Biomedical technology and increase in life expectancy

Biomedical technology and increase in life expectancy

Biomedical technology and increase in life expectancy

Major successes:

Recombinant technology, production of recombinant proteins, such asEpo, IFN, Growth Hormone, Tpo mimetics, G-CSF and others

Treatments for anemia, viral infections, growth retardation, thrombocytopenia,neutropenia of chemotherapy

Replacement therapy for missing enzymes, such as glucocerebrosidases

Targeted therapy based on mutations/translocations: imatinib in CML

• Epo is used in treatment of anemia of kidney disease and ofchemotherapy induced anemia in cancer- controversy!

• G-CSF is used to mobilize hematopoietic stem cells and for thetreatment of neutropenia in chemotherapy of for severe congenitalneutropenia.

• Thrombopoietin fusion proteins are used for the treatment ofthrombocythopenia in autoimmune thrombocytopenia purpuraand hepatitis C induced thrombocythopenia.

• Interferon alpha is used in leukemia/ cancer treatment, in the treatment of viral infections such as HCV, while interferon betais used in the treatment of relapsing remitting multiple sclerosis.

Proteins as Drugs: The Case of Cytokines

Syed et. al., Nature 1998, 395, 511

Stem cells CFU-GEMM Early BFU-E Late BFU-E CFU-E Red cell

IL3, SF,GM-CSF+/- Epo

IL3, SF,GM-CSF+/- Epo

3 Days

7-9 Days

Colonies from: CFU-GEMM BFU-E CFU-E

Constantinescu et al., Trends in Endocrinology and Metabolism 1999, 10, 18-23

Development of Aranesp

A Novel Highly N-glycosylated Protein with Enhanced Stability- lessfrequent injections

In vivo activity of EPO isoforms

Adapted from Egrie J, et al. Blood. 1997;90:56A. Abstract 243.

EPO isoform

In vivoactivity in mice

0 5

0

15 20 25 30Day of study

Incr

eas

e in

He

mat

ocr

it (%

)

Isoform 14Isoform 13rHuEPO (9-14)Isoform 12Isoform 11Isoform 10Isoform 9Isoform 8Placebo

15

10

25

10

Incr

eas

ing

se

rum

hal

f-lif

e

Incr

eas

ing

re

cep

tor a

ffin

ity

EPO = erythropoietin

5

20

Effect of mutations on EPO bioactivity

>70% active

20%–70% active

<20% active

<2% active

9G8A normal

9G8A increased

9G8A high

Aranesp development strategy

• Introduce N-linked glycosylation consensus sequences (Asn-Xxx-Thr/Ser) into r-HuEPO by site directed mutagenesis

• Identify individual variants that have the desired properties

• Test optimal combinations of variants

Aranesp development questions

• Would the glycan addition be efficient?

• Would the molecules be properly folded and stable?

• Would the ability to stimulate erythropoiesis be retained?

• Would in-vivo activity be increased?

Aranesp: molecular structure

Biomedical technology and increase in life expectancy

Targeted therapy: the example of chronic myeloid leukemia,

HER2-positive breast cancer, EGFR-positive colon cancer

Genomics Informed Medicine

NGS approaches Massive Parallel Sequencing - a very dynamic field-choice of test:

Whole Exome Sequencing WES- only coding regions explored,700 €/sample.

Whole Genome Sequencing WGS-entire genome explored, approx10,000 €/sample.

RNA-seq- determines sequence and levels of gene expression, 800 €/sample.

Chip-seq- determines DNA sequences bound to proteins,700 €/sample

FIRST GENOME SEQUENCING: SANGER METHOD, 1.5 BILLION US $

Myeloproliferative Neoplasm(Bcr-Abl Positive)

First disease with chromosomal translocation tr 9:22, BCR-ABL

First disease with activated tyrosine kinase activity: ABL

First disease with successful targeted therapy: imatinib

First example of resistance emergence to inhibitors: ABL mutations, i.e. T315I

Blast crisis(>20% balsts in blood and marrow

Chronic Myeloid Leukemia

(Accelerated phase)10-20% blasts in blood and marrow

(Chronic leukemia=chronic phase)<5% blast cells in blood and marrow

National Cancer Institute, USA SEER Fact sheets CML

Platelets

Pro-megakaryocyte

BFU-E

CFU-Megamegakaryoblast

CFU-E

Erythrocyte

Reticulocyte

Proerythroblast

CFU-GM

CFU-Mmonoblast

CFU-Gmyeloblast

Pro-monocyteMeta-

megakaryocyte

*Hematopoietic stem cell

CFU-GEMM

peripheral blood

Tissue

Macrophage

Monocyte Segmented

neutrophil

N./E./B.band

Meta-myelocyte

Myelocyte

eosinophil basophil

Normoblast

Bone marrow

Tpo

Tpo

Tpo

Tpo

BCR-ABL*

**

Amplified in CML

*

Y1294

Kinase Activation in BCR-ABL

Adapted from Smith KM, et al. Molecular Cell. 2003;12 :27-37.

