of isomeric forms. The primary goal of this study was to compare degradation of rapamycin and its ring-opened isomer, secorapamycin, in aqueous solution under identical conditions. Reaction kinetics and mechanisms were studied in 30/70 vol/vol acetonitrile-water mixtures containing either MeCOONH 4 (apparent pH 7.3) or NaOH (apparent pH 12.2). Degradation kinetics was well described by the first-order rate law. For rapamycin in 237 and 23.7 mM solutions of MeCOONH 4 , apparent half-lives of 200 h and 890 h were obtained. When compared to the latter value, the rapamycin half-life was reduced by 3 orders of magnitude in the pH 12.2 solution. Under all conditions studied, secorapamycin degradation was significantly slower than that of the parent compound. Both specific and general base catalysis was observed for reactions of rapamycin and secorapamycin. Two primary products of rapamycin degradation were identified as individual isomers of secorapamycin and a hydroxy acid formed via lactone hydrolysis. No evidence for the interconversion between the products was obtained. In highly basic solutions, both products undergo fragmentation and water addition reactions.

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Identification of a new metabolite of macrolide immunosuppressant, like rapamycin and SDZ RAD, using high performance liquid chromatography and electrospray tandem mass spectrometry

Cited by 32 publications

References 14 publications

Identification of a Novel Route of Extraction of Sirolimus in Human Small Intestine: Roles of Metabolism and Secretion

Identification of a Novel Route of Extraction of Sirolimus in Human Small Intestine: Roles of Metabolism and Secretion

CYP3A4-Transfected Caco-2 Cells as a Tool for Understanding Biochemical Absorption Barriers: Studies with Sirolimus and Midazolam

Degradation of rapamycin and its ring-opened isomer: Role of base catalysis

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