rev. 20221212

Ministero della Salute

DIREZIONE GENERALE DELLA RICERCA E DELL’INNOVAZIONE IN SANITA’

Ufficio 3

Convenzione tra il Ministero della salute, il destinatario istituzionale Fondazione Policlinico San ▇▇▇▇▇▇ e, per conoscenza, il PI della ricerca ▇▇▇▇▇▇ ▇▇▇▇▇▇▇▇, per la regolamentazione dello svolgimento dei progetti di ricerca finalizzata, relativi al bando della ricerca finalizzata 2021, esercizi finanziari 2020-2021, afferenti alla tipologia progettuale - Ordinari della ricerca finalizzata (RF) - “Theory enhancing”.

Convenzione progetto RF-2021-12374476 Premesso che

ai sensi di quanto disposto dall’art. 12 e dall’art. 12 bis del decreto legislativo 30 dicembre 1992 n.502, come modificato e integrato dal decreto legislativo n. 229/1999, concernenti il finanziamento da parte del Ministero della salute dei progetti di ricerca presentati dai destinatari istituzionali, individuati dalla normativa stessa, si rende necessario - ai fini dello svolgimento dei progetti di ricerca finalizzata, relativi agli anni finanziari 2018-2019 e approvati dal Comitato Tecnico Sanitario – sezione c), nella riunione del 5 giugno (progetti SG), nelle riunioni del 22 e 30 settembre 2020 (progetti RF-CO-GR), disciplinare i conseguenti rapporti di collaborazione e finanziari;

l’art.7 del decreto ministeriale 8 aprile 2015, recante il riordino degli uffici di livello dirigenziale non generale del Ministero della salute, ha individuato gli uffici in cui si articola la Direzione generale della ricerca e dell’innovazione in sanità, individuando, fra le altre, le specifiche competenze assegnate all’ufficio 3 della stessa;

con il Decreto Direttoriale dell’1 marzo 2022, registrato dall’Ufficio Centrale di Bilancio in data 4 marzo 2022, al n. 247, con il quale il ▇▇▇▇. ▇▇▇▇▇▇▇ ▇▇▇▇▇▇▇▇▇ è stato autorizzato, tra l’altro, all’esercizio del potere di spesa sul capitolo di spesa 3398, piano gestionale 3 piani gestionali per i residui passivi perenti, limitatamente agli importi destinati agli IRCCS;

con Decreto del Ministro dell’economia e delle finanze 31 dicembre 2021, recante “Ripartizione in capitoli delle unità di voto parlamentari relative al bilancio di previsione dello Stato per l’anno finanziario 2022 e per il triennio 2022–2024”;

con Decreto 12 novembre 2021 del Ministro della salute di concerto con il Ministro dell'istruzione dell'università e della ricerca, registrato all’Ufficio di controllo di bilancio con visto numero 592 del 22 novembre 2021, e alla Corte dei Conti in data 13 dicembre 2021 con numero 3018, è stato approvato il bando della ricerca finalizzata 2021, per gli anni finanziari 2020-2021, in cui confluiscono le somme disponibili di euro 50.000.000,00 (cinquantamilioni/00) riferite all’anno finanziario 2020 ed euro 50.000.000,00

(cinquantamilioni/00) riferite all’anno finanziario 2021, per complessivi euro 100.000.000,00 (centomilioni/00) ripartiti tra le diverse tipologie progettuali menzionate nel richiamato bando;

in data 27 dicembre 2021 è stato pubblicato sul portale del Ministero della salute il bando della ricerca finalizzata 2021, relativo agli anni finanziari 2020-2021;

nella riunione del 28 ottobre 2022 il Comitato tecnico sanitario sezione c), preso atto della regolarità del processo di valutazione, ha approvato la graduatoria finale distinta per ciascuna delle sezioni del Bando e delle tipologie progettuali, nonché il finanziamento da destinare ai progetti collocatisi in posizione utile;

con il Decreto direttoriale in data 28 ottobre 2022 - registrato dall’Ufficio centrale di bilancio in data 18 novembre 2022 al numero 1057 - con il quale sono state approvate le graduatorie relative alle previste sezioni e tipologie progettuali del Bando per la Ricerca Finalizzata anno 2021, relativo agli anni finanziari 2020- 2021, con indicazione, per ciascun progetto collocatosi in posizione utile ai fini del finanziamento, della somma a carico di questo Dicastero per la realizzazione del progetto medesimo;

nel suddetto provvedimento è menzionato il progetto RF-2021-12374476 denominato “Development of innovative inhalatory targeted liposomal formulations for the treatment of inflammatory driven pulmonary fibrosis” - Destinatario istituzionale - Fondazione Policlinico San ▇▇▇▇▇▇ – utilmente collocato nella specifica graduatoria “Ordinari della ricerca finalizzata (RF)”) ed al quale è stato attribuito un finanziamento complessivo di €450.000,00 (quattrocentocinquantamila/00).

TANTO PREMESSO SI STIPULA E CONVIENE QUANTO SEGUE TRA

Il Ministero della salute

rappresentato dal ▇▇▇▇. ▇▇▇▇▇▇▇ ▇▇▇▇▇▇▇▇▇ – Direttore dell’Ufficio 3 della Direzione generale della ricerca e

dell’innovazione in sanità;

E

Il Destinatario istituzionale Fondazione Policlinico San ▇▇▇▇▇▇

(nel prosieguo denominato Destinatario istituzionale)

rappresentato da - ▇▇▇▇. ▇▇▇▇▇▇▇ ▇▇▇▇▇▇▇▇

Articolo 1

1. Le premesse sono parte integrante della presente convenzione.

Articolo 2

1. La presente convenzione regola l’affidamento da parte del Ministero della salute – Direzione generale della ricerca e dell’innovazione in sanità - al Destinatario istituzionale del progetto di ricerca finalizzata, RF-2021-12374476 dal titolo “Development of innovative inhalatory targeted liposomal formulations for the treatment of inflammatory driven pulmonary fibrosis”.

2. Il principal investigator è individuato nella persona dott./dott.ssa ▇▇▇▇▇▇ ▇▇▇▇▇▇▇▇, codice fiscale ▇▇▇▇▇▇▇▇▇▇▇▇▇▇▇▇.

Articolo 3

1. Il finanziamento è di €450.000,00 (quattrocentocinquantamila/00) a valere sui fondi del capitolo 3398/1 ed i seguenti perenti 3398/83-84-87

Articolo 4

1. Il Destinatario istituzionale ed il principal investigator svolgono congiuntamente il progetto di ricerca secondo quanto riportato nel piano esecutivo e finanziario presentato in ottemperanza a quanto previsto dal Bando per la ricerca finalizzata 2021 relativo agli anni finanziari 2020-2021 di cui in premessa ed allegato alla presente convenzione di cui è parte integrante.

Articolo 5

1. La presente convenzione ha la durata di tre anni prorogabile eventualmente di ulteriori 12 mesi come previsto dal successivo articolo 11.

2. L’attività di ricerca, da svolgersi nell’arco temporale della vigenza della convenzione, deve avere inizio improrogabilmente entro e non oltre il 30 aprile 2023, comunicando la data effettiva con nota sottoscritta digitalmente dal proprio rappresentante legale e dal principal investigator della ricerca almeno 20 giorni prima dell’inizio effettivo.

3. Il Destinatario istituzionale entro e non oltre 30 giorni dall’invio della presente convenzione da parte del Ministero della Salute per la sottoscrizione provvede alla restituzione della convenzione firmata dal legale rappresentate e controfirmata dal principal investigator, tramite il sistema di monitoraggio del WFR, accompagnato dalla comunicazione del codice CUP del progetto e i codici CUP e codici fiscali delle singole Unità operative;

4. La presente convenzione, vincolante all'atto della sottoscrizione per il destinatario istituzionale ed il principal investigator, diventa efficace per il Ministero a seguito della registrazione da parte dell’organo di controllo.

5. Il Destinatario istituzionale, entro e non oltre 20 giorni precedenti la scadenza del termine di cui al comma 2 del presente articolo, pena la decadenza dal finanziamento, è tenuto a trasmettere - con nota sottoscritta digitalmente dal proprio rappresentante legale e dal principal investigator della ricerca - la seguente documentazione, soggetta a verifica da parte del Ministero al fine di autorizzare l’avvio del progetto:

a) il parere positivo del comitato etico e/o l’autorizzazione di cui all’articolo 31 del

decreto legislativo n.26 del 4 marzo 2014, ove previsti;

b) le dichiarazioni indicanti le unità operative coinvolte nel progetto nonché l’accettazione degli Enti che svolgono funzioni di unità operativa e dei relativi responsabili di unità operativa dell’accettazione dei termini della presente convenzione;

c) la dichiarazione con la quale il destinatario istituzionale attesta che il principal investigator svolgerà la propria attività, relativamente al progetto in questione, esclusivamente presso la struttura del S.S.N. all’uopo individuata dal destinatario istituzionale medesimo. La dichiarazione di cui al presente comma dovrà essere controfimata dal Princiapl Investigator interessato;

d) il certificato AIRE (Anagrafe degli italiani residenti all’estero) nel caso di collaborazione con ricercatori italiani residenti e operanti all’estero;

e) La traduzione in lingua italiana della proposta progettuale senza apportare alcuna modifica alla versione in inglese allegata alla presente convenzione;

6. Il monitoraggio e la verifica del raggiungimento degli obiettivi del progetto di ricerca di cui alla presente convenzione sono affidati alla Direzione generale della ricerca e dell’innovazione in Sanità, Ufficio 3.

