AgeLab Pharma GmbH, Hamburg, Germany *Dermatology Clinic, University Hospital, Eppendorf, Hamburg

Amal K. Mukhopadhyay, Niels Grabe, Karsten Neuber *

Recombinant DNA Technology and Functional Genomics have given us an unprecedented possibility to diagnose, and sometimes even totreat many diseases which were so far categorised as of idiopathic origin.

In the post-human genome era, with nearly the entire human genome sequence being revealed, the proteomics technology has moved to a centre stage in the drug discovery processes.

Growth in Pharma companies has been drivenby the technologies emerging from biotech industries

Targetidentification

Targetvalidation

LeadIdentification

Leadoptimisation

Preclinicaldevelopments

Moving bottlenecks in the discovery value chain

Adapted from Recap.com, L.E.K. analysis

"Life would be infinitely happier if we could only be born at the age

of eighty and gradually approach eighteen."

-- Mark Twain, Autobiography with Letters

Mega-Trend Aging

Predicted changes in the burden of disease from 1990 to 2020

Lower Respiratory infection 1 1 Ischaemic heart diseaesesDiarrheal diseases 2 2 Unipolar major depressionPerintalassociated conditions 3 3 Road traffic accidentsUnipolar major depression 4 4 Cerebrovascular diseasesIschaemic heart diseaeses 5 5 Chronic obstr. lung diseaseCerebrovascular diseases 6 6 Lower respiratory tract inf.Tuberculosis 7 7 TuberculosisMeasles 8 8 WarRoad traffic accidents 9 9 Diarrheal diseasesCongenital anomalies 10 10 HIV

1990 2020

Non melanoma skin cancer

stomach

Bladder

Colon

Prostate

Respiratory tract / Lung

Rectum

Stomach

Uterus

Breast

Non-Hogkin Lymphoma

Melanoma

Kidney/Urinary Tract

Stomach

Ovary

Non melanoma skin cancer

Colon

Rectum

Melanoma

Bladder

Respiratory tract / Lungs

Ten most common cancers in men Ten most common cancers in women

Common Types of Cancers ( Rheinland-Pfalz)

AgeLab: Organspecific Ageing with a focus on skin

Section

Young skin

Section:

Aged skin

Molecular analysis of ageing process Alterungsprozessen

Young Old Sick

Protein-profile

Samples

Difference

Kd

220

9766

45

30

20,1

14,3

Increased

Decreased

1

7

8 9

10

11

12

14

15

16

20

21

22

23

24 25

26

27

28

2930

31

3233

34

35

36

37

38

39

1718

19

40 41

Y

Kd

220

9766

45

30

20,1

14,3

Increased

Decreased

1

7

8 9

10

11

12

14

15

16

20

21

22

23

2425

26

27

28

29

30

31

3233

34

35

36

37

38

39

17

18

19

40 41

O

Examples:

Decreased in Ageing :

Spot 2 and 3 , pI 7.6 and 7.8; Kd 20 ; identified as a CRP-1

Decreased in ageing

Spot 7, pI 6.3 / 32kD) : identified as anEnzyme/ Dehydrogenase

I ncreased in aged prostate :

Spot 17: pI 6.8 / 30 kD ;

Diff erent members of a protein family

Decreased in ageing : Spots 10 (pI 6.0; 25 kD) , 11 (pI 5,5 ; 30kD) , 12 (pI 5.8 ; 18 kD):

Variants of the same proteins ( Enzyme / Reductase )

Spots 14,15,19

Pooled Standard

2D gel co-separationIPGphor, Ettan DALT six

Image acquisitionThyphoon 9400

Cy2/Cy3 co-detection & normalisation

Cy2/Cy5 co-detection & normalisation

Differential In-gel Analysis (DIA)DeCyder Software

3D protein profile

Cy3 Cy5 Cy2

Mix samples

Sample A control

Sample B treated

DIGE Cy-dye labelling

2D sample preparation2D Clean-Up Kit2D Quant Kit

DIGE - experimental workflow

DeCyder differential in gel (DIA) analysis

Result of full automatic spot detection:

- 2153 spots detected- 270 spots decreased*- 170 spots increased*

* at least 2-fold change of max spot volume; no spots excluded by filtering

DeCyder DIA analysis - Example 1

Spot 658 increased, volume ratio 3.93

DeCyder DIA analysis - Example 2

Spot 714 increased, volume ratio 6.02

DeCyder DIA analysis - Example 3

Spot 1685 increased, volume ratio 7.28

The ProteomeWorks „ Workflow“

IMAGING PDQUEST

2-D GELSOR

BLOTS

SAMPLEPREP

ProteinLynx PepSeq

PROTEINDIGEST

STATION

MASS SPECMALDI-TOF

MS/MSESI-MS

SWISS-PROTTrEMBL,etc.HTML Links

SPOTCUTTER

Marker-Proteins for Aging

6 years

6 years 34 years 78 years

39 years

M 1

M 2

81 years

Regulatory Network of

Cells

Systems-BiologyBioinformatics based diagnostics

Protein-Chip

• Minimal-invasive Micro-biopsy for sample collection

• Suitable for epidermal diseases- Dermatitis- Eczema- Epidermal skincancer- Skin cancer precursor cell abnormalities.

New Diagnostic procedure

- Chip is coated with specific antibodies against identified markers

- AgeLab has identified more than 50 different potential marker proteins.

–Apoptosis regulatory proteins (13)–Cell cycle regulatory proteins (9)–Nitric oxide (NO) signaltransduction

regulatory Proteins (5)–Intermediate Filament-Proteins (13) –Chaperones (7) –Tumor Suppressors (8)

Chip-Substrat

AgeLabs diagnostic Protein-Chip

• Tissue sample was labelled with fluorescent dye and after incubation, the chip was scanned in a fluorescence scanner.

Protein Chip Development

Early detection of epidermal skin cancer

• Currently punch-biopsy followed by histology is the only procedure for diagnosis of actinic keratoses, a pre-cancerous stage. The procedure is painful, leaves scar and the outcome is unfortunately not absolutely

dependable. • With the help of AgeLab's procedure which is free from

any scar development, a diagnostic procedure is being offered for the first time which is patients-friendly and dependable results can be expected.

• Early diagnosis of pre-cancerous stage in skin becomes possible with the help of the protein chip based micro-

diagnostic procedure developed by AgeLab.

Euro-India Biotech-Conference, Hamburg, September, 2004

Contact email: am@agelabpharma.de

Expression of Thanks

University of Hamburg

Prof. F.A. Leidenberger

AgeLab GmbH:

Dr. Matthias Koenig

Mr. Björn. Ehlers,

Ms. Monika Kistler

Special thanks are due to Amersham Biotech, Freiburg

and Bio Agency, Hamburg