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Wirkstoffentwicklung gegen Volkskrankheiten -
Neues aus der AD-Forschung
Prof. Hans-Ulrich Demuth, Halle (Saale)
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Largest University – Biotech complex in former East Germany
The Biocenter - Probiodrug
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• 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
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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
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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
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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
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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
(�)
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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
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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
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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
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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
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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
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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
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Morbus Alzheimer
2000199919981967
William Utermohlen: Diagnosis in 1995
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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)
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“Unmet medical need” AD – Amyloid-Cascade
Interkalators
- failed
Active or
passive
Immunisation
- failed, continued
Blocking Aβ-Formation
- failed, but continued
D‘Andrea, 2004
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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
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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
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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 %
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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
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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
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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
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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
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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
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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
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pE-Aβ induced Aβ oligomers memorize initial templates
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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
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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
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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
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� 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
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� 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
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Halle (Saale) – in der Mitte Deutschlands
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Probiodrug-Ingenium Teams – Halle & München
Halle a.d. Saale München
Lange Nacht der Wissenschaften 1. Juli 2011 – Hans-Ulrich Demuth
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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