Ricerca e sviluppo di nuovi farmaci per le malattie dimenticate. Maria Paola Costi Dipartimento di Scienze della Vita, Università di Modena e Reggio Emilia.

Presentazione sul tema: "Ricerca e sviluppo di nuovi farmaci per le malattie dimenticate. Maria Paola Costi Dipartimento di Scienze della Vita, Università di Modena e Reggio Emilia."— Transcript della presentazione:

1 Ricerca e sviluppo di nuovi farmaci per le malattie dimenticate. Maria Paola Costi Dipartimento di Scienze della Vita, Università di Modena e Reggio Emilia

2 WHO roadmap inspires unprecedented support to defeat neglected tropical diseases 03 February 2012 | Geneva The publication of a roadmap that lays out the vision for ending the misery caused by neglected tropical diseases has inspired public and private partners to unite in the fight against some of the most devastating diseases.

3 Nei paesi in via di sviluppo le malattie infettive sono prima causa di morte Global Health Ideas Finding global health solutions through innovation and technology Archive for the ‘Philanthropy’ Category

4 Quali malattie sono considerate trascurate oggi? Difficile una classificazione rigida, è possibile considerare i diversi ambiti operativi: Ricerca di nuovi farmaci Malaria e Tubercolosi (P.Oliviero—1-2012, IMN. Milano) Kinetoplastidae (Leishmanisis e Tripanosomiasi) Elmintiasi (filiriasi, schistosomiasi-praziquantel) (12% dei malati, 1,3%delle risorse)

5 La scoperta di un nuovo farmaco (non esiste un unico modello) Un farmaco speciale: Specifico Facile da somministrare Stabile Poco costoso (1 $ una terapia) Da somministrare in associazione Protezione brevettuale?? controversie

6 Progettazione e sviluppo di un candidato farmaco

7 Nello sviluppo di un farmaco l’azienda farmaceutica decide in base al ritorno economico. Come si collocano i farmaci contro le malattie dei paesi più poveri? Sviluppo di un farmaco: 1-2 billioni di $ Questo modello non può funzionare nell’ambito delle malattie NTD oggi. La big pharma o al SME non può sviluppare da sola questo farmaco. PDP product development partnership e PPP private public partnership Cambiare modello o cambiare processo e partnering.

8 Per sviluppare un farmaco 1-2 billioni di $ (1 000 000 000 o 10 9 )

9 Distribuzione di fondi per le malattie neglette Global Health Ideas Finding global health solutions through innovation and technology Archive for the ‘Philanthropy’ Category Chi si preoccupa di pìù e chi produce di più?

10 La situazione è confusa: Servono nuovi farmaci meno tossici Lo sviluppo di resistenza può essere un problema Il costo per lo sviluppo di un nuovo farmaco dalla ricerca di base alla clinica è di oltre 1 billione di $, ma nessuno investe questa somma. Non c’è sufficiente massa critica per ottenere un farmaco efficace.

11 Strategie per sviluppare un farmaco per le malattie neglette (A) label extension, extending the indications of existing drugs for other conditions to tropical diseases; (B) piggy-back discovery, in which the discovery of new drugs is focused on one or a few classes of well-studied and validated targets; (C) de novo drug discovery. These strategies collectively seek to exploit two possible sets of drug targets: those that have been validated in other organisms and diseases, and those that have not – perhaps because they are unique to neglected- disease pathogens – but that nevertheless have potential as novel sites of action. PLoS Negl Trop Dis. 2010 Aug 24;4(8):e804. Identification of attractive drug targets in neglected-disease pathogens using an in silico approach. Crowther GJCrowther GJ, Shanmugam D, Carmona SJ, Doyle MA, Hertz-Fowler C, Berriman M, Nwaka S, Ralph SA, Roos DS, Van Voorhis WC, Agüero F.Shanmugam DCarmona SJDoyle MAHertz-Fowler CBerriman MNwaka SRalph SARoos DSVan Voorhis WC Agüero F

12 Il farmaco ideale parasitological cure in all the phases of the disease, effective in single or few doses, low cost for the patients, no collateral or teratogenic effects, no need for hospitalization, and no induction of resistance. oral administration

13 Identificazione del target: un enzima? una proteina di membrana? Un recettore? Un gene? tramite PLoS Negl Trop Dis. 2010 Aug 24;4(8):e804. Crowther GJCrowther GJ, Shanmugam D, Carmona SJ, Doyle MA, Hertz- Fowler C, Berriman M, Nwaka S, Ralph SA, Roos DS, Van Voorhis WC, Agüero F.Shanmugam DCarmona SJDoyle MAHertz- Fowler CBerriman MNwaka SRalph SARoos DSVan Voorhis WCAgüero F Division of Allergy and Infectious Diseases, Department of Medicine, University of Washington, Seattle, Washington, United States of America. crowther@u.washington.edu Identificazione del target

