1

Wirkstoffentwicklung gegen Volkskrankheiten -

Neues aus der AD-Forschung

Prof. Hans-Ulrich Demuth, Halle (Saale)

2

Largest University – Biotech complex in former East Germany

The Biocenter - Probiodrug

3

• Prof. Dr. H.-U. Demuth, Diplom-Biochemiker 1978 >170 Paper > 200 Patente• Studium, Promotion, Habilitation in Halle/S.• Ltr. Abt. Wirkstoffbiochemie am

Leibnitz-Instuitut f. Naturstoff-Forschung Jena, 1994-2000• GF F&E Probiodrug GmbH (Vorstand der AG) seit 1997• GF Ingenium Pharmaceuticals GmbH, München, 2007• Clustersprecher Biotechnologie Mitteldeutschland, 2006

• Drittmittel > 15m€ (BMBF, DFG, BMWi, LSA, EU C)

Einleitung/Vorstellung

4

Probiodrug - Integrierte Biopharmazeutische

Forschungsfirma – von Targetvalidierung –> Phase 2

Targetidenti-

fication / validation

Assay / Hit /Lead

MarketClinics

Phase 1

Clinics

Phase 2

Clinics

Phase 3

Pre-

clinics

MolecularBiology

IP

Enzymology PeptideChemistry

MassSpectrometry

MedicinalChemistry Bioanalytics RegulatoryPharmacology

CellBiology

5

Diabetes – Therapeutische Herausforderung

7-10% der Weltbevölkerung ist diabetisch

∼ 95% Typ 2 Diabetes, “Altersbedingter

Diabetes”

Spätfolgen:

Angiopathie (CVK)

Nephropathie (Nierenleiden)

Retinopathie (Augenleiden)

Neuropathie (z.B. Geschwüre)

US-Behandlungskosten:

>100 Milliarden $ pro Jahr

6

0

50

100

150

200

250

300

350

400

-50 0 50 100 150 200

time after glucose challenge (min)

blo

od

glu

co

se

(m

g/d

l)

Diabetes – Glukose Last

Blut Glukose nach OGT (75g Glukose)

Lange Nacht der Wissenschaften 1. Juli 2011 – Hans-Ulrich Demuth

7

Neuer Wirkungsmechanismus

DP 4 – Hemmung verbessert die Glukosekontrolleakut(chronisch)

� Insulin

� GlukoseDünndarm

LeberMagen

Bauchspeicheldrüse

GIP1-42

GLP-17-36 amide

GIP3-42

GLP-19-36 amide

DPIV

(�)

8

Humanversuche: GLP-1

DP 4-Hemmung verstärkt die GLP-1 Aktivität in Patienten

0 100 200 3000

10

20

30

P32/98

Placebo

Zeit (min)

Ko

nze

ntr

atio

n G

LP

-1 (

pm

ol/l

)

Lange Nacht der Wissenschaften 1. Juli 2011 – Hans-Ulrich Demuth

9

Humanversuche: Insulin

n=24

0 60 120 180 240 3000

200

400

600P32/98

Placebo

Zeit (min)

Pla

sma

In

sulin

(p

mo

l/l)

*

*

*

*

DP 4-Hemmung verstärkt die Insulinreaktion in Patienten

10

0 60 120 180 240 3005

10

15

20

25

PlaceboP32/98

*

Zeit (min)

Pla

sma G

luko

se (

mm

ol/l

)

Humanversuche: Glukosegleichgewicht

DP 4-Hemmung normalisiert die Blutzuckerwert in Patienten

11

Die „süße“ Revolution in der Diabetes Therapie

• Entdeckung 1994 –

• Patent 1996

• Humanstudien seit 2000

• Patientenverfügbarkeit 2006/2007

Entwicklungszeit ca. 12 Jahre

Januvia®, Janumet® – Merck & Co. (MSD) Galvus® - Novartis

12

2004 - After 7 Years PBD Track Record in T2D

Innovative target identification and drug development

Now up to 40 companies develop inhibitors of DP 4 for type 2 diabetes

All depend on Probiodrug (Prosidion Ltd.) patents

DP 4-inhibitors have a predicted turnover of 2billion US$ in 2010

13

Refokussierung 2004 –„Me too“ war das Motiv

024681012141618202224262830323436384042

Probiodrug

Novartis AGNovo FerringTufts University

BMS

Merck & Co.

