SCOPERTA E SVILUPPO DEI FARMACI

SCOPERTA E SVILUPPO DEI FARMACI Studi pre-clinici Studi clinici Fase registrativa FASE I (soggetti sani, ~20-80) Richiesta di commercializzazione Scoperta e selezione delle molecole FASE II (pazienti, ~100-200) Valutazione delle autorità sanitarie (EMEA) Studi su animali Richiesta autorizzazione alla sperimentazione FASE III (pazienti, ~1000-3000)

PROGRAMMA DEL CORSO DRUG DISCOVERY DRUG DEVELOPMENT ESEMPI Identificazione e validazione del bersaglio Identificazione di composti guida ottimali High-throughput screening Studio in vitro delle proprietà farmacocinetiche Studi tossicologici preliminari DRUG DEVELOPMENT Studi pre-clinici Modelli in vitro Modelli in vivo (efficacia, tossicità, farmacocinetica) Studi clinici Uso degli animali da esperimento nella ricerca farmacologica e nello sviluppo dei farmaci ESEMPI Modelli sperimentali per lo studio di farmaci antitumorali e antiangiogenici Modelli sperimentali per lo studio di farmaci attivi sul sistema cardiovascolare

DRUG DISCOVERY

Approccio “compound-based” composti naturali

Chemical process for natural product discovery Chemical process for natural product discovery.   The natural product is extracted from the source, concentrated, fractionated and purified yielding essentially a single biologically active compound. Identification of known compounds, thereby avoiding replication of previous efforts, has been greatly aided by directly coupled HPLC-mass spectrometer (LC-MS) systems and natural-product databases57. De novo structure determination of compounds that are novel has been revolutionized by advances in spectroscopic techniques, particularly in high-resolution nuclear magnetic resonance technologies. Although the determination of complex structures is technically challenging, it is no longer a major impasse in the drug discovery process. In those cases in which the biological activity profile meets criteria for potency and selectivity, preliminary structure–activity relationship (SAR) studies are conducted and the purification process is scaled up. Once the feasibility of modulating biological response through synthetic modification is established, the hit is declared a lead and proceeds onward for additional optimization by traditional medicinal chemistry.

Generic scheme for bioassay-guided fractionation Generic scheme for bioassay-guided fractionation.   Several cycles of fractionation are usually needed to obtain a pure compound. Multiple active components can illuminate structure–activity relationships. HP20, a solid-phase adsorber; HPLC, high-performance liquid chromatography; IR, infrared spectroscopy; LC/MS, liquid chromatography/mass spectrometry; NMR, nuclear magnetic resonance; UV, ultraviolet.

Approccio “compound-based” composti naturali analoghi di agonisti endogeni Approccio “target-based”

TARGET DISCOVERY

QUALI BERSAGLI?

QUALI BERSAGLI? • enzimi • recettori • canali ionici; proteine di trasporto • ormoni; fattori di crescita • DNA/RNA; ribosoma; fuso mitotico • meccanismi chimico-fisici di natura diversa

Figure 1 | Overview of molecular- and system-based approaches to target discovery.   Target discovery is composed of three steps: the provision of disease models/tissues, target identification and target validation. The 'molecular' approach (a) uses techniques such as genomics, proteomics, genetic association and reverse genetics, whereas the 'systems' approach (b) uses clinical and in vivo studies to identify potential targets. During validation, modulation of gene expression and/or protein function in both cell and animal models is used to confirm the role of the target prior to passing into the drug discovery pipeline.

Figure 2 | Correlative technologies used in target identification Figure 2 | Correlative technologies used in target identification.   Techniques such as genomics (a), proteomics (b) and genetic association (c) attempt to identify novel targets through the measurement of the differential expression of messenger RNA, protein or genetic polymorphisms in diseased and normal tissue. SDS–PAGE, sodium dodecyl sulphate-polyacrylamide gel electrophoresis

Figure 2 | Correlative technologies used in target identification Figure 2 | Correlative technologies used in target identification.   Techniques such as genomics (a), proteomics (b) and genetic association (c) attempt to identify novel targets through the measurement of the differential expression of messenger RNA, protein or genetic polymorphisms in diseased and normal tissue. SDS–PAGE, sodium dodecyl sulphate-polyacrylamide gel electrophoresis

Figure 2 | Correlative technologies used in target identification Figure 2 | Correlative technologies used in target identification.   Techniques such as genomics (a), proteomics (b) and genetic association (c) attempt to identify novel targets through the measurement of the differential expression of messenger RNA, protein or genetic polymorphisms in diseased and normal tissue. SDS–PAGE, sodium dodecyl sulphate-polyacrylamide gel electrophoresis

Figure 3 | Phenotype-driven target identification Figure 3 | Phenotype-driven target identification.   Technologies such as forward (a) and reverse (b) genetics involve the identification of potential disease targets through modulation of a disease phenotype. Forward genetics involves random modulation of the phenotype using in vitro biological and chemical library screen or in vivo chemical mutagenesis and the subsequent identification of the gene (phenotype to gene), whereas reverse genetics entails gene manipulation and examination of the phenotype (gene to phenotype). ENU, ethylnitrosurea.

