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Transcript of Problemi aperti nella scoperta e nello sviluppo di … GARATTINI.pdf · Problemi aperti nella...
Problemi aperti nella scoperta
e nello sviluppo di farmaci
Milano 24 Gennaio 2017
SILVIO GARATTINI
DISCOVERY AND DEVELOPMENT
ARE INTIMATELY CONNECTED
A DISCOVERY IS A PROMISE WHICH
BECOMES A REALITY ONLY
AFTER DEVELOPMENT
Post-marketing
surveillanceRegistration
Pre-clinical
Clinical
synthesis
Lead compound
structure
optimization
screening
Drug candidate
Chemical library
Chemistry
Formulated pre-drug
Prospective drug
Drug
validation
proof of concept
delivery
side effectsToxicity
(acute, muta, chronic, embryo, organ)
Pharmaco-economics
Phase I
Phase II
Phase III
Classic
Combinatorial
Target oriented
Function oriented
High-throughput
Efficacy
(models: in vitro)
Formulation
Mechanism of Action(models: in vitro, in vivo)
PK(ADME)
Efficacy(models: in vivo)
Dose, PK, PD
Efficacy
Superiority
Translation
Health Policy
Concept Target definitionTarget
(diseases, function,
cell type, pathway, molecule)
Field Action Modality Product
Variable
Mario Negri
Registered pharmaceutical
DRUG DISCOVERY AND DEVELOPMENT
• THE PRESENT SITUATION
Scannell et al., 2012
DRUG DISCOVERY AND DEVELOPMENT
• THE PRESENT SITUATION
• OPEN PROBLEMS
• UNMET NEEDS OF PATIENTS
Stiggelbout et al., 2012
53 ANTITUMORALI
Salas-Vega et al., 2016
FDA
EMA
OVERALL SURVIVAL (OS)
24
23 OS > 3 mesi
6 OS < 3 mesi
NICE 17 (UK)
HAS 10 (Fr)
PBAC 06 (Australia)
NESSUN EFFETTO
OPPURE OS NON
DETERMINATO
DRUG DISCOVERY AND DEVELOPMENT
• THE PRESENT SITUATION
• OPEN PROBLEMS
• UNMET NEEDS OF PATIENTS
• NEED TO HAVE BETTER TEST PREDICTIVE
OF CLINICAL EFFICACY
Drug screening: a change in paradigm
Historically, drugs were discovered through identifying
the active ingredient from traditional remedies or by
serendipitous discovery. Later chemical libraries were
screened in intact cells or whole organisms (functional
screening) to identify substances that have a desirable
therapeutic effect.
FROM OLD APPROACHES…..
ISOLATED
HEART
COMBINATION OF TARGET AND FUNCTION
RECEPTOR b-ADRENERGIC
ANTAGONIST
BRADYCARDIA
CYCLOOXYGENASE
ASPIRIN
TROMBOXANE
PLATELETS
COMBINATION OF TARGET AND FUNCTION
FROM OLD APPROACHES…
Drug screening: a change in paradigmSince sequencing of the human genome, which allowed rapid
cloning and synthesis of large quantities of purified proteins, it has
become common practice to use high-throughput screening of
large compounds libraries against isolated biological targets which
are hypothesized to be disease modifying in a process known as
reverse pharmacology (targeted screening). Hits from these
screens are then tested in cells and animals for efficacy. Even more
recently, scientists have been able to understand the shape of
biological molecules and to use that knowledge to design drug
candidates.
