Human Metabolism of a Novel Chemotherapeutic: Illuminating the Mechanisms of P450-Catalyzed Deboronation

Transcription

1 uman Metabolism of a ovel Chemotherapeutic: Illuminating the Mechanisms of P450-Catalyzed Deboronation J. Scott Daniels Pharmacokinetics, Dynamics and Metabolism Pfizer, Inc. Chesterfield, M

2 Multiple Myeloma (MM) plasma cell malignancy - 5 cases in 100,000 people - YR 2005 : 16,000 new cases - 50,000 with MM in USA Multiple Myeloma (amplified proteasome activity) (1) uncontrolled division (2) traffic to bone (3) cell adhesion (4) M protein (MMRF)

3 MM Cells Dependent on 26S Proteasome cell cycle control β6 β7 β1 β2 cell differentiation β3 β5 β4 antigen processing C 3 Threonine protease stress response courtesy of Dr. M. Groll Ludwig-Maximilians-University of Munich

4 Bortezomib : Transition State Analog of 26S 26S 26S C 3 bortezomib B C 3 B enzyme K i (nm) 20S proteasome 0.62 Leuk. elastase 2,300 cathepsin G 630 chymotrypsin 320 thrombin 13,000 approved in 2003 for treatment of multiple myeloma * currently approved for 2 nd line treatment undergoing multiple clinical trials Bioorg. Med. Chem. Lett. 1998, 8, ; Clin. Cancer. Res. 2004, 10, * relapsed, refractory

5 uman Disposition of Bortezomib for intravenous injection ~ 83% protein bound high V d ; long elim. t 1/2 not subjected to drug efflux (e.g., Pgp) does not inhibit efflux in Caco-2 cells o obsvd inhibition of P450 (M1/M2-2C19) P450 phenotype (3A4>2C19>1A2>2D6>2C9) renal elimination of metabolites

6 B 2 Bortezomib (1) 2 M25, M26 M27, M28 M23, M24 M1 M3 M33 M2 M6 M34 M5 M4 M8 uman Metabolism of Bortezomib B 2 Bortezomib (1) 2 M25, M26 M27, M28 M23, M24 M1 M3 M33 M2 M6 M34 M5 M4 M8 major biotransformation: deboronation P450-mediated?? Drug Metab. Dispos. 2005, 33,

7 Routes of Borane/Boronate xidation (.C. Brown) A R B R B retention B() 3 R peroxidation Fe B B R B X X no stereocontrol RX reduction + R autooxidation X = 2 or peroxyl radical o published accounts of boronic acid metabolism (i.e., P450)

8 uman Liver Metabolism of Bortezomib UV M3 M5 M1 o observed interconversion Relative Absorbance M6 M8 Bortezomib M2 M1 M2 ear equal ratio of M1:M Time (min) What is the mechanism of this novel P450 reaction?

9 ESI-LC/MS to Detect Isotope ( 18 ) Incorporation 1 B LM 2 18 M1/M2 Imine intermediate unlikely (no incorporation) LM 18 2 ( ) [M+a] Incorporation M1/M2 18 [M+a] + Use of 18 2 resulted in the formation of 18 -labeled M1 and M2.

10 Stability of Bortezomib in Formulation ambient air & temp ultraviolet (hν). 2 Relative Abundance TIC bortezomib Fenton-type erratic rxn: stability behavior. V. Stella Fe(II)S 4, 2, buffer (p 7.4). J. Pharm. Sci. 2000, 89(6), M / 2 2 produced M2 M3 M34 fortified solutions of bortezomib with M n+ Ascorbate & EDTA reactive oxygen species mediate deboronation

11 xidase Activity of P450: RS Formation ( uncoupling ) Chem. Res. Toxicol. 2001, 14(6),

12 P450 2E1 Metabolism of Bortezomib the leaky CYP Relative Abundance TIC 2E1 reaction M3 2E1 reaction + SD/catalase bortezomib M1 M /3 time LMs convert to M1 & M2

13 Evidence of Radical Intermediate? Relative Abundance M15 a/b S M15 a/b M + GS ADP GS conjugates metabolites obtained from rat bile Time (min) 1 / 13 C MR-confirmed mics regiochemistry of carbinolamides drug Δ Extracted Ion (TIC) carbinolamides bortezomib MeC LC/MS/MS

14 Zen and the art of P450 Catalysis The real purpose of the scientific method is to make sure ature hasn t misled you into thinking you know something you don t actually know. If you get careless or go romanticising scientific information, giving it a flourish here or there, ature will soon make a fool out of you. Robert Pirsig Zen and the Art of Motorcycle Maintenance (Copyright 1974, Pirsig)

15 3A4 Metabolism of Bortezomib 2 metabolites Relative Abundance M5 TIC M1 M2 M A4 reaction 3A4 reaction + SD/catalase M3 M5 M6 M6 M8 M8 M34 partial contribution by RS? M1 M2 M34

16 Enzyme-bound oxidants contributing? X Fe (III) Fe (V) peroxo-iron oxo-iron FeTPP:PhI I Fe 6-fold increase in M1:M2 over control (PhI) see references within Chem. Res. Toxicol. 2001, 14, 611. oxometalloporphyrin biomimetic

17 Summary of this ovel P450 Reaction bortezomib 1 B 2 + P450 P dismutation 3A4 SD/Catalase results M1/M M1 secondary metabolites 2 / 3 reduction + M2 P450 secondary metabolites peroxycarbinolamides = putative radical Chem. Res. Toxicol. 2006, 19(4),

18 [We can teach you metabolism; we can t teach you chemistry.] G.T. Miwa Thank you Gerald. Sincerely, A Grateful Chemist

19 Acknowledgements Millennium DMPK Lawrence Gan (biotransformation) Professor Kent Gates (Missouri) Jason Labutti Vinita Uttamsingh, Chuang Lu, Cindy Xia and Teresa Pekol Mark Milton, Darrell ix (nonclinical PK) Shimoga Prakash, Yuexian Li (stable isotope synthesis) Professor Paul rtiz de Montellano (UCSF) Professor eal Castagnoli Jr. (Va Tech) VELCADE ncology Team Mark Williamson, Larry Dick and Chris Tsu