7. La scheda del piano finanziario è vincolante relativamente al solo totale del finanziamento assegnato e al riparto iniziale tra unità operative, mentre ha valore meramente indicativo per quanto riguarda la ripartizione tra voci di costo e le motivazioni a giustificazione di tali costi. Il Destinatario istituzionale si impegna a rispettare le quote percentuali previste dal bando per le varie voci di costo che saranno calcolate, a consuntivo, sulle spese rendicontate, al netto di eventuali economie riscontrate sul finanziamento assegnato e sulle sole spese eleggibili, dopo verifica da parte del Ministero della salute.

8. Le parti convengono che le comunicazioni relative al progetto di cui trattasi siano effettuate attraverso il sistema di monitoraggio delle ricerche denominato Workflow della ricerca a disposizione dei

Destinatari istituzionali. Il Ministero si riserva di attivare, qualora reso disponibile, il sistema di rendicontazione on-line sulla piattaforma del Workflow della ricerca, e lo stesso sarà vincolante dalla data di comunicazione della relativa disponibilità.

9. Il Destinatario istituzionale attraverso il proprio rappresentate legale, nonché il principal investigator devono firmare digitalmente tutti gli atti inerenti alla ricerca.

Articolo 6

1. La prima rata del finanziamento è pari a €225.000,00 (duecentoventicinquemila/00) e la procedura per il pagamento della stessa è avviata solo a seguito dell’accertamento da parte del Ministero degli avvenuti adempimenti di cui al comma 2 e 4 dell’articolo 5 della presente. La predetta rata è imputata sull’esercizio finanziario 2022.

2. La seconda rata del finanziamento è pari ad €135.000,00 (centotrentacinquemila/00) ed è erogata dopo la trasmissione da parte del Destinatario istituzionale della relazione intermedia di cui al successivo art. 7 e solo a seguito della valutazione positiva della stessa da parte del Ministero. La predetta rata è imputata sull’esercizio finanziario 2025.

3. La terza rata, a saldo del finanziamento, è pari ad €90.000,00 (novantamila/00). Essa è corrisposta una volta accertata la sussistenza dei requisiti di cui al successivo articolo 9 e solo a seguito della valutazione positiva della relazione finale da parte del Ministero. La predetta rata è imputata sull’esercizio finanziario 2027.

4. A garanzia della coerenza con l’inizio dell’attività dichiarata, il Destinatario istituzionale si impegna ad anticipare le risorse economiche necessarie, nell’eventualità in cui le somme da corrispondersi da parte del Ministero siano in regime di perenzione.

5. Laddove non vengano rispettati i termini di cui alla presente convenzione, che non consentano la tempestiva erogazione dei fondi, il Destinatario istituzionale esonera il Ministero da eventuali ritardi nell’erogazione delle somme spettanti.

Articolo 7

1. Allo scadere dei 18 mesi dall’inizio dell’attività della ricerca e comunque non oltre i trenta (30) giorni da tale termine, il Destinatario istituzionale trasmette al Ministero la relazione intermedia sullo stato d’attuazione della ricerca - sottoscritta digitalmente dal legale rappresentante del Destinatario istituzionale e dal principal investigator - contenente la descrizione delle attività svolte dalle singole unità operative da cui risulti il regolare svolgimento del progetto secondo quanto riportato nel piano esecutivo . Tale relazione deve essere accompagnata da un documento di sintesi, a cura del principal investigator, che illustri, nella globalità, lo stato di avanzamento dei lavori, inclusa la descrizione delle attività realizzate da eventuali Enti co-finanziatori.

2. Il Ministero ha facoltà, previa comunicazione preventiva al destinatario istituzionale, di attivare le procedure per la sospensione del finanziamento ed il recupero delle somme erogate, comprensive degli eventuali interessi legali maturati, qualora il Destinatario istituzionale non adempia a quanto previsto entro i termini di cui al comma 1 del presente articolo.

3. il Ministero, previa comunicazione preventiva al Destinatario istituzionale, ha facoltà di non erogare la seconda rata di finanziamento, subordinandola all’eventuale esito positivo del giudizio in ordine alla relazione finale, qualora la relazione intermedia, all’esito dell’istruttoria, non sia considerata idonea a dimostrare che siano stati pienamente raggiunti gli obiettivi medio termine o emerga che essa sia stata condotta non in piena conformità con quanto previsto nel piano esecutivo approvato. In tal caso il Ministero potrà procedere alla erogazione della seconda rata contestualmente al saldo. Laddove non vengano rispettati i termini di cui alla presente convenzione, che non consentano la tempestiva erogazione dei fondi, il Destinatario istituzionale esonera il Ministero da eventuali ritardi nell’erogazione delle somme spettanti.

4. Il Ministero, previa comunicazione preventiva al Destinatario istituzionale, può sottoporre alle valutazioni al Comitato tecnico sanitario sez. c), un dossier, qualora la relazione intermedia, all’esito della istruttoria ministeriale, non consenta di esprimere un compiuto motivato parere. La decisione

del suddetto Comitato è vincolante per il Destinatario istituzionale ai fini del prosieguo della convenzione.

Articolo 8

1. A partire dal 6° mese successivo all’avvio del progetto e fino a 12 mesi prima della scadenza del progetto, il Destinatario istituzionale, con nota firmata dal proprio rappresentate legale e dal principal investigator - può apportare modifiche al piano esecutivo, coerenti con gli obiettivi progettuali, o alla distribuzione di fondi tra le unità operative, solo se approvato dal Ministero con espresso e formale atto preventivo di assenso e purché non comportino un aumento del finanziamento a carico del Ministero. Non è consentito oltre tale periodo avanzare richieste di modifica. In caso di una eventuale necessità di una ulteriore modifica progettuale è possibile presentare tale modifica solo dopo 6 mesi dall’approvazione da parte del Ministero dell’ultima modifica progettuale.

2. La distribuzione delle somme tra le diverse voci di costo, nell’ambito di ogni singola unità operativa, è consentita sotto la responsabilità del Destinatario Istituzionale che ha presentato il progetto e che dovrà verificare il rispetto delle percentuali previste dal bando.

3. Qualsiasi proposta emendativa deve essere adeguatamente motivata dal principal investigator per documentare che quanto richiesto risulti indispensabile per assicurare il raggiungimento degli obiettivi a suo tempo prefissati.

4. Solo dopo l’approvazione del Ministero, il Destinatario istituzionale potrà procedere a dare attuazione alle modifiche di cui al comma 1 del presente articolo. In caso di eventuali inadempimenti al presente articolo il Ministero ha facoltà di procedere sia alla risoluzione della convenzione, dandone comunicazione al Destinatario istituzionale, sia alla sospensione del finanziamento nonché al recupero dell’importo erogato.

Articolo 9

1. Fatta salva l’eventuale concessione di proroga della durata delle attività progettuali, al termine di trentasei mesi - e comunque non oltre trenta (30) giorni dopo la data fissata per il termine della ricerca

– ai fini dell’erogazione del saldo, il Destinatario istituzionale, con nota firmata digitalmente dal rappresentante legale, trasmette contestualmente al Ministero la seguente documentazione, redatta dal principal investigator e recante la firma digitale dello stesso:

a) la relazione finale della ricerca contenente quanto posto in essere da eventuali Enti co- finanziatori, documenti, per ciascuna unità operativa, la coerenza delle attività svolte con il programma esecutivo approvato e gli obiettivi raggiunti;

b) copia dei lavori pubblicati su riviste impattate a seguito dello svolgimento della ricerca di cui

all’articolo 1 della presente;

c) la rendicontazione delle spese sostenute con i fondi ministeriali, utilizzando se disponibile il sistema di rendicontazione on-line del WFR;

d) indicazioni della URL del repository pubblico dove sono resi disponibili i dati grezzi progettuali e quelli utilizzati per le pubblicazioni scientifiche correlate.

2. Tutta la soprarichiamata documentazione deve essere redatta utilizzando esclusivamente la modulistica reperibile sul sistema Workflow della ricerca.

3. La documentazione di supporto deve essere a disposizione del Ministero presso il Destinatario istituzionale, che deve provvedere alla relativa custodia.

4. Il Ministero provvede ad applicare una decurtazione pari al 10% della rata del saldo, qualora la documentazione di cui alle lettere a) b) c) d) del comma 1 del presente articolo sia trasmessa al Ministero in un periodo compreso tra il trentunesimo ed il sessantesimo giorno dalla data di conclusione del progetto.