ATP

Cat. domain

Y1294 PP

1. Phosphorylation

*Courtesy Prof. John GoldmanImperial College

CGP57148B: 2-phenylaminopyrimidine derivative

N

N

N N

H

N

H

O

N

N

� Potent inhibition of Abl-K, c-kit and PDGF-R

� Salts are soluble in water

� Orally bioavailable

� Not mutagenic

Cellular permeability

No PKC inhibition

TK inhibitory activityStability to hydrolysis

Solubilisation

1992

Courtesy Prof. John Goldman

Imperial College

Bcr-Abl

Y = TyrosineP = Phosphate

Bcr-Abl

ATP

Substrate

PPP

P

Y

Mechanism of action of imatinib – (2000)

Courtesy Prof. John GoldmanImperial College

Substrate

Imatinib

Bcr-Abl

Y = TyrosineP = Phosphate

Bcr-Abl

ATP

Substrate

PPP

P

Y

Mechanism of action of imatinib – (2000)

Cancer. Jun 15, 2012; 118(12): 3123–3127. Huang et al.

Estimations of the prevalence of CML due to success of therapy :

70,000 in 2010,

112,000 in 2020,

144,000 in 2030,

167,000 in 2040

181,000 in 2050.

Specific Inhibition of BCR-ABL in CML, KIT in GIST and PDGFR inLeukemias by Imatinib Derivatives Will Save Millions of Lives Worldwide

Specific Inhibition of BCR-ABL in CML, KIT in GIST and PDGFR inLeukemias by Imatinib Derivatives Will Save Millions of Lives Worldwide

BCR-ABL1 inhibitors (2011):Imatinib, nilotinib, dasatinib, bosutinib

Bosutinib(SK-606)

Nilotinib(AMN107)

Dasatinib Bosutinib(SK-606)

Imatinib(Phos. IC 50)

PDGFR72 nM >

Kit99 nM >

BcrAbl221 nM >

Src>1000 nM

Nilotinib(Phos. IC 50)

BcrAbl20 nM >

PDGFR75 nM >

Kit209 nM >

Src>1000 nM

Dasatinib(Phos. IC 50)

Src0.1 nM >

BcrAbl1.8 nM >

PDGFR2.9 nM >

Kit18 nM

Bosutinib(Phos. IC 50)

Src3 nM >

BcrAbl85 nM >

PDGFR>3000 >

Kit>10000 nM

1. Manley PW, et al. Proc Am Assoc Cancer Res 2007;48:772.2. Weisberg E, et al. Cancer Cell 2005;7:1129.3. Remsing Rix LL, et al. Leukemia 2009;23:477.

Kinase targets of the inhibitors

Threonine

Steric hindrance: Replacement of threonine by isoleucine at 315 (T315I)

Gorre et al, Science 2001

isoleucineThreonine

ImatinibImatinib Imatinib

Isoleucine

Ponatinib (AP 24534)

New Field: Precision Medicine

• Every patient is different : unique sequences.

• Clones are genetically different and co-exist in cancer

• Sub-clones emerge after treatment by selection or by new mutations

• Treatment efficacy depends on genetic make-up

• Sequencing-based prognosis can adjust severity of treatment

Myeloproliferative Neoplasms

Polycythemia Vera(Vaquez 1892)

Essential Thrombocythemia (Epstein and Goedel, 1934)

Myelofibrosis(Heuck 1879)

These 3 syndromes are 5 fold more prevalent

than chronic myelogenous leukemia31

32

The Homologous V617F Mutation Activates JAK1 and Tyk2

JAK2 Inhibitors Also Target Other Kinases and May Induce Resistence

JAK2 Inhibitor Manufacturer Target Clinical Activity IC50, nMCurrent Stage of Clinical Development

INCB018424 (Ruxolitinib, Jakafi)

Incyte / NovartisJAK1, JAK2, JAK3, TYK2

Decreased spleen size, improved quality of l ife, decreased inflammatory cytokine levels. No significant effect on JAK2V617F allele burden. PhaseIII trial evidence of increased survival.

JAK1: 2.7 JAK2: 4.5 JAK3: 322

Approved FDA, EMEA

TG101348 SAR302503

TargeGen / SanofiJAK1, JAK2, JAK3

Dose-dependent reduction in spleen size and leukocytosis, no thrombocytopenia.

JAK1: 105 JAK2: 3 JAK3: 996

Phase 1/2, Phase 3

XL019 ExelixisJAK1, JAK2, JAK3, TYK2

Decreased spleen size only in patients with JAK2V617F or MPL mutations, decreased pruritus, decrease in circulating blasts in peripheral blood.

JAK1: 132 JAK2: 2 JAK3: 250

Development halted

CEP-701 (Lestaurtinib)Cephalon FLT3, JAK2 Decreased spleen size.JAK2: 1 JAK3: 3 Phase 2

SB1518 S*BIOJAK1, JAK2, JAK2

Reduction of leukocytosis, hepatosplenomegaly, and phospho-STAT5.

JAK1: 1276 JAK2: 22 JAK3: 1392

Phase 1

CYT387Cytopia / YM Biosciences / Gilead

JAK1, JAK2Decrease of spleen size, decrease of transfusion requirements (decrease in severity of anemia), broad anti-cytokine effects.

JAK2: 11 JAK1: 18 JAK3: 155

Phase 1/2

JAK2 Inhibitor Ruxolitinib Is Approved for the Treatment of Int 2 and High Risk Myelofibrosis and is Tested in Pancreatic Cancer and Autoimmune Allopecia

SC1

Slide 33

SC1 Title for this slide?Sandipan Chatterjee; 06/06/2013

JAK2 Inhibitor Cures Autoimmune Allopecia

Nature Medicine and Columbia University

Guessing The Future...............

The problem with the future is that:

It is No Longer What It Used to Be