Comparison of the in vitro metabolism of the macrolide immunosuppressants sirolimus and RAD

Jacobsen, Wolfgang; Serkova, Natalie J.; Hausen, Bernard; Morris, Randall E.; Benet, Leslie Z.; Christians, Uwe

doi:10.1016/s0041-1345(00)02116-3

Cited by 108 publications

(103 citation statements)

References 2 publications

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“…These two metabolites showed low formation rates accounting for approximately 10% of sirolimus depletion by pooled HLMs. Sirolimus is indeed subject to an extensive oxidative metabolism leading to at least 12 identified metabolites (Jacobsen et al, 2001), some of which are further metabolized. Therefore, sirolimus metabolism was further investigated indi- Hydroxylation of Sirolimus Demethylation of Sirolimus …”

Section: Discussionmentioning

confidence: 99%

Metabolism of Sirolimus in the Presence or Absence of Cyclosporine by Genotyped Human Liver Microsomes and Recombinant Cytochromes P450 3A4 and 3A5

Picard¹,

Djebli²,

Sauvage³

et al. 2006

Drug Metab Dispos

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ABSTRACT:Sirolimus is an immunosuppressive drug currently used alone or in combination with cyclosporine. Both drugs undergo extensive metabolism by the CYP 3A enzymes. This study aimed at comparing the activity of recombinant CYP (rCYP) 3A4 and 3A5 toward sirolimus, investigating the effect of cyclosporine on the metabolic rate of these two cytochromes P450 (P450s), as well as the impact of the CYP 3A5*3 polymorphism on that of human liver microsomes (HLMs). Two distinct approaches were used; i.e., the measurement of (1) hydroxy-sirolimus and desmethyl-sirolimus production, and (2) sirolimus depletion by the in vitro half-life method. rCYP 3A5 exhibited a lower intrinsic clearance (CL int ) for both hydroxylation (0.11 versus 0.24 l/pmol P450/min) and depletion of sirolimus (0.64 versus 2.36 l/pmol P450/min) than rCYP 3A4. Similar CL int values for hydroxylation, demethylation, and depletion were found when comparing a pool of HLMs carrying at least one CYP 3A5*1 (active) allele with a pool of HLMs not expressing CYP 3A5. This was further confirmed for sirolimus depletion using individual microsome preparations (p ‫؍‬ 0.42). A deeper inhibitory effect of cyclosporine on the CL int of sirolimus depletion was found for rCYP 3A4 than for rCYP 3A5 (i.e., ؊44% versus ؊8% at 0.62 M, 750 g/l cyclosporine), and sirolimus metabolism was slightly less inhibited for HLMs expressing CYP 3A5 than not (؊38% versus ؊56%). In the absence of cyclosporine, the CYP 3A5*3 polymorphism may not influence significantly sirolimus metabolism at the hepatic level. However, strong CYP 3A4 inhibition by cyclosporine could unveil the influence of this polymorphism.

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Section: Discussionmentioning

confidence: 99%

Metabolism of Sirolimus in the Presence or Absence of Cyclosporine by Genotyped Human Liver Microsomes and Recombinant Cytochromes P450 3A4 and 3A5

Picard¹,

Djebli²,

Sauvage³

et al. 2006

Drug Metab Dispos

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“…7). The metabolite structures were identified as described previously (Jacobsen et al, 2001). Briefly, metabolites generated from human liver microsomes were isolated by HPLC, and structural identification was carried out by ion trap mass spectrometry and analysis of the fragmentation pattern.…”

Section: Methodsmentioning

confidence: 99%

“…The metabolism of sirolimus has been extensively characterized in human and intestinal microsomes as well as in cDNA-expressed cytochrome P450s (Lampen et al, 1998;Jacobsen et al, 2001). Sirolimus metabolism has been found to be mediated primarily by CYP3A4 with some contribution from CYP3A5 and CYP2C8 (Jacobsen et al, 2001).…”

Section: Transport Of Midazolam and Sirolimus Across Mdr1-mdck And MDmentioning

confidence: 99%

“…Midazolam, a preoperative anesthetic agent, is a classic CYP3A4 substrate that has been shown to undergo extensive gut metabolism (Paine et al, 1996) and does not exhibit bidirectional transport by P-gp (Kim et al, 1999;Polli et al, 2001). The immunosuppressive drug sirolimus is a dual CYP3A4 and P-gp substrate that is known to be transformed by CYP3A4 to numerous hydroxylated and demethylated metabolites (Jacobsen et al, 2001) and has been shown to be an inhibitor of P-gp (Arceci et al, 1992). Conflicting evidence regarding its status as a P-gp substrate has emerged; some investigators have shown high net secretion (18-fold) (Crowe and Lemaire, 1998) and others have shown net absorption (Dias and Yatscoff, 1994;Wacher et al, 2002) across monolayers of Caco-2 cells.…”

mentioning

confidence: 99%

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

Cummins¹,

Jacobsen²,

Christians³

et al. 2003

J Pharmacol Exp Ther

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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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“…In vitro studies supported by clinical data established that everolimus is metabolized by cytochrome P450 3A (CYP3A; ref. 23). This is a concern for drug-drug interactions, as coadministered drugs or food components may drastically alter CYP3A activity, and therefore the systemic levels of orally administered everolimus, resulting in either undertreatment, or serious (life-threatening) side effects of this potentially highly toxic drug.…”

Section: Introductionmentioning

confidence: 99%

P-Glycoprotein, CYP3A, and Plasma Carboxylesterase Determine Brain and Blood Disposition of the mTOR Inhibitor Everolimus (Afinitor) in Mice

Tang

Sparidans

Cheung

et al. 2014

Clinical Cancer Research

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Comparison of the in vitro metabolism of the macrolide immunosuppressants sirolimus and RAD

Cited by 108 publications

References 2 publications

Metabolism of Sirolimus in the Presence or Absence of Cyclosporine by Genotyped Human Liver Microsomes and Recombinant Cytochromes P450 3A4 and 3A5

Metabolism of Sirolimus in the Presence or Absence of Cyclosporine by Genotyped Human Liver Microsomes and Recombinant Cytochromes P450 3A4 and 3A5

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

P-Glycoprotein, CYP3A, and Plasma Carboxylesterase Determine Brain and Blood Disposition of the mTOR Inhibitor Everolimus (Afinitor) in Mice

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