5. Il Ministero provvede ad applicare una decurtazione pari al 20% della rata del saldo, qualora la documentazione di cui alle lettere a) b) c) d) del comma 1 del presente articolo sia trasmessa al Ministero in un periodo compreso tra il sessantunesimo ed il novantesimo giorno dalla data di conclusione del progetto.

6. Il Ministero, previa comunicazione preventiva al Destinatario istituzionale, attiva le procedure per la sospensione del finanziamento e la conseguente economia della rata finale nonché per il recupero delle somme già erogate, comprensive degli interessi legali maturati, qualora la documentazione di cui alle lettere a) b) c) d) del comma 1 del presente articolo non sia trasmessa al Ministero entro il novantesimo giorno dalla data di conclusione del progetto.

7. Il Ministero si riserva la facoltà di chiedere informazioni ed eventuale documentazione integrativa al Destinatario istituzionale, che deve fornire riscontro entro e non oltre i successivi 15 giorni, qualora:

- la relazione finale non sia considerata idonea a dimostrare il regolare svolgimento della ricerca, in conformità di quanto previsto nel piano esecutivo e nel piano finanziario approvati;

- la rendicontazione risulti incompleta o incongruente sia sui dati contabili sia sulle descrizioni.

8. Il Ministero provvederà ad emettere la valutazione finale sulla base di quanto acquisito agli atti, in caso di mancato o esaustivo riscontro da parte del Destinatario istituzionale delle richieste di cui al precedente comma 7.

9. Il Ministero comunica al Destinatario istituzionale il parere negativo in ordine alla relazione finale e conseguentemente in ordine alla erogazione del saldo ed ha facoltà di chiedere la restituzione delle somme già erogate comprensive degli interessi legali maturati, in caso di mancato riscontro oppure laddove dalla istruttoria della documentazione integrativa emerga che sono stati disattesi gli obiettivi di cui al piano esecutivo.

10. Il Ministero, previa comunicazione preventiva al Destinatario istituzionale, può sottoporre al Comitato tecnico sanitario sez. c). un dossier, qualora la relazione finale all’esito della istruttoria ministeriale, non consenta di esprimere un compiuto motivato parere. La decisione del suddetto Comitato è vincolante per il Destinatario istituzionale ai fini del prosieguo della convenzione.

11. Il Ministero, previa comunicazione preventiva al Destinatario istituzionale, attiva le procedure per la sospensione del finanziamento e la conseguente messa in economia delle rate residue, nonché per il recupero delle somme già erogate, comprensive degli interessi legali maturati, in caso di mancato rispetto da parte del PI dell’orario di lavoro contrattuale, ovvero di quello minimo stabilito tramite convenzione con altro Ente, durante il periodo di svolgimento della ricerca. A tal fine, il Ministero si riserva la facoltà di richiedere al Destinatario Istituzionale i tabulati relativi alla durata dell’orario di lavoro giornaliero svolto dal PI, rilevato con sistema di misurazione oggettiva; il Destinatario istituzionale dovrà fornire riscontro entro e non oltre 15 giorni dalla richiesta.

Articolo 10

1. Il Ministero della salute – Direzione generale della ricerca e dell’innovazione in sanità, in via autonoma o sentito il Comitato Tecnico Sanitario, ha facoltà di chiedere chiarimenti e può disporre verifiche in ogni momento e anche durante lo svolgimento della ricerca.

Articolo 11

1. Il termine della ricerca può essere prorogato dal Ministero per un periodo massimo di mesi 12 dalla data di scadenza, solo a seguito di formale, motivata e documentata istanza firmata digitalmente dal legale rappresentante del Destinatario istituzionale e del principal investigator. A detto periodo possono essere applicate eventuali deroghe a seguito di provvedimenti della Direzione generale della ricerca e dell’innovazione in sanità per eventi emergenziali.

2. La richiesta di cui al comma 1 può essere avanzata dopo la presentazione della relazione intermedia di cui all’articolo 7 e fino a 12 mesi precedenti il termine del progetto con formale e motivata istanza da parte del Destinatario istituzionale e del principal investigator che avrà efficacia solo dopo l’approvazione da parte del Ministero.

Articolo 12

1. La proprietà degli studi, dei prodotti e delle metodologie sviluppati nell’ambito del progetto è regolamentata dalla normativa vigente in materia, salvo particolari accordi stipulati tra le parti firmatarie del presente atto, ferma restando la possibilità dei soggetti istituzionali del Servizio Sanitario Nazionale di fruirne, previa richiesta alle parti firmatarie.

2. Nel caso in cui il contraente intenda trasferire ad altri soggetti qualsiasi diritto, anche parziale, relativo alla ricerca in questione, ai risultati della stessa o ad eventuali brevetti derivati deve darne preventiva comunicazione al Ministero.

3. Qualsiasi documento prodotto, ivi comprese le pubblicazioni scientifiche inerenti al progetto di ricerca oggetto della presente convenzione – per i quali deve essere assicurato l’accesso non oneroso al Dicastero - deve contenere l’indicazione del finanziamento ministeriale e del codice del progetto finanziato.

4. Il Ministero non riconosce l’eleggibilità dei costi delle pubblicazioni sui propri fondi qualora in dette pubblicazioni non si faccia espressa menzione del finanziamento ministeriale e del codice progetto.

5. Il Ministero provvede ad una decurtazione pari al 10% dell’intero finanziamento, nel caso in cui il Destinatario istituzionale al termine delle attività progettuali non inoltri documentazione relativa a quella indicata alla lettera b) del comma 1 dell’articolo 9.

6. Il Ministero provvede ad una decurtazione pari al 10% dell’intero finanziamento, nel caso in cui il Destinatario istituzionale al termine delle attività progettuali non inoltri documentazione relativa a quella indicata alla lettera d) del comma 1 dell’articolo 9.

7. Il Ministero provvede ad una decurtazione pari al 5% dell’intero finanziamento, nel caso in cui il Destinatario istituzionale al termine delle attività progettuali inoltri documentazione relativa a quella indicata alla lettera b) del comma 1 dell’articolo 9 priva della menzione del Ministero della salute e del codice progetto.

8. Il Ministero provvede ad una decurtazione pari al 5% della rata del saldo, nel caso in cui il Destinatario istituzionale al termine delle attività progettuali inoltri documentazione relativa a quella indicata alla lettera b) del comma 1 dell’articolo 9 dalla quale risulti che solo alcune pubblicazioni prodotte recano la menzione del Ministero quale istituzione finanziatrice e del codice progetto.

9. Le parti convengono che il Ministero della salute possa dare direttamente diffusione, anche attraverso il proprio sito web, dell’estratto della proposta progettuale e dei risultati della ricerca sia in forma completa che sintetica e delle pubblicazioni scientifiche da essa derivate.

Articolo 13

1. I beni e gli strumenti necessari per l’esecuzione del presente progetto di ricerca possono essere posti a carico dei fondi ministeriali qualora acquisiti a mezzo leasing, noleggio ovvero in comodato d’uso, per un periodo pari alla durata del progetto.

2. È fatto divieto di utilizzare i fondi del Ministero della salute per l’acquisto diretto di apparecchiature

e materiale inventariabile.

3. Relativamente ai progetti RF-CO-GR, per il pagamento di quote parte stipendiali è riconosciuto un contributo fisso al limite di euro 40.000,00 l’anno per “full time equivalent”, nei limiti del 50% del finanziamento complessivo del progetto ovverosia della quota totale rendicontata a carico del Ministero della salute e riconosciuta eleggibile.

4. Relativamente ai progetti SG, per il pagamento della borsa di studio del ricercatore proponente, è

riconosciuto un contributo fisso al limite di euro 90.000,00 per l’intera durata del progetto.

Articolo 14

1. Le parti contraenti prendono atto che il finanziamento del presente progetto di ricerca afferisce alla gestione dei fondi per il finanziamento delle attività di ricerca o sperimentazione, “Ricerca Scientifica” capitolo 3398/1 ed i seguenti perenti 3398/83,84 e 87, ad esclusione degli importi destinati agli IRCCS, di pertinenza del centro di responsabilità Direzione generale della ricerca e dell’innovazione in sanità, dello stato di previsione del Ministero della salute, in relazione a quanto disposto dal decreto legislativo n. 502/1992 e successive modifiche ed integrazioni.

Articolo 15

1. Le parti si impegnano all’osservanza, per quanto di rispettiva competenza, delle disposizioni inerenti alla tracciabilità dei flussi finanziari di cui all’art.3 della legge 13 agosto 2010 n.136, e successive modifiche ed integrazioni.

Letto, confermato e sottoscritto con firma digitale, ai sensi dell’art. 21 del decreto legislativo 7 marzo 2005, n.82.