GSK

RochePointServierG

uilfordDana-FaberAm

gen Inc.FondatechEisaiPfizerSanofiKyowa Hakko

Welfide

BI TanabeTaisho

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Morbus Alzheimer

Fakten & Zahlen 2011 (USA)

Todesfälle zwischen 2000 und 2008~ 5.4 Millionen Fälle in den USA

– 1 von 8 über 65 Jahre alter Personen– 1 von 2 über 85 Jahre alter Presonen

– 200 000 früher Beginn (~ 35-50 Jahre)

– 6. führende Todesursache– Überlebensrate 4-8 Janre nach Diagnose– 40% der Zeit in schwerem Stadium– 75% Pflege zu Hause– $183 Milliarden Gesundheitsausgaben

15

Morbus Alzheimer

2000199919981967

William Utermohlen: Diagnosis in 1995

16

A specific pattern in Molecular Neuroimaging

[18F]-DG-TEP

Pooled sensitivities and

specificities (9 studies) of

86% for temporo-parietal

hypometabolism

(Patwardhan, 2004)

PET-PiB. Increased radio-ligand

retention in AD compared

to control subjects

(Klunk, 2004)

17

“Unmet medical need” AD – Amyloid-Cascade

Interkalators

- failed

Active or

passive

Immunisation

- failed, continued

Blocking Aβ-Formation

- failed, but continued

D‘Andrea, 2004

18

Classical amyloid hypothesis. Plaques trigger neuron loss –

true?

There is no simple correlation between

plaques and severity of the Alzheimer‘s

syndrome

There is no simple correlation between

plaques and severity of the Alzheimer‘s

syndrome

Source: Alex P.

Osmand

Amyloid Plaques

NeurofibrillaryTangles

19

DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIAT

ββββ-secretase γγγγ-secretase

Aβ1 Aβ11 Aβ40 Aβ42Human plaques contain

significant amounts of

N-truncated and

modified Aβ-peptides

Amyloid Precursor Protein

Modified pE-driven Amyloid Hypothesis

• Most abundant in AD brain deposits• Seeding aggregation• Toxic mixed oligomer formation –

neuronal loss• Correlates with cognition deficites in

tg mice

20

Major Aββββ species in sporadic AD – up to 50%

pGluAβ = pE-Aβ - is Abundant in AD Deposits

Paper Sample Preparation Analytical

Method

% pGluAββββ

Mori et al.,

1992

J. Biol. Chem.

FA-soluble fraction, Endo-Lys Digestion,

PGAP-Digestion

HPLC, MS Plaques: 15-20 %

Kuo et al.,

1997

BBRC

Separation of plaques and vasculature, SDS

wash followed by FA extraction, Tryptic

digestion and separation by RP-HPLC

HPLC, MS Plaques: 51 %

Vessels: 11 %

Harigaya et al.,

2000

BBRC

Grey matter dissolved in 70 % FA without

previous Triton or SDS extraction

Western

Blotting

ELISA

Plaques 23-26 %

Aββββ3(pE)-42

Vessels: 4-20 %

Sergeant et al.,

2003

J. Neurochem.

FA extraction followed by 2D PAGE and MS

analysis

SDS PAGE, MS approx. 16%

in total brain tissue

Günthert et al., 2006

Neuroscience

Laser Dissection of Plaques

Solubilization using FA

Laser Dissection,

MS

Plaques: 15-45 %

Wu et al.,

2010

SfN, San Diego,

Poster 457.30/M10

New pGluAbeta specific pAβ/ELISA/methods

Soluble Aβ was 11% NA, 28% AD-brain,

insoluble 47% but of Aβ42 more than 95% is

pEAβ42

ELISA, IP &

SELDI-TOF-MS

Plaques: 47 %

21

pE-Aββββ

Selective Hippocampal Neurodegeneration in Transgenic Mice

Expressing Small Amounts of Truncated Aβ is Induced by

Pyroglutamate-Amyloid-β-Formation

Alexandru et al., Sep 2011, J Neuroscience

pE-Aβ -Formation triggers Neuronal Loss and Neuroinflammation

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� Acyltransferase, Zn dependent, 42 kDa, 329 aa, two isoenzymes

� Located in neuroendocrine tissues, secretory tissues & B-lymphocytes

� Catalyzes the cyclization of N-terminal glutamine & glutamate residues

• Decreased proteolysis

• Increased hydrophobicity

• Increased tendency to aggregate

O

NH

H2N

NH2O

peptide

O

NH

peptide

HN

- NH3

O

NH

H2N

OHO

peptide

O

NH

peptide

HN

- H2O

Gln pGlu

Glu pGlu

O

Oslow

Glutaminyl Cyclase (QC) is Responsible for pE-Formation

23

QC / isoQC are Part of a Vicious Cycle in AD

QC

Aβ3-40/42

Aβ3(pE)-40/42

APP

Aβ1-40/42

Oligomers, ADDL

Neuronal Dysfunction

Cell loss, Dementia

Upregulation of QC and substrates (e.g. CCL2)

Early Inflammatory response

Pro-CCL2(unstable)

pGlu-CCL2

(stable)

isoQC

Stimulus- Aβ- Lesion

24

Significant reduction of pGluAβ and Aβ deposits in 5xFAD/QCKO mice

compared to 5xFAD and 5xFAD/hQC

Significant reduction of pGluAβ and Aβ deposits in 5xFAD/QCKO mice

compared to 5xFAD and 5xFAD/hQC

- 5xFAD * - Crossbreeding with QC-transgenic and QC-knock out animals

- age 6 months

B: Percentage of cortical area occupied by total Aβ-(top) or p-Glu-Aβ (bottom) deposits

