Crizotinib (Xalkori®)

A Small Molecule Tyrosine Kinase Inhibitor By: Graham McLaren Cancer Overview

11 Lung Cancer

ALK+ EML4-ALK Fusion (~2-7% of NSCLC) Small Cell Lung Adenocarcinoma Cancer (15%) (~50%) Other Oncogene Lung Cancer Cancers Non Small Cell Squamous-Cell Lung Cancer Carcinoma (NSCLC) (85%)

Large-Cell Lung Carcinoma

9 Non Small Cell Lung Cancer

• Non Small Cell Lung Cancer (NSCLC) is the most common cause of cancer-related deaths • EML4-ALK Fusion present in ~2-7% of NSCLC cases – 10,000 new cases per year in the United States alone – Most common in younger, never smokers

9 Overview

• Fights ALK+ Cancers – EML4-ALK oncogene (NSCLC) • Translocation in short arm of 2 • Encodes a protein that can undergo ligand independent dimerization – Is therefore constitutively active and unregulated – ALK-NPM (ALCL) • Present is anaplastic large cell lymphoma • Other tyrosine kinases – c-MET • Competitively binds to ATP binding site – Shuts down pro-survival second messenger signaling Background on EML4-ALK

• A translocation on the short arm of • Fusion between: – The N-terminal end of the echinoderm microtubule- associated protein-like 4 (EML4) • Several truncations observed – Always contains coiled-coil domain – Everything downstream of intron 20 of the anaplastic lymphoma kinase (ALK) gene • encodes the entire intracellular tyrosine kinase domain • EML4-ALK fusion is an oncogene

1,4 EML4-ALK Translocation

10 What does ALK do?

• The function of ALK in healthy individuals is not fully understood. – May play an important role in embryonic neurogenesis. • It is present in high levels in utero and shortly after birth but then declines to very low levels that are maintained throughout adulthood. – May be partially responsible for the regeneration of axons in damaged motor neurons.

1,8 The ALK Tyrosine Kinase Makes a Good Target • Vulnerable – Attacking it has anti-tumor effects • Not necessary (in mice at least) – ALK knockout mice (mice without any ALK ) were found to be viable and fertile. • If ALK proteins serve few functions in adults then inhibiting healthy ALK proteins should produce few side effects • Unintended side benefits – ALK knockout mice tended to perform better than their wild type counterparts in experimental models of clinical depression. • Possible research into ALK inhibitors as an antidepressant.

1 What does EML4-ALK do?

• The EML4-ALK fusion gene codes for a protein called, cleverly, the EML4-ALK protein. – Oncoprotein – Is capable of using EML4’s coiled-coil domain to dimerize proteins and constitutively activate ALK’s tyrosine kinase domain. • Type of trans-autophosphorylation • No ligand is needed

6 11 Signaling Pathways

• 3 main, slightly disputed pathways – Ras/Raf/MEK/ERK pathway (agreed) – PI3K-AKT pathway (disputed) – STAT3 pathway (disputed) Ras/Raf/MEK/ERK pathway

• Long cascade that ends with the phosphorylation on of extracellular signal- regulated kinase (ERK) • Phosphorylation of ERK is responsible for: – Increased cell proliferation – Decreased apoptosis

6,8 PI3K-AKT pathway

• Phosphorylation and activation of Protein Kinase B (PKB or AKT) – Triggers other signaling cascades • Increase growth • Increase proliferation • Evade apoptosis

1 11 STAT3 pathway

• Abridged JAK/STAT pathway • When active, the STAT3 pathway induces the production of a protein called ‘survivin.’ – Survivin prevents apoptosis by inhibiting caspase proteins – Allows cells to live longer than healthy cells

6,8 6 Crizotinib

Crizotinib Facts Crizotinib Structure • Chemical Formula

– C21H22Cl2FN5O • IUPAC Name – 3-((R)-1-(2,6-dichloro-3- fluorophenyl)ethoxy)-5-(1- (piperidin-4-yl)-1H-pyrazol-4- yl)pyridin-2-amine • Molecular Weight – 450.34 g/mol

9 Where did Crizotinib come from?

• Discovered by accident… kind of – Pfizer was working on a c-MET inhibitor • c-MET is another tyrosine kinase receptor – Found a drug with great pharmacodynamics but poor pharmacokinetics – Pfizer continued to tweak the drug until they came up with crizotinib – Crizotinib binds to the lipophilic ATP binding pocket of RTK – Marketed as Xalkori®

3 Crizotinib Timeline

• Crizotinib was developed in 2005 • Target was discovered in 2007 • Target validated in 2009 • FDA approved in 2011

5,6 Cost

• Costs $9,600 per month without insurance • With insurance, co pay can be as low as $30 Why Crizotinib works

• Binds efficiently (IC50 = 20nM) • The 2-aminopyridine NH group forms a Hydrogen bond with the hinge residue Glu 1197 while the ring nitrogen hydrogen bonds with the hinge residue Met 1199. • The alpha-methyl group aids the structure of the benzyl group and makes favorable hydrophobic interactions in the lipophilic pocket. – Only the R configuration will fit.

3,4 2 2 What that all means. ATP CANNOT BIND 6 No ATP, No Autophosphorylation

• With the EML4-ALK protein no longer active, the aforementioned signaling cascades are all inhibited. • This leads to decreased cell proliferation and increased apoptosis. • Clinically, an objective response rate (ORR) of 55% was seen with 3 complete responses. (n=255)

4,9 Shortcomings

• Crizotinib works very well, very quickly… – 80% with objective response saw improvement within the first 8 weeks • But, it also looses its effectiveness quickly. – The average length of response was about 42 weeks. – Resistance formed through secondary mutation • Usually consists of a larger amino acid residue being substituted into the pocket, preventing crizotinib from binding • Author’s speculation that the cleft in which the 3-benzyloxy group sits would be a fitting place for the substitution.