Roma, (data della sottoscrizione come quella dell’ultima firma digitale apposta)

per il Ministero della salute ▇▇▇▇. ▇▇▇▇▇▇▇ ▇▇▇▇▇▇▇▇▇ Direttore dell’Ufficio 3

Direzione generale della ricerca e dell’innovazione in sanità

per il Destinatario istituzionale Fondazione Policlinico San ▇▇▇▇▇▇ ▇▇▇▇. ▇▇▇▇▇▇▇ ▇▇▇▇▇▇▇▇

Codice fiscale ▇▇▇▇▇▇▇▇▇▇▇▇▇▇▇▇

Il presente atto è sottoscritto per presa visione

il principal investigator - ▇▇▇▇▇▇ ▇▇▇▇▇▇▇▇

Codice fiscale ▇▇▇▇▇▇▇▇▇▇▇▇▇▇▇▇

CAVALLINI SAN ▇▇▇▇▇▇

RF-2021-12374476

MDC primary: Cardiologia-Pneumologia

MDC secondary: Trapiantologia

Project Classification IRG: Project Classification SS:

Cardiovascular and Respiratory Sciences Lung Injury, Repair, and Remodeling - LIRR

Project Keyword 1:

Project Keyword 2:

Project Keyword 3:

Pulmonary fibrosis and interstitial lung diseases: Includes granulomatous diseases (such as sarcoidosis), idiopathic pulmonary fibrosis, interstitial pneumonias, autoimmune lung diseases, and lymphangioleiomyomatosis. This would also include involvement of mesenchymal stem cells, epithelium dysfunction, and epithelial-mesenchymal transition.

X

X

X

treatment liposomes

Project Request: Animals:

Humans:

Clinical trial:

The object/s of this application is/are under patent copyright Y/N:

Patent number: n°102021000022253

Type patent owner: Public Inst. Company Patent owner: Policlinico San ▇▇▇▇▇▇ / Universita di Torino

Operative Unit not SSN: OU2

Co-PI: Codullo Veronica

Person in charge for the animal experiment: ▇▇▇▇▇▇ ▇▇▇▇▇

Retired personnel: None within three years of project start

Overall Summary

The study aims to develop an innovative nano-platform for the treatment of lung fibrogenic disorders (LFD). The approach involves local delivery by inhalation of drug-loaded liposomes, coated with hyaluronic acid (HA) to directly target CD44+ pathogenic cells. We aim to expand and develop the formulation "XHALIP¿ (patent pending) by the following steps: 1) Characterization of safety and bioavailability in healthy and lung fibrogenic disorders (LFD) mice; 2) Evaluation of the pharmacokinetics and uptake by human LFD fibroblasts and macrophages and on healthy/LFD mice; 3) testing of anti- fibrotic/-inflammatory activities of the most promising XHALIP on mouse LFD models and translational studies on lung cells/tissues from LFD patients. This research will allow XHALIP to scale up to human testing. The multidisciplinary consortium with clinical, chemical, pharmacological biological expertise, and the preliminary basis of available stable formulations, guarantee the achievement of the aims.

Background / State of Art

In inflammatory LFD chronic allo- or auto specific immune insults lead to mesenchymal cell (MC) proliferation and ExtraCellular Matrix (ECM) deposition involving lung interstitial spaces and small airways. Bronchiolitis obliterans syndrome (BOS), a lung or allogeneic bone marrow transplant complication, and interstitial fibrosis in collagen vascular diseases (CTD-ILD) represent the most severe disorders and, albeit rare, they bear a bad prognosis and significant mortality. Few therapeutic options are available: immune modulators (conventional or small molecules such Janus Kinase Inhibitors: JKI) and the oral antifibrotic tyrosine kinase inhibitor (TKI) Nintedanib, which shows a slight improvement of clinical progression but is burdened by significant toxicity. Among the different options to improve its therapeutic index, a liposomal formulation linking HA represents an interesting and innovative strategy to improve targeted lung delivery of TKI or JKI. The tissue amount of HA, an ECM component synthesized by MC, is related to inflammation, influences MC migration and immune cell activation. HA plays a key role in fibrosis by interacting with its receptor, CD44. As recently demonstrated, primary MC cell lines isolated from Broncho-alveolar lavage (BAL) of patients (pts) with CTD-ILD and BOS highly express CD44, while normal, non-inflamed epithelium does not. A nanoparticle dependent targeting to CD44 thus could be a promising approach to maximize delivery of TKI and other anti

It’s available a Systematic Review on this topic? No

Hyphotesis and Specific AIMS

Hyphotesis and Significance

To satisfy the unmet need in the field of LFD, and from the evidence of a high CD44 expression by primary MCs, in the last years novel therapeutic approaches for LFD have been designed on the basis of the following considerations of nanocarriers: 1)Local delivery allows to reach high drug concentration in the lung avoiding systemic toxicity; 2)they permit to deliver to lung cells hydrophobic/toxic drugs otherwise difficult to administer as free drugs by inhalation; 3) they can be functionalized to induce an active uptake and targeting of pathogenic cells, sparing broncho-alveolar epithelium. A first evidence of the validity of this approach has been gained both in vitro on different lung cell populations (MC, epithelial cells, macrophages and other immune cells) and in vivo (mouse models of bleomycin induced pulmonary fibrosis (BM) and of heterotopic tracheal transplantation (HTT-M)). When anti CD44 coated imatinib loaded gold nanoparticles were administered intratracheally (see as reference Publication list from PI and Co-PI).

The exploitation of this approach has been hampered by the potential toxicity of gold after chronic intratracheal administration due to its poor biodegradability. To overpass this limitation, a liposomal formulation named XHALIP was therefore designed and fully characterized. We here provide limited data on the in vitro and in vivo activity of XHALIP since details are still confidential during patent evaluation. XHALIP was designed jointly by Turin University (Department of Drug Science Technology ▇▇▇▇ ▇▇▇▇▇▇ ▇▇▇▇▇▇▇¿s Lab) and IRCCS San ▇▇▇▇▇▇ Foundation (PI lab) which presented a patent file on

August 2021 (n° 102021000022253).

XHALIP are functionalized on the surface by HA in order to specifically target CD44+ cells and loaded in their internal aqueous core with either a TKI (including imatinib and nintedanib) or a JKI (ruxolitinib). When tested on MC and immune cells XHALIP showed a high stability in hydrophilic solutions or plasma and a significantly higher uptake by CD44+ than by CD44- cells. XHALIP were also able to significantly: inhibit MC proliferation and viability, decrease in vitro collagen production by MC, and inhibit molecular drug targets, and were lacking intrinsic pro-inflammatory activity. Finally, encouraging preliminary results were obtained in the BM by Unit2. Unit 1 and 2 are already performing preliminary toxicity studies with XHALIP, Unit 3 has been recently involved in the research group thanks to its expertise in the field of pharmacology, pharmacokinetics and nanomedicine. Our consortium includes: clinical competences from Unit 1 to obtain human cells and tissues for in vitro tests with the Ethics already set up; a longstanding experience in animal models mirroring human diseases (HTT-M and BM) from Unit 2 (UniMe) and finally of pharmacokinetics and tissue and cell tracking systems (Unit 3 IRFMN). Unit 1 will provide XHALIP, under the supervision of UniTo that agrees in the presentation of present project.Since the last months, in collaboration with Unit 1, all groups have been working on the issue of pulmonary delivery of NPs in both healthy and LFD mice. In particular, we were able to select the intranasal and inhalatory way of NP administration to obtain the better lung/filter organs ratio. Very interestingly, intranasally administered fluorescent NPs were able to cross the lung barrier and to rapidly segregate in lung immune cells, mainly macrophages .Moreover, in the last years UO3 optimized a combined visualization of both fluorescent NPs and of the drug conjugated to them by a serial sectioning of different organ tissues (tumors, brains, lungs) alternating MALDI mass spectrometry imaging (MALDI- MSI) and and fluorescent microscopy acquisitions. This has an extreme biological relevance since it will enable us to monitor different time-points both the kinetics of NP penetration and the processes of drug release and its potential degradation or clearance.

Preliminary Data

Over the last years Units 1 and 2 have been performing preliminary toxicity studies with XHALIP, whereas Unit 3 has been recently involved in the research group thanks to its expertise in the field of pharmacology, pharmacokinetics and nanomedicine. A series of preliminary results have been recently provided by our team. Below we have summarized the most significant unpublished preliminary results (PR, see attached supporting picture):

PR 1: Based on previous experimental data from our group, a biocompatible formulation of XHALIP was designed according to the following process: drug-loaded liposomes were prepared by hydrating a lipid film composed of 1,2- dipalmitoyl-sn-glycero-3-phosphocholine, cholesterol and L-¿-phosphatidylglycerol (70:30:3 molar ratio) in citrate buffer pH

4.5. The obtained suspension was then extruded and gel filtration with HEPES buffer pH 7.4 was used to change the external buffer. To further encapsulate the drug into liposomes the transmembrane pH gradient method was used. XHALIP decorated liposomes were prepared as described (▇. ▇▇▇▇▇▇▇, 2013). In these conditions, drug encapsulation efficiency value is about 85%, liposome size around 200 nm and zeta potential -30 mV. After 4-week storage at 4°C the formulations still conserve at least 90%-95% of the initial drug content and over this period no appreciable size and zeta potential change, precipitation or aggregation are observed.