14 Table 2 Preliminary genome-wide prioritization of Leishmania major targets. RankingGene_name Gene productWeight 1LmjF29.0820cysteine peptidase C (CPC),CPC cysteine peptidase, Clan CA, family C1, Cathepsin B-like416 2LmjF05.0350trypanothione reductase386 2LmjF06.0860dihydrofolate reductase-thymidylate synthase386 2LmjF23.0050cyclophilin, putative,peptidyl-prolyl cis-trans isomerase, putative386 2LmjF25.0910cyclophilin a386 2LmjF06.0120cyclophilin386 2LmjF18.0270protein kinase, putative,glycogen synthase kinase, putative386 8LmjF36.1960phosphomannomutase, putative366 8LmjF23.0270pteridine reductase 1366 10LmjF30.2970glyceraldehyde 3-phosphate dehydrogenase, glycosomal351 10LmjF12.0220hydroxyacylglutathione hydrolase, putative,glyoxalase II, putative351 10LmjF24.0850triosephosphate isomerase351 13LmjF27.1870trypanothione synthetase, putative341 13LmjF06.0560deoxyuridine triphosphatase, putative,dUTP diphosphatase341 15LmjF21.0250hexokinase, putative336 15LmjF25.1320serine/threonine protein phosphatase, putative336 15LmjF19.0550methionine aminopeptidase, putative,metallo-peptidase, Clan MG, Family M24 336 15LmjF34.1260mitochondrial DNA polymerase I protein A, putative336 15LmjF30.0880adenosine kinase, putative336 15LmjF33.1630cyclophilin, putative336 15LmjF10.0890FKBP-type peptidyl-prolyl cis-trans isomerase, putative336 15LmjF04.1160fructose-1,6-bisphosphatase, cytosolic, putative336 15LmjF23.0950cytosolic leucyl aminopeptidase,metallo-peptidase, Clan MF, Family M17 336 15LmjF32.1580phosphomannose isomerase, putative336 25LmjF36.2380sterol 24-c-methyltransferase, putative326 25LmjF36.2390sterol 24-c-methyltransferase, putative326

15 Druggability e validazione del target Proof of concept

16 16 Target ValidationGenetic and chemical evidence of essentiality DruggabilityKnown drug-like inhibitors and active site potentially druggable (clinical precedent) Assay FeasibilityAssay ready in plate format and protein supply assured within appropriate timelines ToxicityNo human homologue present or human homologue known to be non-essential Resistance PotentialNo known isoforms within same species and not subject to escape from inhibition Structural InformationLigand-bound structure of target (or ligand in closely related homologue) at high resolution (< 2.3 Å) Target Assessment – Traffic Light System ● ● ● Red No or weak evidence Amber Partial evidence Green Adequate evidence

17 Identificazione dei primi composti da sviluppare (HITS)

18 Fase iniziale (Early phase) drug discovery (HIT-affinità <100 microM) con metodi computazionali Virtual screeningHighthrouput screening (HTS)

19 valutazione biologica sul target e sul parassita Valutazione in laboratorio Highthrouput screening (HTS)

20 Ottimizzazione del composto iniziale a Lead (Medicinal chemistry development), un composto attivo verso i parassiti, non tossico verso le cellule umane, chimicamente stabile.

21 Arrivare alla farmacolgia sull’animale.

22 Ciclo iterativo della fase iniziale di drug discovery (Lead-affinità nanoMolare) La sintesi : parallela, combinatoriale, microwave, etc..

23 Farmcologia animale e fase clinica approvazione FDA post-marketing

24 Fase clinica

25 I farmaci NTD in uso

26 I bersagli

27 Specificità del target e selettività nel parassita

28 Esempio: famaci anielmintici (più diffusa malattia infettiva al mondo)

29 Altri esempi

30

31 La ricerca e sviluppo

32 The challenge 300 death/day Leishmania T. cruzi T.brucei

33 The Disease Chagas Disease or American Trypanosomiasis Each year over 8 million people in the Americans contract the Chagas disease. Chagas occurs in two stages and kills more people in the region than any other parasite-borne disease, including malaria. It is caused by the parasite Trypanosoma cruzi transmitted primarily by insects known as “kissing bugs”. The existing treatments are not satisfactory and can have toxic side effects. What patients urgently need are affordable, safe, and efficacious diagnostics tools and treatments for children and adults as well as a drug that treats both stages of Chagas.