9 Pharmacokinetics

• Administration – 250mg taken orally, twice daily • Absorption – Peak plasma levels are reached in 4-6 hours after dosing • ~131ng/mL after a single dose – Steady state is reached in about 15 days • ~256ng/mL minimum – The bioavailability ranged from 32-66% – Absorbs best at low pH

4 Pharmacokinetics

• Distribution – The VD is 1772 L • Indicative of extensive uptake by tissues • Of the drug left in circulation, 91% was bound to plasma proteins – Half life ~42 hours • Metabolism – Primarily metabolized by chytochrome P450 (CYP3A4/5) • Oxidized the piperidine ring, • Dealkylation – Conjugation

4 Pharmacokinetics

• Elimination – 63% through feces • 53% unchanged – 22% through urine • 2.3% unchanged

4 Side Effects

• Most are manageable • Over half (62%) report low grade visual disturbances – Light trails, vitreous floaters and blurred vision • GI disturbances were generally low grade – Nausea, diarrhea, vomiting and constipation • 1.6% of patients reported life-threatening interstitial lung disease • Cases of hepatotoxicity (7%)

4 Contraindications

• Avoid taking crizotinib with any CYP3A substrates, inducers or inhibitors • Avoid taking crizotinib with anything that can raise the stomach pH – Antacids – Crizotinib is best absorbed at low pHs • Because crizotinib is such a new drug, many drug interactions are untested

4 Why am I interested?

• My mom has been taking crizotinib since New Years • First diagnosed in 2006 – stage 3 adenocarcinoma – Surgery, chemo and radiation put it into remission • Scan shows masses in brain Fall 2010 – Erlotinib (Tarceva) and whole brain radiation shrunk both • Stage 4 diagnosed December 2012 after finding her sacrum full of disease – Palliative radiation to the area relived pain and crizotinib was started early January 2013 – Currently doing well – Bought a dog on Tuesday (23 April 2013) Acronym Table

Acronym Meaning NSCLC Non-Small Cell Lung Cancer ALK Anaplastic Lymphoma Kinase EML4 Echinoderm Microtubule-Associated Protein-Like 4 TKR Tyrosine Kinase Receptor ALCL Anaplastic Large Cell Lymphoma NPM Nucleophosmin References

• 1 - Ardini, E., P. Magnaghi, P. Orsini, A. Galvani, and M. Menichincheri. "Anaplastic Lymphoma Kinase: Role in Specific Tumours, and Development of Small Molecule Inhibitors for Cancer Therapy." Cancer Letters 299.2 (2010): 81-94. • 2 - "Biological Macromolecular Resource." RCSB . Web. 03 Apr. 2013. • 3 - Cui, J. Jean, Michelle Tran-Dubé, Hong Shen, Mitchell Nambu, Pei-Pei Kung, Mason Pairish, Lei Jia, Jerry Meng, Lee Funk, Iriny Botrous, Michele McTigue, Neil Grodsky, Kevin Ryan, Ellen Padrique, Gordon Alton, Sergei Timofeevski, Shinji Yamazaki, Qiuhua Li, Helen Zou, James Christensen, Barbara Mroczkowski, Steve Bender, Robert S. Kania, and Martin P. Edwards. "Structure Based Drug Design of Crizotinib (PF-02341066), a Potent and Selective Dual Inhibitor of Mesenchymal– Epithelial Transition Factor (c-MET) Kinase and Anaplastic Lymphoma Kinase (ALK)." Journal of Medicinal Chemistry (2011): 110818151402053. • 4 - Curran, Monique P. "Crizotinib in Locally Advanced or Metastatic Non-Small Cell Lung Cancer." Adis Drug Profile 72 (2012): 99-107. • 5 - Gandhi, Leena, and Pasi A. Janne. "Crizotinib for ALK- Rearranged Non– Small Cell Lung Cancer: A New Targeted Therapy for a New Target." National Center for Biotechnology Information. U.S. National Library of Medicine, 30 Apr. 2012. Web. 03 Apr. 2013. References

• 6 - Okamoto, Isamu, and Kazuhiko Nakagawa. "Echinoderm Microtubule- Associated Protein-like 4-Anaplastic Lymphoma Kinase-Targeted Therapy for Advanced Non-Small Cell Lung Cancer: Molecular and Clinical Aspects." Cancer Science 103.8 (2012): 1391-396. • 7 - Pillai, Rathi N., and Suresh S. Ramalingam. "The Biology and Clinical Features of Non-Small Cell Lung Cancers with EML4-ALK Translocation." Current Oncology Reports 14 (2012): 105-10. • 8 - Roskoski, Robert, Jr. "Anaplastic Lymphoma Kinase (ALK): Structure, Oncogenic Activation, and Pharmacological Inhibition." Pharmacological Research 68 (2013): 68-94. • 9 - Shaw, Alice T., Uma Yasothan, and Peter Kirkpatrick. "Crizotinib." Nature Reviews 10 (2011): 897-98. • 10 - Soda, Manabu, Young Lim Choi, Munehiro Enomoto, Shuji Takada, and Yoshihiro Yamashita. "Identification of the Transforming EML4–ALK Fusion Gene in Non-small-cell Lung Cancer." National Center for Biotechnology Information. U.S. National Library of Medicine, 02 Aug. 2007. Web. 03 Apr. 2013. • 11 - Wecker, Lynn, Lynn M. Crespo, and Theodore M. Brody. Brody's Human Pharmacology: Molecular to Clinical. Philadelphia, PA: Mosby/Elsevier, 2010. Print.