Francesco Parlati, Ph.D.

Transcription

1 Novel Pharmacodynamic Assays to Measure Glutaminase Inhibition Following Oral Administration of CB-839 Francesco Parlati, Ph.D. Calithera Biosciences South San Francisco, California

2 Disclosure Information AACR Annual Meeting 214 Francesco Parlati I have the following financial relationships to disclose: Stockholder in Calithera Biosciences Employee of Calithera Biosciences - I will not discuss off label use and/or investigational use in my presentation.

3 Glutamine is an Important Fuel for Tumor Cells glucose glucose glutamine glutamine Glutaminase TCA Cycle glutamate CB-839 Tumor Cell Most tumor cells depend on both glutamine and glucose for growth and survival These fuels generate energy and provide a source of key biosynthetic buildings blocks The enzyme glutaminase catalyzes the first step in glutamine breakdown Glutaminase inhibition suppresses growth in glutamine-dependent tumor cells CB-839 is a novel, selective glutaminase inhibitor in clinical trials

4 CB-839 Inhibits Glutaminase (GLS) GLS Both forms inhibited by CB-839 GLS2 Not inhibited by CB-839 GLS is widely expressed Enzyme IC 5 (nm) GAC 24 KGA 29 GLS2 > 5 KGA GLS Mito GAC up-regulated in tumors Minimal catalytic domain Alternatively spliced C-terminus GAC GLS2 Mito 7% sequence identity to KGA/GAC Primarily expressed in liver Gross et al., Molecular Cancer Therapeutics (214)

5 M C F 1 A H C C 7 H s T M D A- M B S U M P T M D A- M B M X - 1 H C C H C C S U M P T B T H C C 3 8 H M C B T - 2 D U H C C H C C H s T H s T M D A- M B M C F 7 JIM T - 1 AU H C C H C C M D A- M B S K - B R - 3 T D N C I- H N C I- H N C I- H N C I- H N C I- H 2 3 N C I- H N C I- H 2 3 A5 4 9 N C I- H N C I- H N C I- H N C I- H N C I- H H C C N C I- H N C I- H N C I- H C h ag o - K - 1 N C I- H N C I- H N C I- H N C I- H N C I- H N C I- H U S U - D H L - 4 D O H H - 2 S U - D H L - 1 S U - D H L - 6 W S U - D L C L 2 JV M - 2 N U - D U L - 1 M in o H u T 1 2 D B R L G R AN T A N C I- H AM O - 1 L K M S B M K M S M O L P - 8 K M S B M M M 1 - S L P - 1 K AR P AS K M S M O L P - 2 U B 1 M M 1 R IM 9 R P M I O P M - 2 K M S P E E JM K M S S K - M M - 2 K M S - 2 K M S K M M - 1 K M S P E d elt a- 4 7 H u N S 1 N C I- H 2 8 N C I- H M S T O H N C I- H S U P - T 1 S U P - B 1 5 B D C M R eh C B IC 5 ( M ) CB-839 Anti-Tumor Activity in Several Cancer Types B re a s t (N = 2 8 ) E R + o r T r ip le - n e g a t iv e H e r 2 + (N = 2 ) (N = 8 ) N S C L C (N = 2 4 ) L y m p h o m a (N = 1 3 ) M y e lo m a (N = 2 7 ) M e s o th e lio m a (N = 4 ) A L L (N = 4 ) > C e ll L in e (C B tre a tm e n t fo r 7 2 h r)

6 M e ta b o lite le v e l (n m o l/1 6 c e lls ) CB-839 Suppresses Metabolic Pathways Downstream of Glutamate CB-839 glutathione glutamine glutamate Glutaminase a-ketoglutarate (a-kg) HCC-186 cells (triple negative breast cancer) fumarate 4 hr treatment in vitro malate TCA cycle oxaloacetate citrate glutamate a-kg aspartate G lu ta m in e G lu ta m a te G lu ta th io n e M a la te C itra te F u m a ra te A s p a rta te ** **** ** 2. *** ***. 2 5 *** ****... DMSO CB-839 (1µM) Gross et al., Molecular Cancer Therapeutics (214)

7 T u m o r V o lu m e (m m 3 ) CB-839 Has Anti-Tumor Activity in a Multiple Myeloma Xenograft Model RPMI-8226 Tumor Growth Pharmacodynamic Response Change in metabolites 4hr after last dose 1 V e h ic le (2 5 % H P B C D ) G lu ta m in e G lu ta m a te C B m g /k g q 1 2 h r **** **** 8 P e a k A re a Vehicle CB D o s in g S ta r t G lu ta th io n e F u m a r a te M a la t e C itra te A s p a rta te * ** ** **** 4 *** 71% TG I P e a k A re a D a y s P o s t-im p la n t

8 Clinical Pharmacodynamic Assay Objectives Directly measure glutaminase inhibition in patient tissues Platelets (surrogate tissue) Tumors (from biopsy samples) Inhibited Glutaminase Active Glutaminase Gln Glu Glu Gln CB-839 allosteric binding site X CB-839 Gln Glu Glu Gln Challenges: CB-839 interaction with glutaminase is reversible Under standard tissue lysis conditions, CB-839 dissociates from glutaminase Goal: stabilize CB-839/glutaminase complex throughout sample preparation