UNDERSTAND THE DISEASE
• UNRAVEL THE UNDERLYNG CAUSE
genes, rnas, proteins, enzymes
neuromediators, immunomediators
metabolites
RETT SYNDROME
COMPRENDERE LA MALATTIA
• RICERCARE LA CAUSA
geni, rna, proteine, enzimi
immunomediatori
metaboliti
Chahrour and Zoghbi, 2007
TARGET
IDENTIFICATION
• TARGET IS A SINGLE
MOLECULE “DRUGABLE”
IDENTIFICAZIONE
DEL TARGET
• TARGET E’ UNA SINGOLA
MOLECOLA POTENZIALMENTE
SVILUPPABILE COME FARMACO
2007 Mecp-2 12 targets
2012 Mecp-2 33 targets
2002 Mutants Mecp-2 3 mouse models
2012 Mutants Mecp-2 12 mouse models
PROGRESS MADE IN
TRANSLATIONAL RESEARCH
TARGET VALIDATION
• TARGET MUST BE
RELEVANT FOR THE
DISEASE, THROUGH STUDIES
IN SILICO, IN VITRO, IN VIVO
VALIDAZIONE DEL TARGET
• IL TARGET DEVE ESSERE
RILEVANTE PER LA MALATTIA
ATTRAVERSO STUDI IN SILICO,
IN VITRO, IN VIVO
Chahrour and Zoghbi, 2007
• IN SILICO, IF THE STRUCTURE OF THE
TARGET IS KNOWN
• FROM NATURAL PRODUCTS BY
EXTRACTING THE ACTIVE PRINCIPLE
• DE NOVO SYNTHESIS
• HIGH-THROUGHPUT SCREENING
• GENETICALLY ENGENEERED
BIOLOGICAL MOLECULES
PROCEDURES TO FIND
A LEAD COMPOUND
DRUG DISCOVERY
• FIND A “LEAD COMPOUND”
SCOPERTA DEL FARMACO
• TROVARE UN PRIMO PRODOTTO
ATTIVO
Normal Dysfunction Loss
JNK role in
spine injuryProcesso di deterioramento delle spine dendritiche
JNK regola il deterioramento sinaptico ed
il mantenimento della funzionalità
PSD-95 < PSD-95 << PSD-95
JNK JNK
Sclip A, Antoniou X, Colombo
A, Pozzi L, Ploia C, Feligioni
M,, Cardinetti D, Veglianese P,
Balducci C, Cervo L, Costa C,
Tozzi A, Calabrese P, Forloni
G, Borsello T. Chronic
inhibition of JNK prevents
synaptic failure and
cognitive impairment in
Alzheimer’s Disease.
JBC 2011
D-JNKI1: a specific JNK inhibitorMolecola
inibitrice
della proteina
JNK
PRECLINICAL TESTING TEST PRECLINICI
ComplementLectin PathwayActivation
MBL targets
• MBL circulates associated with serine
proteases (MASPs)
• MBL acts as a soluble pattern
recognition receptor (PRR)
• MBL targets include:
• pathogen-associated molecular patterns
(PAMPs). i.e. carbohydrates exposed on the
surface of a pathogen
• “altered self” or damage-associated
molecular patterns (DAMPs), i.e. dying or
damaged cells through recognition of
changes in glycosylation pattern on the cell
surface
• Binding of MBL to its targets leads
to complement activation
• MBL is a circulating serum protein
made by the liver
MBL
Non ischemic vessels
MBL immunostaining
in the ischemic brain
(MBL, Vessels, Nuclei)
MBL is deposited on brain vessels after ischemia
- Polyman-2 binds MBL with high affinity
- the binding is dose-dependent
Binding studies by surface plasmon resonance
Polyman-2 was selected for the in vivo studies
Orsini et al., 2012
Polyman-2 induces a significant reduction in neurological deficits and
ischemic volume with a wide therapeutic window
Effect of Polyman-2 in ischemic mice (30 µM, iv)
neurological deficits
infarct volume
48h
tMCAo
0’ 30’3h 6hor 18h 24hor or 30hor
Mean ± SD, n=7-12, One way ANOVA
followed by Dunnett test; ***p<0.001, **p<0.01, *p<0.05
63%
47%
Orsini et al., 2012
OLD NEW
FUNCTIONAL MOLECULAR
SCREENING SCREENING
EFFECT ON EFFECT ON
FUNCTION TARGET
MECHANISM OF EFFECT ON
ACTION FUNCTION
Pros• Robust: by definition, the cellular, ex vivo or in vivo model is representative of
the target pathology
• Effective: screening programs are likely to result in the identification of
significant pharmacological hits
• Process-oriented: screening tests do not require knowledge on the underlying
molecular mechanisms and pharmacological targets, target definition generally
follows identification of the hit compound
• Accounting for biological complexity: the approach takes into consideration
biological complexity
Cons• Time consuming: medium - low-throughput screening strategies
• Expensive: it requires previous validation of the models. Cell cultures and
animals are expensive !
