Ibrutinib

Development of an Irreversible Inhibitor of Bruton’s Tyrosine Kinase:

Ibrutinib

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Outline

•  Part I: Background of disease biology and in vitro and in vivo efficacy of PCI-32765

•  Part II: Mechanism of Action and Structural

Development and Design •  Part III: Current Treatments,

Pharmacokinetics & Future Directions

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Part I: Background of disease biology and in vitro and in vivo efficacy of PCI-32765

Christine Cuthbertson

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Non-Hodgkin Lymphomas

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•  Production of abnormal white blood cells originating from lymph nodes

•  Classified by how cells look under the microscope, chromosome

features and the presence of certain proteins on the surface of cells •  B-cell lymphomas make up the largest proportion (85%) of non-

Hodgkin lymphomas (NHL) •  Ibrutinib has been approved for 3 types of NHL so far, and is in

clinical trials for 3 more, as well as some leukemias

American Cancer Society, Types of non-Hodgkin lymphoma

B-cell Differentiation

1.  B-cells receive signals •  “T-cell help” •  establish germinal

centers (GC)

1.  Somatic hypermutation to increase affinity for antigens

2.  Differentiate if:

•  functional B-cell receptor (BCR)

•  BCR has high antigen affinity

Kuppers, Nat. Rev. Cancer, 2005 5

Lymph node

B-cell Receptor Signaling

6 Mraz and Seda, Eur. J. Haematol., 2014

Mantle cell lymphoma (MCL) ●  Usually begins with lymph node enlargement1

o  can spread to other tissues such as the bone marrow, liver, spleen and GI tract ●  Symptoms include: anemia; GI, pulmonary and/or CNS

complications; and leukocytosis1

●  Distinguished by overexpression of cyclin D11

o  caused by translocation of chromosomes 11 and 14 ●  71,850 cases of NHL expected for 20152

o  MCL represents about 6% of NHL1

1.  Leukemia & Lymphoma Society, Mantle Cell Lymphoma Facts, 2014 2.  American Cancer Society, Cancer Facts & Figures 2015 American Society of Hematology Image Bank 7

Chronic lymphocytic leukemia (CLL)

●  Usually found by blood work from unrelated health problem or routine check-up with high lymphocyte count1

●  Symptoms include: weakness, weight loss, enlarged lymph nodes, pain or sense of “fullness,” even after a small meal1

●  Cause is unknown; inheritance of CLL is very rare1

o  most common genetic abnormality is deletion of part of chromosome 13

●  54,270 cases of leukemia expected for 20152

o  ~36% will be CLL

1.  American Cancer Society, Leukemia--Chronic Lymphocytic, 2015 2.  American Cancer Society, Cancer Facts & Figures 2015 American Society of Hematology Image Bank 8

IC50 values and fold selectivity for inhibition of enzymatic activity by PCI-32765

Honigberg et al., PNAS, 2010

●  33P-ATP filtration binding assay

●  Modest selectivity

for kinases tested against

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*Kinases with Cys homologous to Cys481 of BTK

PCI-32765 potently and selectively inhibits BTK

Honigberg et al., PNAS, 2010 10

PCI-32765 inhibits B-cell signaling and activation

Honigberg et al., PNAS, 2010 11

PCI-29732

PCI-32765

Compound exposure: Continuous 1 hr

PCI-32765 selectively inhibits B-cell signaling and activation


Shows 10 nM is sufficient to occupy all BTK active sites in primary B cells in culture

“By combining fast irreversible binding to BTK with rapid in vivo elimination, PCI-32765 defines a unique approach to improve selectivity for BTK in vivo relative to reversibly inhibited off-target

kinases.”