PR2: Imatinib-loaded XHALIP were tested in vitro on primary MC from patients with CTD-ILD or BOS, lung-derived CD44+/- epithelial cell lines, and macrophages. Uptake of HA-conjugated liposomes by CD44+ cells was significantly higher than that of uncoated vehicles (Fig1 PR2). CD44-negative epithelial cells showed a significantly lower liposome uptake, while alveolar macrophages were actively internalizing this formulation. In vitro cell treatment caused a significant inhibition of cell proliferation (fig2 PR2), a significant inhibition of collagen production and of the expression of phospho-c-abl (Fig3 PR2).

PR 3: Intranasal XHALIP administration leads to a quick and massive penetration in lung parenchyma.

PR 4: Integrated spectroscopy and imaging enabled us to follow the fate of both nanoparticles and associated drugs in mouse sections over time.

PR5: Research lab of Unit1 has recently established thin cut lung slice cultures. (Fig1 UPR5 )

PR6: preliminary experiments in the mouse model of bleomycin-induced pulmonary fibrosis have been conducted by Unit 3 with positive encouraging results. Specific data are not available since confidential due to pending patent extension.

Picture to support preliminary data

Immagini last FINALIZZATA meloni pdf (2).pdf

Specific Aim 1

Our project will be carried out through three, tightly related, aims. AIM 1: XHALIP characterization and bio-nano interaction

As previously explained, the project makes use of nanocarriers already extensively characterized in vitro in terms of colloidal stability, loading capacity and drug release. The different XHALIP encapsulating different agents (Imatinib, Nintedanib and Ruxolitinib) have been already assessed and will be made available for the study by Unit 1 with the support and supervision of ▇▇▇▇ ▇▇▇▇▇▇▇¿s laboratory at UniTo. Two different nanoformulations will be employed: drug-unloaded fluorescent liposomes (F-LIP) for distribution studies and drug loaded liposomes (XHALIP) for safety/toxicology studies.

F-LIP will be firstly delivered by three administrations to a small group of healthy animals: aereosolization, intratracheal and intranasal route to choose the one which is associated to the highest and even lung distribution. The best method will be used for further experiments. Further experiments will be performed on healthy animals and in a small subgroup of BM animals (to assess whether in pathologic conditions we assist to a different permeability and distribution).

The safety of these liposomal formulations will be tested after an acute and a chronic treatment (following Acute Toxicity EOCD 425 and Chronic toxicity EOCD 452 protocols) at Unit 2 and 3. To discriminate between a potential toxicity due to the nanocarriers and the drug incorporated, another group of mice receiving the empty liposome and three groups receiving the three drugs respectively will be enrolled. To determine the impact of the different formulations observational, haemato- chemical and histopathological analyses will be carried out.

In addition to the toxicity analyses, we will evaluate the potential risk due to bio-accumulation of both liposomes and drugs in the main off-target organs (liver, spleen and kidneys) combining histology (liposomes will be labeled with a dye detectable by confocal laser excitation) and mass spectrometry (the three drugs will be HPLC/MS/MS). Moreover, by the overlapping of serial micrometric sections alternatively processed for the fluorescence detection (by virtual slider Olyvia- Olympus scanning) and the Maldi-Imaging (Hamamatsu Instruments) we will be able to follow the fate of carrier and cargo in different organs and at different time-points.

Finally we will also explore in vitro different-fluorescent XHALIP preparations on a model of alveolo-capillary barrier (Unit1) in order to analyze the ability to interact with mucous/surfactant layers and to cross the alveolo-capillary interface.

Specific Aim 2

Organ and cell lung Targeting

The second aim of this project is the evaluation of the lung lung penetration, the release and the potential accumulation in macrophages of the formulations selected by the safety studies. The in vivo study will be carried both in healthy and LFD mice at Unit 3 and 2 respectively. Firstly, an acute (single) treatment will be observed at different time-points (from 15 mins to 24 hours) to determine the kinetics of penetration of XHALIP in lungs, the ability to interact with target cells and the organ distribution of both liposomes and incorporated molecules. These analyses will be carried out by the procedures described in AIM 1. Both histology and pharmacokinetic analyses will be performed. Subsequently, in lungs collected from the animals used for safety studies, we will evaluate the amount of drug(s) and the interaction with target cells (for this study the animals will be sacrificed 1 hour after the last administration). This phase will be pivotal to select the best formulation able

to improve the lung delivery of drugs and minimizing the side-effects in off-target organs.

In order to get an adequate statistical robustness, according to the 3R principles, the sample-size of each experimental group will be calculated (with the aim of avoiding any unjustified and unethical use of an excessive number of animals). In order to make our study more translational, a comparison between the behavior of primary MC, lung epithelial and lung endothelial cells, macrophages, lymphocytes and neutrophils deriving from either mice or LFD patients will be carried out and comparing the uptake ability, the subcellular localization, interaction with molecular drug targets, and drug interference in response to pro-inflammatory or pro-fibrotic stimuli. Finally, Unit 1 will perform an analysis of F-LIP uptake and XHALIP activity on specific lung cells on thin cut lung slice (TCLS) cultures obtained from normal mouse and human lungs and from lungs of patients with LFD (explanted in case of lung Transplantation or Re-transplantation). These studies will be performed with confocal microscopy and electron microscopy + histology + immune histochemical examination of TCLS cultures. The results that will emerge from this part of the project will be crucial for defining the best treatment schedule and most relevant cellular targets in animal models of diseases (AIM 3).

Specific Aim 3

Efficacy assessment in lung fibrosis models

To this aim two well-characterized animal LFD models( the Bleomycin lung fibrosis Model and the Heterotopic trachea transplantation model respectively) will be used.The two highest performing XHALIP formulations (selected in 1st and 2nd phase of the project as for lung biodistribution and safety profile) will be administered to animals following specific administration time points (every 5 days to sacrifice starting from day 10 after intratracheal Bleomycin administration or from day 10 after heterotopic tracheal transplantation). The HTTM will require local administration of a liposome suspension into a subcutaneous pouch, while in BM model the best administration way among aereosolization, intratracheal and intranasal selected in AIM1 will be adopted. These time points have been selected in order to reflect at best human treatment needs (diagnosis of both these disorders is usually made at a relatively late time points when the fibrogenic process has already started) and considering the drug releasing profile of XHALIP. Liposome dosage will be selected according to results obtained in AIM1 and 2. As a control, the same dose of the selected free drug will be administered by parenteral/subcutaneous route. A vehicle-treated group will be included as control. In each model, the efficacy of the therapy will be evaluated by assessing a number of endpoints: mortality, histological damage by HE and Masson staining for architectural damage and fibrosis, Collagen determination, myeloperoxidase and Malondialdehyde staining and immunohystochemistry for TGF beta, lymphocyte, neutrophil and macrophage infiltration as well as Western blot analysis for several factors including, among others, TRAF-6, NF-kb, IKKb, mmp 9, mmp2, p-JNK, p-p38 and specific drug molecular targets analysis.

In addition, the expression of several cytokines and mediators involved in the development of the inflammatory /fibrogenic

response and endothelial mesenchymal transition (EMT) will be evaluated by RT-qPCR. The evaluation of these expression patterns at time 0 and at end of treatment with different XHALIP formulation will help in defining the best treatment schedules.

From this project we expect to both provide further indications on the potential translation of XHALIP and give an important boost to the development of innovative devices in the field of materials science and nanomedicine. If successful, the results that will emerge from this study may pave the way towards a future clinical translation.

Experimental Design Aim 1

XHALIP will be produced for the study by Unit 1 with the support and supervision of Prof ▇▇▇▇▇▇▇'▇ laboratory at UniTo. Two

different nanoformulations will be employed: drug-unloaded fluorescent liposomes (F-HALIP) for distribution studies and drug-loaded liposomes (XHALIP) for safety/toxicology studies (some XHALIP will also be produced as fluorescent with Rhodamine linked to the outer layer). F-HALIP will be firstly delivered to a small group of healthy animals to compare 3 different administration routes, namely aerosolization, intratracheal, and intranasal instillation with the aim of choosing the formulation with the highest and more uniform intrapulmonary distribution. The most efficient method will then be used for further experiments that will be performed in a small subgroup of BM animals to assess whether pathologic conditions may lead to different permeability and distribution. To discriminate the potential toxicity of empty nanocarriers, mice receiving F- HALIP will be then compared with 3 groups each receiving a XHALIP formulation loaded with one of the three drugs. To determine the impact of the different formulations, biochemical and histopathological analyses will be performed after the sacrifice of mice. We will measure and discriminate the biodistribution of both liposomes and drugs after synchronous administration of F-HALIP and XHALIP in all organs by an innovative approach combining serial micrometric sections alternatively processed for fluorescence detection (F-HALIP labeled with a dye detectable by confocal laser excitation) and Matrix Assisted Laser Desorption/Ionization (MALDI) imaging, where the spatial distribution of the chemical compounds incorporated into the liposomes will be detected by mass spectrometry. Moreover, a quantitative measurement of drug accumulation will be carried out by HPLC/MS/MS. This approach will allow us to follow the fate of carrier and cargo in different organs at different time-points. Finally Unit 1 will explore in vitro different F-HALIP preparations on a model of alveolo-capillary barrier in order to analyze their ability to interact with mucous/surfactant layers and to cross the alveolo- capillary interface.

