ELSEVIER
`Comparison of the In Vitro Metabolism of the Macrolide
`Immunosuppressants Sirolimus and RAD
`
`W. Jacobsen, N. Serkova, B. Hausen, R.E. Morris, L.Z. Benet, and U. Christians
`
`RAD [40-O-(2-hydroxyethyl)-rapamycin] is a novel mac-
`
`rolide immunosuppressant that is structurally related
`to sirolimus.1 Because of its immunosuppressive synergistic
`interaction,2 RAD is under clinical
`investigation as an
`immunosuppressant, in combination with cyclosporine, for
`posttransplantation therapy. It was our goal to study the
`oxidative metabolism of sirolimus and RAD, to identify the
`structures of their metabolites, and to evaluate the impact
`of the 40-O-2-hydroxyethyl group on the in vitro metabo-
`lism of RAD in comparison with sirolimus.
`
`METHODS
`
`Sirolimus or RAD was incubated with human liver microsomes.
`The metabolites were separated and isolated using HPLC and their
`structures elucidated using MS/MS in combination with collision-
`induced dissociation and ion-trap MS (MSn). The cytochrome P450
`(CYP) enzymes involved were identified using isolated cDNA-
`expressed human CYP enzymes. Sirolimus, RAD, and their me-
`tabolites were quantified using LC/LC-MS.
`
`RESULTS AND DISCUSSION
`
`A total of 12 sirolimus and 13 RAD metabolites were
`isolated and the structures of 10 sirolimus (46-, 24-, 25-, 12-,
`11-, 14-, and 49-hydroxy; 39-O-, 27-O-, and 16-O-desmethyl
`sirolimus) and 11 RAD metabolites (as for sirolimus 1
`40-O-deshydroxyethyl RAD) were proposed based on the
`analysis of MS fragmentation patterns. CYP3-A4, -3A5,
`and -2C8 were the major CYP enzymes involved in siroli-
`mus and RAD metabolism (Fig 1). Based on the metabo-
`lism sites, five groups of metabolites were differentiated.
`CYP enzymes showed different preferences for these me-
`tabolism sites (Fig 1). The RAD 40-O-2-hydroxyethyl group
`not only inhibited 39-O-demethylation, the major metabo-
`lism reaction of sirolimus, but also decreased hydroxylation
`
`in the C10 to C14 region, which is the major hydroxylation
`region of sirolimus (Fig 1). In addition to the metabolic
`pathways similar to sirolimus, RAD was dehydroxyethyl-
`ated by CYP-3A, resulting in rapamycin. Based on intrinsic
`clearance, it was estimated that dehydroxyethylation is only
`a minor, most likely clinically insignificant, RAD metabolic
`pathway. In vitro, 5.1% was converted to rapamycin. How-
`ever, if extrapolated to the in vivo situation, this is most
`likely an overestimation, because our in vitro assay ignored
`that: (a) there are probably more RAD metabolites gener-
`ated than have been isolated in our study; (b) there is most
`likely significant metabolism of rapamycin to its inactive
`metabolites in vivo; and (c) there may be significant degra-
`dation of both RAD and rapamycin in vivo. The total
`intrinsic formation clearance of RAD metabolites by hu-
`man liver microsomes was 2.7-fold lower than that of
`sirolimus (mean 682 vs 1810 mL/gzmin). We conclude that,
`although structurally similar, in vitro metabolism of RAD
`and sirolimus shows significant differences.
`
`REFERENCES
`1. Sedrani R, Cottens S, Kallen J, et al: Transplant Proc 30:2192,
`1988
`2. Schuurman HJ, Cottens S, Fuchs S, et al: Transplantation
`65:32, 1997
`
`From the Department of Biopharmaceutical Sciences, Univer-
`sity of California (W.J., N.S., L.Z.B., U.C.), San Francisco,
`California; and the Laboratory of Transplantation Immunology,
`Stanford University School of Medicine, (B.H., R.E.M., U.C.),
`Stanford, California.
`Address reprint requests to Dr Uwe Christians, Biopharma-
`ceutical Sciences, University of California, 533 Parnassus Ave-
`nue, Room U-66C, San Francisco, CA 94143-0446. E-mail:
`uwec@ucsf.itsa.edu.
`
`0041-1345/01/$–see front matter
`PII S0041-1345(00)02116-3
`
`514
`
`© 2001 by Elsevier Science Inc.
`655 Avenue of the Americas, New York, NY 10010
`
`Transplantation Proceedings, 33, 514–515 (2001)
`
`NOVARTIS EXHIBIT 2059
`Breckenridge v. Novartis, IPR 2017-01592
`Page 1 of 2
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`

`

`IN VITRO METABOLISM
`
`515
`
`Group 2
`CYP3A4 > 3A5 ·
`
`hydroxylation
`0 dealkylation
`= Group 5, CYP3A4 > CYP3A5
`
`Group 3
`CYP2C8 » 3A4 > 3A5
`
`RAD
`
`~
`-
`
`Group 4
`CYP3A4
`
`Sirolimus: C(40)-OH
`
`400
`
`..J
`(.)
`
`Sirolimus
`
`3[l8~/I'Jscin
`27-0-des
`desalkyl
`15-0-des
`48-0H
`OH-piper"II
`14<?~ffiper'I
`11-0H
`2s1.6~H
`24-0H
`46-0H
`
`hydroxy
`
`Metabolite
`Fig 1. Comparison of in vitro metabolism of sirolimus and RAD by human liver microsomes. (Top) Major drug metabolism sites and
`cytochrome P450 enzymes. (Bottom) Comparison of intrinsic formation clearances of individual sirolimus and RAD metabolites.
`Numbering of the molecules follows IUPAC guidelines.
`
`RAD
`
`NOVARTIS EXHIBIT 2059
`Breckenridge v. Novartis, IPR 2017-01592
`Page 2 of 2
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