Functional screening: pros and cons
Pros• Simple: availability of the molecular target favors design of simple in
vitro screening tests
• Effective: easy and rapid identification of pharmacological hits
• Time saving: high-throughput screening strategies
• Cost effective: the approach cuts down on the costs of hit identification
Cons• Over-simplifying: the molecular target must be an essential
determinant of the pathological process considered, otherwise the
identified pharmacological hits are unlikely to be significant
Targeted screening: pros and cons
DRUG DISCOVERY AND DEVELOPMENT
• THE PRESENT SITUATION
• OPEN PROBLEMS
• UNMET NEEDS OF PATIENTS
• NEED TO HAVE BETTER TEST PREDICTIVE
OF CLINICAL EFFICACY
• UNDERSTANDING THE COMPLEXITY OF
BIOLOGICAL SYSTEMS
Estelamari Rodriguez,
The Molecular Oncology Report
Vol 1, 2007
INHIBITORS
Angiostatin
Cartilage-derived
Endostatin
IFN-a
IFN-b
PAIs
PF4
Prolactin fragment
Proliferin-related
protein
Protamine
Thrombospondin
TIMPs
INDUCERS
Angiogenin
FGFs
G-CGF
IGF-1
Interleukin-8
PD-ECGF
Placenta growth factor
Pleiotrophin; Proliferin
Prostaglandins E1, E2
Scatter factor/HGF;
TAT
TGF-a, TGF-B
TNF-a
VEGF
ANGIOGENESIS SWITCHQUIESCENT VESSELS
Angiogenesis is regulated by a balance between angiogenic factors and
inhibitors. Disruption of this balance and acquisition of angiogenic
potential by tumor cells is called the “angiogenic switch”
HEART MIOCYTES
HER-2
STIMULATES GROWTH
BREAST CANCER
HEART
FAILURE
REDUCED
GROWTH
TRASTUZUMAB
HER-2 ANTIBODY
• PHARMACOKINETICS
ABSORPTION, METABOLISM, EXCRETION
CONCENTRATION AT THE TARGET
ACTIVE METABOLITES
• IDENTIFICATION OF ACTIVE DOSE AND
OPTIMAL DURATION OF TREATMENT
• TOXICOLOGICAL TESTING
DIFFERENT TYPE OF CELLS
MUTAGENESIS ACUTE AND CHRONIC
TOXICITY CANCEROGENESIS
REPRODUCTION TOXICITY
• FROM A MOLECULE TO A PRODUCT
PURITY, STABILITY, OPTIMAL ABSORPTION
PRECLINICAL TESTS
• PHASE 1 HUMAN VOLUNTEERS (20 – 100)
MAXIMAL TOLERATED DOSE
• PHASE 2 PATIENTS (100 – 500)
ESTABLISH PRELIMINARY EFFICACY
• PHASE 3 RANDOMIZED CONTROLLED TRIAL
(1,000 – 5,000) COMPARATIVE
THERAPEUTIC OUTCOME
DRUG APPROVAL BY EMA
• PHASE 4 OPTIMIZATION OF THERAPY
CLINICAL TESTING
DRUG DISCOVERY AND DEVELOPMENT
• THE PRESENT SITUATION
• OPEN PROBLEMS
• UNMET NEEDS OF PATIENTS
• NEED TO HAVE BETTER TEST PREDICTIVE
OF CLINICAL EFFICACY
• UNDERSTANDING THE COMPLEXITY OF
BIOLOGICAL SYSTEMS
• PERSONALIZED MEDICINE
Zucchetti et al., 2012
INHIBITOR OF FGFR-1 and VEGF 1-3 (30mg oral)
DRUG
ABSORPTION
DISTRIBUTION
PROTEIN BINDING
GENETICS
METABOLISM
ACTIVE
METABOLITES
INACTIVE
METABOLITES
TRANSPORT
GENETICSRECEPTORS
TRANSDUCTION
EFFECT
STATE OF TISSUE
OR ORGAN
SUPERFAMILY OF CYTOCHROME P450
CYP 2D6
GENETIC POLYMORPHISM
(78 VARIANTS)
DETERMINES
POOR, INTERMEDIATE, EXTENSIVE, ULTRARAPID
METABOLIZERS
IRINOTECAN
CYP 3A 4/5INACTIVE OXIDIZED
METABOLITES
SN-38
ACTIVE METABOLITE
CA
RB
OX
YL
ES
TE
RA
SE
-2
UGT 1 A 1
SN – 38G
POLYMORPHISM
(UGT A1 * 28/27/6)
REDUCED FUNCTION
INDUCED
HIGH TOXICITY
MALDI imaging
MIN
MAX
Section of a tumour
treated in vitro
Section of a tumour
treated in vivo
MALDI IMAGING MASS SPECTROMETRY
The efficiency of in vitro methods is limited
PTX
nt
5x8
+60mg/Kg
PTX m/z 284.2
NT
PTX m/z 284.2
Tumore della mammella
trattato con Paclitaxel
3D reconstruction of tumors.
Malignant pleural
mesothelioma
3D reconstruction of
tumors.
Melanoma