Experimental Design Aim 2

The in vivo study will be performed both in healthy and LFD mice at Units 3 and 2 respectively. Firstly, an acute (single) treatment will be followed at different time-points from 15 minutes to 24 hours to determine the kinetics of penetration of XHALIP in lungs, their ability to interact with target cells and the organ distribution of both liposomes and incorporated molecules. Both histology and pharmacokinetic analyses will be performed. Subsequently, from lungs collected from the animals used for safety studies (see experimental design aim 1), we will evaluate the amount of drug(s) and the interaction with target cells (for this study the animals will be sacrificed 1 hour after the last administration). This phase will be pivotal to select the formulation providing the most efficient lung delivery of drugs and lowest side-effects in off-target organs. In order to make our study more translational, a comparison between the behavior of primary MC, lung epithelial and endothelial cells, macrophages, lymphocytes and neutrophils deriving from both mice and LFD patients will be performed to compare uptake ability, subcellular localization, interaction with molecular drug targets, and drug interference in response to pro- inflammatory or pro-fibrotic stimuli. Finally, Unit 1 will perform an analysis of F-HALIP uptake and XHALIP activity on specific lung cells on thin-cut lung slice (TCLS) cultures obtained from normal mouse and human lungs and from lungs of patients with LFD (explanted in case of lung Transplantation or Re-transplantation). These studies will be performed with confocal microscopy, electron microscopy, conventional histology and immunohistochemistry of TCLS cultures. The results that will emerge from this part of the project will be crucial for defining the best treatment schedule and most relevant cellular targets to be translated in animal models of diseases (AIM 3).

Experimental Design Aim 3

Animal models will be prepared as follows

Animals: CD1 mice (6-12 weeks old) for BM model and C57BL/6 and BALB/c mice (6¿12 weeks old) for HTT model, will be housed in a controlled environment and will be provided with standard rodent chow and water. Procedures involving animals and their care will be conducted in conformity with the Italian regulations on protection of animals used for experimental and other scienti¿c purpose (D.M. 116192) as well as with the EEC regulations (O.J. of E.C. L 358/1 12/18/1986).

BM: Bleomycin administration will be performed as previously described (▇▇ ▇▇▇▇▇ et al., 2016). Bleomycin sulphate (0.1 lU per mouse) is delivered by an intratracheal administration (▇▇▇▇▇ et al., 2017). Experimental groups: 1) Bleomycin: Mice receiving bleomycin administration and treated daily with F-HALIP; 2) Control group: mice receiving intratracheal instillation of saline (0.9% w/v) instead of bleomycin, and treated daily with F-HALIP; 3) Treatment group: Mice receiving bleomycin and treated with XHALIP every 5 days to sacrifice starting from day 10 after intratracheal bleomycin administration.

HTT: Tracheas from C57BL/6 mice will be implanted into Balb/c mice (allogeneic) or C57BL/6 mice (syngeneic) as previously described (▇▇▇▇ ▇▇▇▇ et al., 2015). Experimental groups: 1) Control group: animals subjected to orthotopic tracheal transplantation and treated with F-HALIP; 2) Treatment group: animals subjected to orthotopic tracheal transplantation and treated with XHALIP every 5 days to sacrifice starting from day 10 after heterotopic tracheal transplantation; 3) Sham-operated mice not subjected to orthotopic tracheal transplantation but treated with F-HALIP. XHALIP will be administered to animals following a specific time schedule (every 5 days to sacrifice starting on day 10 after intratracheal bleomycin administration or heterotopic tracheal transplantation). HTTM will require local administration of a liposome suspension into a subcutaneous pouch, while in BM model the best administration route between aereosolization, intratracheal and intranasal instillation selected in AIM1 will be adopted. This schedule has been designed in order to reproduce at best human treatment requirements, since diagnosis of both disorders is usually made at a relatively late stage when the fibrogenic process is already established, and taking into consideration the profile of drug release from XHALIP. Liposome dosage will be decided according to the results obtained in AIM1 and 2. As a control, the same dose of the selected free drug will be administered by parenteral/subcutaneous route. A

In each model, the efficacy of the therapy will be evaluated by assessing the following endpoints: mortality, histological

damage by HE, Masson staining for architectural damage and fibrosis quantification, immunohistochemical stains for collagen, myeloperoxidase, TGF beta, lymphocyte subset characterization, neutrophils and macrophages count, as well as Western blot analysis for several factors including, among others, TRAF-6, NF-kb, IKKb, mmp 9, mmp2, p-JNK, p-p38 and specific drug molecular targets (▇▇▇▇▇▇▇▇▇▇▇▇ et al., 2015),(▇▇▇▇▇ et al., 2014),(▇▇ ▇▇▇▇▇ et al., 2016). In addition, the expression of mRNA levels of several cytokines and mediators involved in the development of the inflammatory/fibrogenic response and endothelial mesenchymal transition (EMT) will be evaluated by RT-qPCR. The evaluation of these expression patterns at time 0 and at the end of treatment with different XHALIP formulations will help in defining the best treatment schedules.

Metodologies and statistical analyses:

Methodologies (describe all measures taken to minimize / avoid bias)

In vitro experiments:

F-HALIP uptake in lungs will be evaluated both by a qualitative (confocal microscopy) and by a quantitative method (flow cytometry). All other variables (cell viability and cell proliferation, endothelial-mesenchymal-transition) will be evaluated by quantitative/semi-quantitative methods as flow cytometry, MTT test, expression studies and WB analysis. In order to avoid bias replicate experiments and adequate negative and positive controls (untreated cells, cells treated with free drugs -not loaded in liposome- HA-uncoated drug-loaded liposomes) will always be included in the experiments. We will also perform replicated experiments on the in vitro model of alveolo-capillary unit and on precision cut lung slices of fresh explanted fibrotic and BOS lungs. Histological and immunohistochemcal evaluation of PCLS with blinded lectures will be provided in order to avoid interpretation bias.

Animal studies:

In order to get an adequate statistical robustness, according to the 3R principles, the sample size of each experimental group will be calculated to avoid any unjustified and unethical use of an excessive number of animals.

1. Longitudinal Biodistribution: In vivo ad ex vivo imaging to track the fate of fluorescent liposome composing the XHALIP in mice will be carried out measuring tissue fluorescence levels by Lumina-X5 IVIS Imager system (PerkinElmer) as previously described (Violatto MB. Et al., 2019.). To maximize the signal/background ratio, liposomes will be functionalized with a dye falling in the infra-red that is the best condition to minimize the tissue autofluorescence.

2. PK Studies: To detect the presence of the therapeutic agents in plasm, lungs and off target tissues drug levels will be measured by HPLC/MS in plasma and different tissues harvested at sacrifice, in order to determine pharmacokinetic profiles (e.g. the half-time of elimination in plasma) and, mainly, the liver-to-plasma ratios as previously described (▇▇▇▇▇▇

A. et al., 2022). Positive standars (the calibration curve of the drug alone) and inner controls (measurements in mice not receiving the drugs) will be used to minimize the experimental bias.

3. Combined Confocal-Maldi imaging: this approach will be carried out to determine the interaction between carriers and drugs in tissues. The evaluation of spatial distribution of both liposomes and therapeutic agents forming the XHALIP will be achieved by serial sectioning of lung and off-target tissues and alternate evaluation of the fluorescence with confocal microscopy and the spectroscopy of analytes (drugs) by ▇▇▇▇▇ ▇▇▇▇▇▇▇, as previously reported (▇▇▇▇▇▇▇▇ S Sci Rep. 2016). The calibration curve associated with the spatial spectroscopy will be drawn using internal standards, whereas untreated animals will be used as inner control in order to avoid confounding results due to the tissue background signals.

Animal models pitfalls and Solutions

To monitor any adverse effects not foreseen following the administration of the substances under examination, the state of health of the animals will be kept under observation by the researchers and by the staff of the animal facility for the duration of the study.

The risks for the operator are linked to the use of laboratory animals (possibility of being bitten or scratched by animals or injuring oneself with sharp objects such as needles). All staff are trained in the use of appropriate personal protective equipment and suitable hoods (eg hoods for the elimination of litter); in addition, the staff is trained in the use of specific operating procedures to minimize the risk of injury.

Methods of data collection (Indicate the data that will be collected, the tools used)

All experimental data will be collected by the three Units according to their coordinating role in the specific WPs. Raw data obtained in each single experiments (conducted in any of the participant Units) will be collected in excel files under the responsability of the WP coordinator, and will be available for review for the whole length of the project.