34 DNDI Leishmania e Tripanosomiasi portfolio (pipeline dei farmaci in via di sviluppo)

35 EARLY PHASE DRUG DISCOVERY per Leishmaniasi- Chagas disease (American Tripanosomiasi) e HAT

36 Nitroimidazoli Nitroimidazoli: danno al DNA, inibizione del metabolismo del glucosio, interferenza con la funzione mitocondriale. Genotossico per la presenza del nitro gruppo. 2-nitroimidazolo, isolato per la prima volta da uno streptomicete nel 1953

37 Candidati in fase pre-clinica e clinica PRECLINICAL 8 Aminoquinolines -in preparation PRECLINICAL

38 Dal benznidazolo al fexindazolo Si trasforma in nitrosoimdazolo e tarsferisce un e- alla Tripanotione reduttasi, enzima essenziale per il T.cruzi-Chagas)

39

40  Antimonial drugs –20mg/kg body wt. for 30-40 days  Pentamidine -severe side effects (interagisce con il DNA, minor groove (AT rich regions) causa rottura del DNA mitocondriale che è circolare.  Amphotericin- high cost has limited its usage JNU Farmaci attivi contro la Leishmania

41 New potential therapies for Visceral Leishmaniasis  Amphotericin B liposome formulation registered in USA and Europe  Oral miltefosine has been registered in India and under phase IV clinical trial. Agisce a livello della membrana cellulare. Ideato come antitumorale.  Parenteral formulation of aminosidine (paromomycin) registered for use in India and is under phase IV clinical trials  Oral sitamaquine has completed phase II trials in India, Kenya and Brazil

42

43 Two mechanisms proposed: 1The nitro-anion radical metabolite, which reacts with the nucleic acids of the parasite, causing a significant breakage in the deoxyribonucleic acid (DNA). This mechanism is similar to that proposed for the antibacterial action of other nitrofuran agents. 2(from nitro to nitrose and then ROS generation) 3It involves the production of superoxide anions, and hence, hydrogen peroxide (both of which are very toxic to the parasite) and inhibition of trypanothione reductase, which is a parasite- specific antioxidant defense enzyme. Another enzyme, ascorbate- linked peroxidase, is also present but at relatively low levels in the organism. Lack of these enzymes leads to the accumulation of hydrogen peroxide to cytotoxic levels, resulting in death of the parasite

44 Eflornithine (α-difluoromethylornithine or DFMO) interferisce con la sintesi delle poliammine

45 Meccanisimo d’azione di eflornitina

46 Per fermare la tripanosomiasi

47 Chi prepara e vende i farmaci Chagas disease The two available drugs are benznidazole and nifurtimox. Main contra-indications are pregnancy and renal or hepatic failure. Especially with nifurtimox, also psychiatric or neuronal disorders (such as seizures). Nifurtimox is free of charge through WHO-Bayer agreement. For ordering, please contact Albajar Vinas P. Tel.:+41-22-7911261; albajarvinap@who.int Benznidazol is produced by Laboratório Farmacêutico do Estado de Pernambuco (LAFEPE). For ordering, please contact LAFEPE, Largo de Dois Irmãos 1117 - Dois Irmãos. 52171-010 - Recife - PE, Brazil. Tel.: +55-81-31831100; sac@lafepe.pe.gov.bralbajarvinap@who.int sac@lafepe.pe.gov.br

48 Esempi di sviluppo di nuovi farmaci (discovery process) Tripanosoma brucei (target N-miristoil transferasi) Leishmania (target pteridina reduttasi- diidrofolato reduttasi)

49 N-myristoil transferasi

50 Protein N-myristoylation is a ubiquitous eukaryotic co- and post-translational modification and is required for membrane targeting and biological activity of many important proteins. The N-myristoylation reaction, i.e., the transfer of myristic acid frommyristoyl-coenzyme A (CoA) to the amino group of N-terminal glycine residues within specific sequence contexts, is catalysed by the enzymemyristoyl-CoA: protein N-myristoyltransferase (NMT). NMT has been shown by gene knockout and RNAi to be essential for viability of the parasites Leishmania major and Trypanosoma brucei, therefore it has been identified as a viable target for the development of novel therapeutics