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PCI-32765 is efficacious in murine models of autoimmune disease

Collagen-induced arthritis model MRL-Fas(lpr) lupus model


●  Began treatment after disease had partially progressed

●  Monitored for 11 days ●  Significant reduction in paw swelling

and joint inflammation at all doses

●  8 wk old mice treated for 12 wk ●  Daily oral dose ●  Significant reduction in proteinuria

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PCI-32765 causes responses in spontaneous canine B-cell lymphomas


----PBMC---- ---LN---

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Dose (mg/kg):

20 20

Dose (mg/kg):

20 20

2.5/5.0

Summary of in vitro and in vivo testing

PCI-32765… 1.  Blocked BCR signaling in human peripheral B cells

with no effect on T cell receptor signaling 2.  Completely suppressed disease in arthritic mice

3.  Inhibited development of kidney disease in MRL-

Fas(lpr) lupus model 4.  Induced objective clinical responses in dogs with

spontaneous B-cell non-Hodgkin lymphoma

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Ibrutinib

Part II: Mechanism of Action and Structural Development and Design

Shireen Ashkar

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The Human Kinome

There are currently 621 protein kinases known to exist within the human genome These kinases phosphorylate proteins, functioning as “on/off” switches within many cell- signaling pathways The human kinome is subdivided into 7 main groups

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Tyrosine kinases

The tyrosine kinases (90 kinases) specifically phosphorylate tyrosine residues on their protein substrates

Tyrosine kinases are divided into: -  Receptor TK’s -  Non-receptor TK’s

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Bruton’s Tyrosine Kinase is a member of the TEC family of non-receptor TK’s

Nature Reviews Molecular Cell Biology, 2014, 5, 33-44 19

The Structure of Bruton’s Tyrosine Kinase (BTK)

Future Med. Chem., 2014 6(6), 675-695 J. Biol. Chem., 2001, 276(44), 41435-41443

BTK is composed of 5 domains The ATP-binding pocket is found in the tyrosine kinase catalytic domain (TK) The other domains function in protein interaction and regulation of BTK activity All 5 domains have been targets of drug design Crystal structure of the kinase domain

in murine BTK (98.3 % identical to human) 20

A closer look at the ATP-binding pocket in the KD

(SYK tyrosine kinase in complex with imatinib)

Nature Reviews Drug Discovery 2009, 8, 892-909 21

Activation of BTK-KD via conformational changes

Phosphorylation

J. Biol. Chem, 2001, 276 (44), 41435- 41443

Activation is initiated by phosphorylation of Tyr-551 (by other signalling proteins) This leads to an exchange of H-bonding from Glu-445/ Arg-544 to Glu-445/ Lys-430 Results in concerted rotation of C-helix inwards, which opens the pocket and allows ATP to bind Subsequent autophosphorylation of Tyr-223 continues the catalytic mechanism

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Inhibition of tyrosine kinases

Non-competitive (allosteric site) Competitive (ATP-binding active site) Reversible Irreversible (covalent) Advantages of a covalent, irreversible kinase inhibitor:

- Higher potency - Better selectivity - Prolonged PD - Suitable for rational design

Gray et al., Cell, 2013

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Reversible Competitive BTK inhibitors

OncoTargets and Therapy, 2013, 6, 161-176

Dasatinib

B43

CGI 1746

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LFM-A13

April 1999 First reversible inhibitor IC50 = 2.5 µM Mechanism: Competes with ATP for binding within the pocket Interacts with the residues within the pocket similarly to ATP

J. Biol. Chem. 1999, 274 (14), 9587-9599 Future Med. Chem. 2014, 6 (6), 675-695

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CGI 1746

November 2010 Reversible IC50 = 1.9 nM Mechanism: Forms H-bonds with the Met-477 residue of the hinge sequence and Lys-430 The bulky tert-butyl group sterically blocks the phosphorylation of Tyr-551 Forces C-helix outwards, locking ATP-pocket in an inactivated conformation

Nature Chemical Biology, 2011, 7, 41-51 Future Med. Chem. 2014, 6 (6), 675-695

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A new direction: ATP itself as the source of lead

ATP B43 PCI-32765 Ibrutinib™

Ibrutinib was developed as an analog of B43, which was designed as a mimic of ATP The earliest of these studies were actually aimed at developing an inhibitor for Lck within T-cells. 27