Genetic Target Validation in Transgenic Mice

* 5xFAD: APPSwFlLon,PSEN1*M146L*L286V)6799Vas/Mmjax

25

Gliosis in Hippocampus after Overexpression of pE-Aβ

WT Aβ/pGlu-

Aβ

TBA2.1 (3 months): Double IF Aβ-6E10 / pE Aβ (red) & GFAP (green) in the hippocampal CA1 region; neurons blue: DAPI nuclear stain

6395

20

x

40

x

6395

6393

20x

HOM pGlu-Aβ

40x

6393

HOM Aβ

20x

6233

40x

6233

26

Aβ3(pE)-40 – Low solubility, rapid aggregation

3 4 5 6 7 8 9 100

100

200

300

400

500

600

700

800

900

1000

1100

Aβ3(pE)-40

Aβ1-40

pH

Pro

tein

[µ

g/m

l]

Solubility of Aβ

Aβ3(pE)-40 Aβ1-40

Schilling et al, Biochemistry 2006, Schlenzig et al, Biochemistry 2009

Aggregation at pH 8.0

0 50 100 150 200 250 3000

100

200

300

400

500

600

Aβ1-40

Aβ3-40

AβpE3-40

0 50 100 150 200 250 3000

100

200

300

400

500

600

Aggregation at pH 7.0

h

h

27

Tau mediated, oligomeric Aβ/pGluAβ induced cell death

E17-18 WT

Neurons

E17-18Tau

KO

Neurons

WT Glia

Vehicle 1-42 pE3-42 5% pE

Treatment with 0.5 µM amyloid oligomers for 24 hours.

Assayed live with calcein-AM

Treatment with 0.5 µM amyloid oligomers for 24 hours.

Assayed live with calcein-AM

Collaboration

with G. Bloom,

C. Glabe,

submitted 2011

28

pE-Aβ induced Aβ oligomers memorize initial templates

29

Oral Treatment with a QC Inhibitor Decreases AβLoad and Improves Cognition

0 4 7

Time (months)

treatment 3-month oral treatment with a QC inhibitor in a

huAPP transgenic AD model

30

Number of

compound

classes *

Compounds

active with nM &

pM binding

constants

>8 >250

Inhibitor

scaffolds

explored

> 50

Metal binding

groups

explored> 100

MBGScaffold

Compounds

synthesized>1500

Structure of

QC inhibitors

hQC compound development status

31

APPSL/hQC: Treatment effect of PQ50 and PQ529

- APPSL/hQC treatment 2-8

months of age, mixed

gender

- Control, verum (PQ50 7.2

g/kg and PQ529 4.8 g/kg

food pellet)

- Analysis of total Aβ (left)

and pEAβ3-42 (right)

- Data presented as mean ±±±±SEM,

one-way ANOVA followed by

Dunnett‘s

n=7-8*

SDS, FA combined

FA

*

PQ529-significant reduction of total Aβ and pGluAβ(3-42), PQ50-strong

trend

PQ529-significant reduction of total Aβ and pGluAβ(3-42), PQ50-strong

trend

32

� Ligand-based virtual screen in MBG-focused libraries

� High-resolution crystal structure of QC, co-crystal structures with 5 inhibitors

� Robust SAR established for structurally distinct lead- and follow-on series

� Lead candidate PQ912:

� Next candidate PQ1228 is approx. 12 months behind (in clinics 2012)

Lead Compound PBD-PQ912 in Phase 1

� Conforms with Lipinski rules

� No (or acceptable) activity against CYPs

� AMES, hERG and PAN lab assays are clean

� High potency with excellent PK properties and

brain availability

� IND enabling GLP tox in two species

� FIH studies initiated, data Q4 2011

33

� Probiodrug owns 50 patent families, >500 applications overall

� 41 patent families alone in QC (AD & inflammation) addressing medical use and composition of matter

� Medical use of QC inhibitors in AD granted worldwide� First patent issued in US in 2008

Claim 1: A method of treating Alzheimer's disease (by)CadministeringCan inhibitor of glutaminyl cyclase ...

Claim 15: A method of inhibiting conversion of N-terminal glutamic acid or glutamine residues to pyroglutamic acid residues C

QC Inhibitors and Methods of Use are Covered by a Strong, Exclusive Patent Portfolio

COM

Animal models

Genes

Diagnostics

Medical use

QC Patent Families

34

Halle (Saale) – in der Mitte Deutschlands

35

Probiodrug-Ingenium Teams – Halle & München

Halle a.d. Saale München

Lange Nacht der Wissenschaften 1. Juli 2011 – Hans-Ulrich Demuth

36

Globales Netzwerk

GERMANY

• Probiodrug AG, Halle (Saale

• University Erlangen, ENU, Prof v Hörsten

• University Göttingen, Profs Bayer & Tittmann

• University Halle, Prof Stubbs

• University Leipzig, PFI, Prof Rossner

• IfN Magdeburg, Prof Reymann

• Butenandt Institute Munich, Prof Haass

• LMU, Ctr Neuropathology, Prof Herms

• University Osnabrück, Prof Brandt

• Ingenium Pharma. GmbH, Martinsried

University of Virginia