51 Meccanismo ed inibizione Frearson et al Trends Parasitol 2007;23:589-95. Wyatt et al Curr Top Med Chem. 2011 Mar 14.

52 52 NMT: A Partial Success Story *Frearson et al. Nature 2010; 464: 728-732 63,332 compounds RNAi shows NMT essential in vivo Hit Series Identified. Potent NMT inhibitors and T. brucei growth inhibitors developed Kinetics & binding mode of DDD85646 understood Series kills parasite by acting on NMT in situ. Cures acute HAT in vivo, but not chronic CNS stage + - drug Series shows clear structure- activity relationship - +

53 53 PS-Q series Hit Discovery Hits to Leads Lead Optimizatio n Target assessment Trypanothione synthetase UDP-Glc-4’- epimerase PTR1/DHFR CRK3 Assay Developme nt Hit Validation UDP-GlcNAc diphosphorylase GSK3 KPST04 BA01 De-N-acetylase PK53 PK50 PLK T. brucei projects on hold or returned to originating laboratory KPST01 Hsp83 No suitable hits from screens Assay method not sufficiently robust Poor structure-activity relationships (SAR) Drop-off from target to cell potency Lack of selectivity PK/PD issues Cytostatic rather than cytocidal Insufficient chemistry resource Failure to progress Reasons for project attrition Trypanothione reductase

54 54 Drop Off in Potency from Target to Cell Glycogen synthase Kinase 3 (GSK3) – GSK3-09 series shows tight SAR – Poor selectivity versus human GSK3 – Inactive against panel of other human protein kinases Series acts on-target – Cell potency modulated by GSK3 expression levels – Over-expression of TbGSK3 short has a toxic effect on cell growth, that can be progressively rescued by DDD85893 up to 3 μM. Raffaella Grimaldi, DDU unpublished.

55 55 Drop-off in Cell Potency (GSK3-09): Intracellular Substrate Concentration Raffaella Grimaldi, DDU unpublished. K i = 2 nM (competitive v ATP) K m ATP = 11  M ATP in assay = 1  M [ATP] in cells = ~2 mM At EC 99 (23.4  M) GSK3 is inhibited 98.5% Wash off experiment after 24 h exposure: 50  M cidal; 15  M not cidal GSK3 IC 50 2.1 nM T.bruce i EC 50 3.9  M At predicted in vivo EC 50, no effect on growth, consistent with GSK3 SKO phenotype

56 Conclusions We have identified two classes of compounds that inhibit specifically PTR1 in the micromolar range (no DHFR or human enzymes inhibition). Some of them are not toxic against MRC5 human cells. We have confirmed the initial finding that specific inhibiton of PTR1 can be synergistic with DHFR inhibition. Potentially Pyrimetamine can be used as a drug also in Leishmania, in combination with PTR1 inhibitors. Same compounds inhibit Lm parasites in sinergy with PYR, while no synergy is observed in T.brucei inhibition. Riluzole and its derivatives can be explored as drug candidates (LABEL EXTENSION?).

57 Target per i farmaci in uso Chagas disease The two available drugs are benznidazole and nifurtimox. Main contra-indications are pregnancy and renal or hepatic failure. Especially with nifurtimox, also psychiatric or neuronal disorders (such as seizures). Nifurtimox is free of charge through WHO-Bayer agreement. For ordering, please contact Albajar Vinas P. Tel.:+41-22-7911261; albajarvinap@who.int Benznidazol is produced by Laboratório Farmacêutico do Estado de Pernambuco (LAFEPE). For ordering, please contact LAFEPE, Largo de Dois Irmãos 1117 - Dois Irmãos. 52171-010 - Recife - PE, Brazil. Tel.: +55-81-31831100; sac@lafepe.pe.gov.bralbajarvinap@who.int sac@lafepe.pe.gov.br

58 Genomica, Proteomica Bioinformatica 6. Biologia Strutturale biofisica Identificazione del target 1. Progettazione Molecular Modelling 2. Chimica (Bio)Farrmaceutica Identificazione di “hits” 3. Screening in vitro Saggi cellulari “Lead” Farmacocinetica Metabolismo 5. Formulazione Candidato Farmaco Ottimizzazione 4. Farmacologia Tossicologia Progettazione e sviluppo di un candidato farmaco HTS validazione Sviluppo clinico e ricerca traslazionale

59 Azioni in corso e passate 1. COST Action CM0801 2. COST Action B22 Fondazione Cassa di Risparmio di Modena (FCRM) Kinetodrugs PRIN2009 COST CM0801 annual meeting Borse distudio COST CM0801 training school Borse distudio COST CM0801 STMS exchange fellowships Borse distudio www.tydockphrma.com