Development of ATP mimic for Lck inhibition In 1996, Pfizer developed a library of pyrazolo-pyrimidines (using an altered synthetic route developed for pyrrolo-pyrimidines in 1988) Their goal at the time was to target Lck within T-cells

Indian J. Chem. 1988, 27B, 778-780 J. Biol. Chem. 1996, 271 (2), 695-701

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In 2000, BASF hypothesized that the tert-butyl group on PP1 was occupying the same space as the ribose of ATP They developed a library of 4-arylaminoquinazolines, and identified compound 2 as a potent inhibitor They reasoned that the phenoxy group was occupying a hydrophobic pocket within the ATP pocket which ATP itself could not reach Replaced the methyl of PP1 with phenoxy and the pyrazole with pyrrole (compound 4) in an attempt to increase the occupation of the hydrophobic pocket

Bioorg. Med. Chem. Lett., 2000, 10, 2167-2170 29

The two most potent inhibitors were

Compound 6 (B43)

Compound 8


The effect of B43 on the conformation (P-loop) of the ATP-binding pocket of BTK

Future Med. Chem., 2014 6(6), 675-695 33

Making B43 a covalent inhibitor: The discovery of Ibrutinib

ChemMedChem 2007, 2, 58-61 Future Med. Chem. 2014, 6 (6), 675-695

In an attempt to increase the potency of B43, the surroundings of the ATP pocket were considered There are 6 cysteine residues in the kinase domain of BTK. Two of these are exposed to solvent and can be targeted by covalent inhibitor molecules Of these two, Cys-481 is closest to the ATP-pocket The idea: taking an already-potent, ATP-competitive inhibitor and adding a moiety that would allow it to covalently bond to the Cys-481 residue nearby This was also expected to increase selectivity since only 9 kinases have a homologous cysteine in such proximity of the ATP-binding pocket

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Strategy of drug design

This idea was introduced in 2006 by Celera Genomics This is the first study that specifically aimed at inhibition of BTK A library of similar structures was developed based on compound 6 and compound 8 from the study at BASF (mostly pyrazole versions of the compounds BASF tested) The most potent scaffold was PCI-29732, a pyrzaole version of compound 6

ChemMedChem 2007, 2, 58-61 35

PCI-29732 (“Compound 1”)

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ChemMedChem 2007, 2, 58-61 37

ChemMedChem 2007, 2, 58-61 38

ChemMedChem 2007, 2, 58-61 39

ChemMedChem 2007, 2, 58-61 40

Synthesis of Ibrutinib as patented by Celera Genomics

(Patent) US20080108636 41

An alternative sythetic route

(Patent) WO2008054827 42

Timeline of events

2006 Celera Genomics discovers Ibrutinib 2007 Celera Genomics sells its BTK inhibitor program (amongst others) to

Pharmacyclics 2013 Pharmacyclics obtains FDA approval of Ibrutinib for MCL 2014 Pharmacyclics obtains FDA approval of Ibrutinib for CLL 2015 AbbVie purchases Pharmacyclics for $ 21 billion

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Part III: Current Treatments, Pharmacokinetics & Future Directions

Tony Nastase

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Current MCL Treatment Options R-CHOP – first-line chemotherapy treatment •  Rituximab

–  B-cell CD20 monoclonal antibody •  Cyclophosphamide

–  cross-linking DNA alkylating agent •  Hydroxydaunorubicin

– DNA intercalating agent •  Oncovin

–  Tubulin-binding agent (anti-mitotic) •  Prednisone

–  Corticosteroid •  Varying efficacy, complex side-effects

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Current MCL Treatment Options HyperCVAD - second-line chemo •  Cyclophosphamide – DNA alkylating agent •  Vincristine – mitotic inhibitor •  Adriamycin – antibiotic-derived intercalator •  Dexamethasone – immunosuppressant •  Cytarabine – nucleoside antiviral/anticancer •  Mesna – antioxidant •  Methotrexate – DHFR inhibitor •  Higher efficacy, debilitating side-effects

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Current MCL Treatment Options

