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