Statistic plan (calculation of statistical data)

Since clinical variables are not included in this study, statistical analysis will be performed straightforward on experimental replicates including appropriate negative and positive controls.

Statistical analysis (describe the main statistical analysis)

When normally distributed variables will be analysed, groups will be compared by means of T-Student test or one-way ANOVA, and group means will be then compared by ▇▇▇▇▇▇▇'▇ test using a statistical package (GraphPad Prism 4.0). A p value of <0,05 will be considered as statistically significant. If variables will not be normally distributed non parametric tests will be applied.

Timing of analysis data (indicate duration of study: duration of enrollment, of therapy, follow-up etc)

Planning of the study waypoints is detailed below.

We do not envisage difficulties in patient recruitment for the ex vivo experiments (BAL lung cell isolation and cultures, precision cut lung slices cultures) since these experiments are already performed routinely in Unit 1 lab on samples obtained from patients referring to UNIT1 for diagnosis and treatment of lung conditions acconding to protocols approved by EC.

Analysis of in vitro and pharmacokinetic/biodistribution experiments will be crucial for the calculation of animal model sample size and planning of dose and time of liposome administration to mice.

Animal experiments will be planned according to the below reported schedule (see also GANTT Chart); statistical analysis will be performed at the completion of experiment sets.

WP 1 liposome preparation (1-30M)

task 1.1 preparation of fluorescent HA-coated and uncoated LIPOSOMES (1-24M); task 1.2 preparation of Imatinib-loaded HA-coated and uncoated Liposomes (7-30M); task 1.3 preparation of nintedanib-loaded HA-coated and uncoated liposomes (10-30M); task 1.4 preparation of ruxolitinib-loaded HA-coated and uncoated Liposomes (10-30M).

WP 2 in vitro assessment of uptake and activity of different XHALIP preparations (1-12M)

task 1.1 uptake by MC, epithelial cells, endothelial cells, macrophages and other inflammatory cells (3-12M); task 2.2 distribution across alveolo-capillary barrier in vitro (3-12); task 2.3 assessment of in vitro activity on different cell types (proliferation, collagen production, migration, viability, cytokine release): (3-12M); task 2.4 assessment of distribution in PCLS cultures (3-30M)

WP 3 pharmacokinetics studies in Animals (3-29M)

task 3.1 Combined Maldi-Confocal Imaging optimization for the tracking of XHALIP in lungs (3-29M); task 3,2 Biodistribution, nanosafety assessment in animals (10-23M); task 3.3 Pharmacokinetics (17-29M)

WP4 Animal Toxicology (8-31M)

task 4.1 acute toxicity studies (8-21M); task4.2 chronic toxicity studies (8-30M); Task 4.3 analysis of cell toxicity (20-31M) WP5: Activity in animal models (8-30M)

Task 5.1 animal model of fibrosis (8-30M); Task 5.2 animal model of BOS (8-30M).

WP6 coordination and dissemination Task 6.1 coordination (1-36M); Task 6.2 dissemination (10-36M)

Expected outcomes

We have previously demonstrated that XHALIP nanoformulation is a reliable and innovative device to maximize the stability and lung macrophage uptake of TK inhibitors drugs. From this project we expect both to provide further indications on the potential translation of XHALIP (Toxicity, PK and Biodistribution, Ativity in animal models BM and HTT) and to give an important boost to the development of innovative devices in the field of materials science and nanomedicine. If successful, the results that will emerge from this study may pave the way towards future clinical translation.

Risk analysis, possible problems and solutions

Aim 1 Protocols of liposome preparation have been fully standatdized. For this reason we do not envisage any potential risk Aim:2.1 Potential risk: Toxicity or bio-accumulation in off-target organs of XHALIPs. Solution: Modify the posology (doses, frequency, route of administration).

Aim:2.2 Potential risk: Reduced release of drug at the late symptomatic phase. Solution: Modify the temporal window by increasing the doses or the frequency of treatment along the clinical progression.

Aim:2.3 Potential risk: Low efficacy due to accelerated clearance of XHALIP or limited penetration in target cells Solution: Modify the liposome surfaces to accelerate the penetration in lungs and in target cells

Aim 3.Potential risk : we do not envisage problems in the assessment of XHALIP efficacy thanks to satisfactory preliminary data in BM; technical problems related to HTT can be forecast, due to the need of XHALIP instillation in the subcutaneous pouch In this case the use of Alzet pump will be considered to guarantee adequate drug delivery.

Significance and Innovation

Significance: Dissimilarly to other projects of preclinical nanomedicine where the goal is to bypass filter organs to increase the accumulation of nanoparticles into the target, we are exploiting the inhalatory way in order to maximize drug load in lungs. Although this proposal focuses on inflammatory-fibrotic lung disorders, in case of positive results this approach could be easily extended to other lung disorders, such as pulmonary hypertension or lung cancer. In this latter case the evidence that XHALIP are efficiently internalized in macrophages will be relevant in the view of modulation of tumor-specific immune responses (often inhibited by tumours).-

Innovation: None of the liposomes registered for inhalation so far (amikacin- or amphotericin B-loaded vehicles) are targeted or addressed to fibrogenic disorders. The development of a targeted TKI formulation adequate for inhalatory administration would represent an absolute innovative step in the field of nano-based therapy.

Description of the complementary and sinergy research team

Our consortium includes: -clinical expertise (Unit1) in diagnosis and management of LFD,crucial to obtain cells and tissues for in vitro tests ( available approval EC); -longstanding experience in mice models which mirror human chronic airway rejection ( HTT-M) and pulmonary fibrosis ( BM) from Unit 2 (UniMe), and -extended experience of pharmacokinetics and tissue and cell tracking systems (Unit 3 IRFMN). Unit 1 will provide XHALIP, under the supervision of UniTo that agrees in the presentation of this project. The present project will involve also some young researchers at the three Units. The coordinator of the project, ▇▇▇▇. ▇▇▇▇▇▇▇▇ ▇▇▇▇▇▇ is a respiratory and lung transplant physician with research experience who already acted as PI of an euronanomed research consortium, the PI of UNIT 2 ▇▇▇▇▇▇▇ ▇▇ ▇▇▇▇▇, as a pharmacologist, has developed and studied previously the 2 animal models and performed animal toxicology studies also in collaboration with Unit 1. The PI of the UNIT 3, Dr ▇▇▇▇▇ ▇▇▇▇▇▇, has a relevant experience in pharmacology and nanomedicine and his group is specialized in nanotracking and pharmacokinetics. He is collaborating with Unit 1 in the last year for the development of a nanotechnological approach to chronic lung diseases . The close relationship between researcher coming from different scientific fields is strongly required to get to a real impact in terms of technological exploitation of nanomedicine products and dissemination of the results.

Training and tutorial activities

The complementary background of the project members will allow the organization of multidisciplinary training sessions to share ideas, knowledge, and skills bridging different expertise from chemistry to medicine. A workshop on nanotechnology and clinical translation will be organized Unit 1. Attendance will be open to University students,hospital researchers and general public. A series of seminars on preclinical imaging and nanokinetics will be held at the Unit 3. In this context, the speakers will consider the chemical problems related to both diagnostics and therapy. A selected number of students and researchers will have the possibility to visit the mouse clinic of Unit 3 and the Radiology department ofUnit 1 to compare in vivo imaging instruments used for mice and patients (e.g. MicroCT, MRI, EcoScan). Finally, symposia and conferences on preclinical/clinical lung disorders will be hosted both at the IRCCS Foundation Policlinico San ▇▇▇▇▇▇ and at Unit 2.

Bibliography

▇▇▇▇▇▇▇ S, ▇▇▇▇▇ C, ▇▇▇▇▇ ▇▇▇▇▇ E, et al. Hyaluronic acid-coated liposomes for active targeting of gemcitabine. European Journal of Pharmaceutics and Biopharmaceutics 2013; 85,373-380

▇▇▇▇▇ E, ▇▇▇▇▇▇▇ M, ▇▇▇▇▇▇ E, et al. Thymosin beta4 reduces IL-17-producing cells and IL-17 expression, and protects lungs from damage in bleomycin-treated mice. Immunobiology 2014;219:425-431.

▇▇▇▇▇ J, ▇▇▇▇▇▇▇, GR, ▇▇▇▇▇▇ C, et al. AQX-1125, small molecule SHIP1 activator inhibits bleomycin-induced pulmonary fibrosis. Br J Pharmacol 2017;174:3045-3057

▇▇ ▇▇▇▇▇ ▇, ▇▇▇▇▇▇▇▇▇▇▇▇ D, ▇▇▇▇▇ R, et al. Ultramicronized palmitoylethanolamide (PEA-um((R))) in the treatment of idiopathic pulmonary fibrosis. Pharmacol Res 2016; 111:405-412

▇▇ ▇▇▇▇▇ ▇, ▇▇▇▇▇▇▇ M, ▇▇▇▇▇ R, et al. Protective effects of ultramicronized palmitoylethanolamide (PEA-um) in myocardial ischaemia and reperfusion injury in VIVO. Shock 2016;46:202-13.