•  Chemotherapy – R-CHOP or HyperCVAD

•  Radioimmunotherapy – Monoclonal antibodies + radionuclide 131I, 90Y

•  Chemo + radiation therapy – R-CHOP or HyperCVAD + Total Body Irradiation

•  Targeted therapy –  Ibrutinib – first in class

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PK Optimization PK was not a significant factor during development •  Originally designed as a “proof of concept tool” •  SAR focus: BTK inhibition, kinase selectivity •  Licensed from Celera in its current form

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Pharmacokinetics – ADME Elimination •  T1/2 – 4 – 6 hrs

–  80% fecal, 8% renal –  <1% Ibrutinib excreted

Absorption •  Rapidly absorbed orally

–  Fraction absorbed ~1

•  Tmax = approx. 1 – 2 hours •  “The absolute bioavailability of

ibrutinib has not been evaluated but is likely low due to extensive first pass metabolism.” -Clin. Pharmacol. Rev. (FDA)

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Pharmacokinetics – ADME

Distribution •  Ibrutinib is 97.3% reversibly protein bound1

–  Fraction unbound (plasma) = 0.027 –  Largely human serum albumin

•  BTK is 100% bound throughout treatment2

•  Steady State Vd = 11 L/kg (~683 L)

–  Abs. bioavail. not tested; expected to be low –  Partition Coefficient = 3.97

1Pharmacyclics, Inc. ImbruvicaTM (ibrutinib) capsules for oral use; US prescribing information. 2013. 2Advani RH, et al. J Clin Oncol. 2013 Jan 1; 31(1):88-94. Epub 2012 Oct 8.

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Advani RH, et al. J Clin Oncol. 2013 Jan 1; 31(1):88-94. Epub 2012 Oct 8. 51


Metabolism •  Primarily CYP3A4

–  Concomitant with strong CYP3A4 inhibitors → 24-fold increase in exposure; not recommended

–  Moderate CYP3A4 inhibitors tolerated with

reduced dose

•  Metabolic hot spots –  Terminal aromatic ring hydroxylation –  Pyrimidine ring opening –  Acryloyl epoxidation

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Major Metabolites Active metabolite

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Rationale for Candidate Selection

Chosen due to •  Pre-clinical safety and efficacy in both

lymphoma and autoimmune models •  Potency (IC50 = 0.5 nM) and selectivity for BTK

in a screening panel of kinases. •  Very few kinases contain homologous Cys481

(covalent target) •  One of FDA’s first Breakthrough Therapy

Designation Pathway for refractory MCL/CLL 55

Rationale for Candidate Selection

Basis for kinase selectivity •  Impact of off-target irreversible

kinase inhibition on efficacy/toxicity unknown

•  Fast irreversible BTK binding •  Fast elimination •  Limits off-target reversible

inhibition of other kinases HER = human epidermal growth factor receptor BLK = B lymphocyte kinase ITK = interleukin 2-inducible T-cell kinase EGFR = epidermal growth factor receptor JAK = Janus Kinase

Off-‐Target Kinases

IC50 (nM)

HER4 0.6 BMX 1 TEC 1 BLK 1 ITK 12 EGFR 12 HER2 22 JAK3 22

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Resistance/Toxicity •  Progression-free survival at 26 months

– 96% in previously untreated patients with CLL – 75% in patients with relapsed/refractory CLL

•  Potential mechanisms of primary or acquired resistance to ibrutinib are currently unknown and likely will become an area of research within the next few years, once ibrutinib is more widely used.

•  Some cases of renal toxicity/failure observed with

Ibrutinib therapy (elevation of creatine levels)

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Combination Therapies

•  MEDI4736 (AstraZeneca) + ibrutinib – Phase I –  MEDI4736 blocks signals that help tumors avoid detection by

the immune system •  Bendamustine and rituximab + ibrutinib – Phase III

–  For refractory CLL and SLL treatment •  GAZYVA® (obinutuzumab) + ibrutinib – Phase III

–  For patients with non-Hodgkin Lymphoma (NHL) and CLL/SLL

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Jan 2015

Feb 2014 Nov 2013

Ibrutinib

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