Figure 3 | Phenotype-driven target identification Figure 3 | Phenotype-driven target identification.   Technologies such as forward (a) and reverse (b) genetics involve the identification of potential disease targets through modulation of a disease phenotype. Forward genetics involves random modulation of the phenotype using in vitro biological and chemical library screen or in vivo chemical mutagenesis and the subsequent identification of the gene (phenotype to gene), whereas reverse genetics entails gene manipulation and examination of the phenotype (gene to phenotype). ENU, ethylnitrosured.

TARGET VALIDATION Tecniche in vitro

Figure 4 | Overview of the techniques used in target validation Figure 4 | Overview of the techniques used in target validation.   The elucidation of target function can be undertaken using a variety of target-validation techniques. Expression of messenger RNA can be modulated either directly (small interfering RNA (siRNA) or antisense) or at the transcriptional level (peptide nucleic acids (PNA), locked nucleic acids (LNA), zinc fingers or ribozymes). Protein function can be modulated through expression of dominant negative and wild-type protein or using blocking/stimulating antibodies or aptamers.

TARGET VALIDATION Tecniche in vitro Tecniche in vivo

TARGET DRUGGABILITY: definisce la capacità di un bersaglio di venire modulato in modo efficace da un ligando a basso peso molecolare con proprietà biochimico-fisiche e farmacocinetiche appropriate perché possa diventare un farmaco adatto all’impiego terapeutico desiderato

HIGH-THROUGHPUT SCREENING: screening (di una serie di composti) allo scopo di identificare degli “hit” in un saggio in vitro, generalmente effettuato in modo automatizzato.

High throughput screening (HTS) This figure outlines the principles behind three assay types used for screening. The examples illustrate their application to kinase assays, but in all cases the methods are applicable to other target types (Table 1). A standard separation-based assay is depicted in a, in which radiolabelled ATP transfers a labelled phosphate moiety from ATP to substrate. The substrate is subsequently captured on a membrane, and separated from the ATP substrate by filtration. The signal is measured by addition of scintillate and quantitated by counting. b | Outlines the general principles of FRET, a homogeneous assay format. In this case, a labelled substrate is phosphorylated allowing it to bind to a second molecule labelled with an acceptor group. The proximity of the two groups allows energy transfer between the donor and acceptor. This causes a shift in wavelength for the signal emitted from the assay and detection of the product without separation from the substrate. c | Another homogeneous assay format in which the conversion of ATP to ADP is coupled to the conversion of NADH to NAD using two enzymes, pyruvate kinase (PK) and lactate dehydrogenase (LD). This decreases the A340 of the assay, which is used to monitor the progress

High throughput screening (HTS) FRET (Förster or Fluorescence Resonance Energy Transfer)

High throughput screening (HTS) This figure outlines the principles behind three assay types used for screening. The examples illustrate their application to kinase assays, but in all cases the methods are applicable to other target types (Table 1). b | Outlines the general principles of FRET, a homogeneous assay format. In this case, a labelled substrate is phosphorylated allowing it to bind to a second molecule labelled with an acceptor group. The proximity of the two groups allows energy transfer between the donor and acceptor. This causes a shift in wavelength for the signal emitted from the assay and detection of the product without separation from the substrate.

High throughput screening (HTS) This figure outlines the principles behind three assay types used for screening. The examples illustrate their application to kinase assays, but in all cases the methods are applicable to other target types (Table 1). c | Another homogeneous assay format in which the conversion of ATP to ADP is coupled to the conversion of NADH to NAD+ using two enzymes, pyruvate kinase (PK) and lactate dehydrogenase (LD). This decreases the A340 of the assay, which is used to monitor the progress

HIT: composto attivo primario dotato di proprietà di legame non promiscue e che supera un valore soglia predeterminato in un dato tipo di saggio

VERIFICA degli “HITS” re-testing generazione di curve dose-risposta test ortogonali screening secondario “maneggevolezza” chimica Valutazione della proprietà intellettuale Test biofisici Hit ranking & clustering re-testing: compounds that were found active against the selected target are re-tested using the same assay conditions used during the HTS. Dose response curve generation: several compound concentrations are tested using the same assay, an IC50 or EC50 value is then generated orthogonal testing: confirmed hit are assayed using a different assay which is usually closer to the target physiological condition or using a different technology. secondary screening: Confirmed hits are tested in a functional assay or in a cellular environment. Membrane permeability is usually a critical parameter. chemical amenability: medicinal chemists will evaluate compounds according to their synthesis feasibility and other parameters such as up-scaling or costs Intellectual Property evaluation: Hit compound structures are quickly checked in specialized databases to define patentability Biophysical testing: Nuclear magnetic resonance (NMR), Isothermal Titration Calorimetry, dynamic light scattering, surface plasmon resonance are commonly used to assess whether the compound binds effectively to the target, the stoïchiometry of binding and to identify promiscuous inhibitors. Hit ranking and clustering: Confirmed hit compounds are then ranked according to the various hit confirmation experiments.