9 R e c o v e ry o f G lu ta m in a s e A c tiv ity Stabilization of the CB-839/Glutaminase Complex Reversibility Experiment Glutaminase pre-treated with CB-839 for 1 h at RT Isolate Glutaminase: CB-839 by gel filtration Dt Assay glutaminase activity KPO 4 KCl Temp. t 1/2 Inhibition at 5 h 15 mm mm RT 45 min % 15 mm 15 mm RT > 5h 95% 15 mm 15 mm 4 o C > 5h 99.7% T im e (m in ) Conditions to maintain glutaminase/cb-839 complex identified

10 Human Platelet Pharmacodynamic Assay Human whole blood treated with CB-839 for 4 h ex vivo at 37 C Isolate platelets (4 C) Homogenize Sample (under conditions that preserve the glutaminase:cb-839 complex) Isolate glutaminase:cb-839 by gel filtration Assay glutaminase activity % G lu ta m in a s e A c tiv ity I C 5 = 2 9 n M [C B ] n M Biochemical IC 5 : 24-29nM Cell-based IC 5 : 1-3 nm 9% glutaminase inhibition at 3 nm CB-839

11 Glutaminase Inhibition in Platelets Correlates with Glutaminase Inhibition in Tumors RPMI-8226 xenografted mice were treated with CB-839 Glutaminase inhibition was measured in platelets and tumors The extent of platelet and tumor glutaminase inhibition was directly correlated Platelets can serve as a surrogate for measuring tumor inhibition in vivo 1 2 R P M I M y e lo m a X e n o g r a ft M o d e l % T u m o r G lu ta m in a s e A c tiv ity m g /k g 2.5 m g /k g V e h ic le % P la te le t G lu ta m in a s e A c tiv ity

12 Pharmacokinetic and Pharmacodynamic Relationship in Xenografted Mice Tumor: HCC-186 (TNBC) CB-839 dose range: mg/kg Time-point: 4 h post-dose Tumor CB-839 T u m o r G lu ta m in a s e A c tiv ity 1 1 Tumor CB-839 (nmol/g tissue) 1 G lu ta m in a s e a c tiv ity (% o f c o n tro l) Plasma CB-839 ( M) P la s m a C B ( M ) T u m o r G lu ta m in e T u m o r G lu ta m a te 6 6 T u m o r M e ta b o lite L e v e l ( m o l/g tis s u e ) 4 2 T u m o r M e ta b o lite L e v e l ( m o l/g tis s u e ) P la s m a C B ( M ) P la s m a C B ( M ) 3 nm CB-839 in plasma is sufficient to maximally inhibit glutaminase in tumors In separate studies, maintaining 3 nm CB-839 in plasma suppresses tumor growth The clinical goal is to maintain greater than 3 nm CB-839 in plasma

13 CB-839 Phase 1 Clinical Trials The objective of these trials is to test the safety/tolerability, pharmacokinetics, pharmacodynamics, and clinical activity of single agent CB-839 Clinical Trial* NCT NCT Specific Indications Solid Tumors: TNBC, NSCLC, RCC & Mesothelioma NHL, Multiple Myeloma, Walde stro s Macroglobulinemia NCT * AML, ALL

14 Glutaminase Inhibition in CB-839 Dosed Patients Oral Dose: 1 mg Pharmacokinetics Glutaminase Inhibition in Platelets Patient 1 [C B ] ( M ) n M % G lu ta m in a s e A c tiv ity T im e (h ) P re -d o s e 4 h P o s t-d o s e Patient 2 [C B ] ( M ) T im e (h ) 3 n M %Glutaminase Activity Pre-dose 4 h Post-dose

15 Conclusions CB-839 has potent single agent anti-tumor activity in animals We developed an assay to monitor glutaminase inhibition in patients Preclinical data show that glutaminase inhibition in platelets can serve as a surrogate for tumor inhibition In animals, maintaining plasma levels of 3 nm CB-839 was sufficient to fully inhibit glutaminase in both platelets and tumors In patients, a 1 mg dose of CB- 3 resulted i % glutaminase inhibition in platelets at 4 hours post dose In ongoing dose escalation trials, monitoring platelet glutaminase inhibition will be an important pharmacodynamic readout

16 Acknowledgements Biology Francesco Parlati Andy MacKinnon Mirna Rodriguez Winter Zhang Ethan Emberley Susanne Steggerda Alison Pan Susan Demo Tania Rogan Pharmacology Matthew Gross Julie Janes Chemistry Jim Li Guy Laidig Lijing Chen Tim Stanton DMPK Weiqun Le Tracy Wang Jing Zhang Jinfu Yang Frances Zhou Poster: #1416 Monday: 8 am-12 pm Hall: A-E Section: 18 Clinical Mai Le Pharm. Dev. Evan Lewis Peter Shwoenek Management Team Susan Molineaux Mark Bennett Eric Sjogren Chris Molineaux