FASE “HIT-TO-LEAD” Stage-by-stage quality assessment to reduce costly late-stage attrition.   Typical important milestones are VALIDATED HIT SERIES (VHS), LEAD SERIES IDENTIFIED (LSI) and clinical candidate selection (CCS), which ensure that only drug candidates with an appropriately high-potential profile are advanced to the next phase.

LEAD: un composto che fa da prototipo per una data struttura chimica che mostra attività e selettività in uno screening rilevante dal punto di vista biochimico o farmacologico. Di solito costituisce la base per un processo di ottimizzazione e sviluppo, allo scopo di identificare un composto candidato per l’impiego clinico.

FASE “HIT-TO-LEAD” Stage-by-stage quality assessment to reduce costly late-stage attrition.   Typical important milestones are VALIDATED HIT SERIES (VHS), LEAD SERIES IDENTIFIED (LSI) and clinical candidate selection (CCS), which ensure that only drug candidates with an appropriately high-potential profile are advanced to the next phase.

GENERAZIONE DI “LEADS” elevata affinità per il target (< 1 µM) possibilità di modificazione chimica libertà da vincoli di proprietà intellettuale mancanza di interferenza con gli enzimi P450 o con le proteine di trasporto della famiglia ABC mancanza di legame all’albumina del siero solubilità in acqua (> 100 µM) stabilità “druglikeness” capacità di permeare attraverso le membrane cellulari attività biologica significativa in un saggio cellulare mancanza di citotossicità selettività verso altri bersagli correlati have compound members that exhibit a high afinity towards the target (less than 1 µM) show chemical tractability be free of Intellectual property not interfere with the P450 enzymes nor with the P-glycoproteins not bind to human serum albumin be soluble in water(above 100 µM) be stable have a good druglikeness exhibit cell membrane permeability show significant biological activity in a cellular assay does not exhibit cytotoxicity not be metabolized rapidly show selectivity versus other related targets

DRUGLIKENESS Regola di Lipinsky (regola del 5): peso molecolare < 500 cLogP < 5 gruppi donatori di legami H < 5 gruppi accettori di legami H < 10 legami ruotabili <10 Log P = log10 del coefficiente di ripartizione (P) tra fase organica (o) e fase acquosa (a) P = [F]o/[F]a

GENERAZIONE DI “LEADS” elevata affinità per il target (< 1 µM) possibilità di modificazione chimica libertà da vincoli di proprietà intellettuale mancanza di interferenza con gli enzimi P450 o con le proteine di trasporto della famiglia ABC mancanza di legame all’albumina del siero solubilità in acqua (> 100 µM) stabilità “druglikeness” capacità di permeare attraverso le membrane cellulari attività biologica significativa in un saggio cellulare mancanza di citotossicità selettività verso altri bersagli correlati have compound members that exhibit a high afinity towards the target (less than 1 µM) show chemical tractability be free of Intellectual property not interfere with the P450 enzymes nor with the P-glycoproteins not bind to human serum albumin be soluble in water(above 100 µM) be stable have a good druglikeness exhibit cell membrane permeability show significant biological activity in a cellular assay does not exhibit cytotoxicity not be metabolized rapidly show selectivity versus other related targets

GENERAZIONE DI “LEADS” elevata affinità per il target (< 1 µM) possibilità di modificazione chimica libertà da vincoli di proprietà intellettuale mancanza di interferenza con gli enzimi P450 o con le proteine di trasporto della famiglia ABC mancanza di legame all’albumina del siero solubilità in acqua (> 100 µM) stabilità “druglikeness” capacità di permeare attraverso le membrane cellulari attività biologica significativa in un saggio cellulare mancanza di citotossicità selettività verso altri bersagli correlati have compound members that exhibit a high afinity towards the target (less than 1 µM) show chemical tractability be free of Intellectual property not interfere with the P450 enzymes nor with the P-glycoproteins not bind to human serum albumin be soluble in water(above 100 µM) be stable have a good druglikeness exhibit cell membrane permeability show significant biological activity in a cellular assay does not exhibit cytotoxicity not be metabolized rapidly show selectivity versus other related targets

GENERAZIONE DI “LEADS” elevata affinità per il target (< 1 µM) possibilità di modificazione chimica libertà da vincoli di proprietà intellettuale mancanza di interferenza con gli enzimi P450 o con le proteine di trasporto della famiglia ABC mancanza di legame all’albumina del siero solubilità in acqua (> 100 µM) stabilità “druglikeness” capacità di permeare attraverso le membrane cellulari attività biologica significativa in un saggio cellulare mancanza di citotossicità selettività verso altri bersagli correlati have compound members that exhibit a high afinity towards the target (less than 1 µM) show chemical tractability be free of Intellectual property not interfere with the P450 enzymes nor with the P-glycoproteins not bind to human serum albumin be soluble in water(above 100 µM) be stable have a good druglikeness exhibit cell membrane permeability show significant biological activity in a cellular assay does not exhibit cytotoxicity not be metabolized rapidly show selectivity versus other related targets

CRITERI PER LA VALIDITà DI MODELLI IN VITRO DI BBB Riproducibilità della permeabilità di composti di riferimento Buona capacità di screening Presenza di giunzioni serrate complesse Espressione di trasportatori Presenza di attività transcitotica