▇▇▇▇ M, ▇▇▇▇ X, ▇▇▇▇ HL, et al. Integrated analysis of transcription factor, microRNA and LncRNA in an animal model of obliterative bronchiolitis. Int J Clin Exp Pathol 2015;8:7050-7058

▇▇▇▇▇▇▇▇ S, ▇▇▇▇▇▇ L, ▇▇▇▇▇▇▇▇▇▇ P, et al. 3D Mass spectrometry imaging reveals a very heterogeneous drug distribution in tumors. Sci Rep 2016;6:37027

▇▇▇▇▇▇▇▇▇▇▇▇ D, ▇▇▇▇▇▇ E, Siracusa R, et al. Protective effect of polyphenols in an inflammatory process associated with experimental pulmonary fibrosis in mice. Br J Nutr 2015;114:853-65.

▇▇▇▇▇▇ A, ▇▇▇▇▇▇▇▇ MB, ▇▇▇▇▇▇▇ E, et al. The mode of dexamethasone decoration influences avidin-nucleic-acid-nano- assembly organ biodistribution and in vivo drug persistence. Nanomedicine 2022;40:102497

▇▇▇▇▇▇▇▇ MB, ▇▇▇▇▇▇▇ E, ▇▇▇▇▇▇▇▇ L, et al. Dexamethasone conjugation to biodegradable avidin-nucleic-acid-nano- assemblies promotes selective liver targeting and improves therapeutic efficacy in an autoimmune hepatitis murine model. ACS Nano 2019;13:4410-4423

Timeline / Deliverables / Payable Milestones

Deliverables

D 1.1 preparation of stable F-HALIP and XHALIP with the 3 drugs [M3¿>30].

D 1.2: Release and sharing of protocols for nanosafety and PK studies of XHALIPs in healthy and diseased mice, and standardization of procedures for the generation of models [M9].

D 1.2: Mid-term report with main results achieved in toxicity screening of XHALIPs in cells and mice [M18]

D 2.1: Release and sharing of protocols of combined in vivo and ex vivo analyses in mice treated with XHALIP [M18].

D 2.2: Final report with the results on the biodistribution and pharmacokinetics of different XHALIP preparations in cells and in healthy and diseased mice [M 36].

D2.3 fFnal report on the toxicity studies of different XHALIP preparations both in vitro and in vivo

D 3.1 Identification of the treatment schedule providing the best disease control according to histo-molecular studies [M 36].

D 3.2 Final report describing the main results of therapy achieved in the chronic treatment of BM and HTT [M 31].

Milestones 18 month

M18 1 conclusive evidence on XHALIP and F-HALIP in vitro uptake and activity on different cell types, including their ability to cross alveolo-capillary barrier and in vitro uptake

Milestones 36 month

M36 1 conclusive evidence on acute and chronic toxicity of different XHALIP preparations M36 2 conclusive evidence on biodistribution and phamacokinetic studies

M36 3 conclusive evidence on the activity of different XHALIP preparations in animal models (BM and HTT) M36 4 conclusive evidence on specific cell uptake and activity on PCLS cultures

Gantt chart

gantt finalizz2022 meloni pi.pptx

Equipment and resources available

Facilities Available

The group of Dr ▇▇▇▇▇▇ (IRCCS-▇▇▇▇▇ ▇▇▇▇▇ Institute) has access to a fully equipped laboratory with modern mass spectrometers.and instruments available for performing HPLC-MS/MS at low and high resolution: API 3000 and API 5500 triple quadrupoles, and a nano-HPLC LTQ-Orbitrap-XL FT/MS and MALDI-Imager-Shimatsu. In addition, a mouse clinic equipped with modern instruments for in vivo imaging in rodents (Magnetic Resonance Imaging, Fluorescence Molecular Tomography, MicroCT, Eco-Scan, Two-Photon Confocal Microscopy) and instruments for imaging of cells and tissues at cellular and subcellular resolution (Olympus Fluoview microscope, Atomic Force Microscope, transmission electron microscope and Super-Resolution Microscope) are available and currently in use. Dr. Meloni¿s Laboratory has an established qualification in nanomedicine research including study of cell uptake and biological activity of new nanocarriers. The lab has also acquired experience in the synthesis of XHALIP according to protocols established and shared with University Of Turin (▇▇▇▇ ▇▇▇▇▇▇▇¿s laboratory). Lab is provided with equipment for biochemistry and molecular biology, cell cultures, cellular and molecular biology, confocal laser scanning microscope (Olympus) and vibratome (Compresstome® VF 210-0Z Vibrating Microtome). Finally Unit2 has an animal facility + all equipment for histochemistry and immunohistochemistry as well as WB or expression studies.

Subcontract

In this project subcontract is not required. ▇▇▇▇ ▇▇▇▇▇▇▇ from the University of Turin agreed in providing full technical assistance to Unit 1 for the synthesis of XHALIP according to the already established protocol that Unit 1 and University of Turin developed over the last three years.

Translational relevance and impact for the National Health System (SSN)

The present project has a concrete translational spillover. XHALIPare a very promising formulation and, according to preliminary data, their further development by means of this research, will provide a strong base for the scale up to human application. Our liposomes will be the first targeted drug-loaded liposomes to be applied for the local treatment ofLFD which, whilst classified as rare diseases, are still associated toa high rate of morbidity and mortality. In addition, there is a strong rational basis for the application of XHALIP in the treatment of other pulmonary disorders, where the use of a TKI: imatinib has been halted by its systemic side effects. Finally, as a consequence of this research, other possible targeted liposomal formulations specifically designed for the inhalatory application could be developed for human application.

▇▇▇▇▇▇ ▇▇▇▇▇▇▇▇

Birth date: 30/09/1961

Institution: Fondazione Policlinico San ▇▇▇▇▇▇

Department/Unit: UOS transplant center

UOCRheumatology

Position Title: Principal investigator

Principal Investigator (PI) Profile

Personal Statement:

F.M has experience as project cocordinators and , thanks to an already established collaboration with both Unit 2 and 3, PI and Co PI (who possess a solid clinical background and provide skills and samples from ex vivo studies) will be able to:

- Coordinate the project

- Provide already set up- stable XHALIP preparations to the consortium

- Perform all ex vivo tests on Human samples (in vitro test on primary cell cultures, on models of alveolar -capillary barrier, and on thin cut lung tissue slices) from patients with CTD-ILD or BOS

Positions and honors

Official H index: 33.0

( autocertificated )

Scopus Author Id:▇▇▇▇▇▇▇▇▇▇ ORCID ID:0000-0003-4014-0617 RESEARCH ID:K-5828-2016

Other awards and honors

From September 2018 to September 2021 secretary of Assembly 8 (Thoracis Surgery and Transplantation) of the European Respiratory Society, Chair Elect since 2021

Member of the ERS Long range planning committee of the Thoracic Surgery and Lung Transplantation assembly Since 2018 she gained the National Scientific Qualification as Full professor in the Discipline of Respiratory Disease

Other CV informations

During her career she developed a scientific interest on the immunological mechanisms of respiratory diseases, PHD was completed at the University of Bern focusing on the biological activity of IL8 in lung fibrosis. Later, she was involved in the following fields: the pathogenesis of interstitial lung fibrosis associated to collagen vascular diseases (CTD-ILD) and in the last 20 yrs, immune and fibrogenic mechanisms of chronic lung rejection. Since 2014 she started developing a local nano- based treatment of these diseases. Present project represent the continuation of a 2017 euro-nanomed ARROW NANO project (PI F.M), that brought to the development of XHALIP. From the clinical point of view F.M is now responsible of the follow up of Lung recipients and , together with Co-PI of CTD-ILD.

* Position: F=First L=Last C=Correspondent O=Other N=Not applicable

** Autocertificated

* Position: F=First L=Last C=Correspondent O=Other N=Not applicable

** Autocertificated

Codullo Veronica

Birth date: 21/08/1980

Institution: Fondazione Policlinico San ▇▇▇▇▇▇

Department/Unit: UOS transplant center

UOCRheumatology

Position Title: CO-PI

CO-PI Profile

Personal Statement:

Clinical expertise in the management of pulmonary fibrosis with an inflammatory background, patient enrolment, in vitro and in vivo data interpretation

Positions and honors

Official H index: 25.0

( autocertificated )

Scopus Author Id:17134491200 ORCID ID:0000-0003-2557-8514 RESEARCH ID:AAC-7384-2022

Other awards and honors

Since 2017 National Qualification of Associate Professor in Rheumatology (ASN)

Other CV informations

Dr ▇▇▇▇▇▇▇ is an expert in pulmonary complications of systemic autoimmune diseases, with a focus on connective tissue diseases. Since 2017 she is in charge of the Scleroderma Unit at IRCCS Policlinico San ▇▇▇▇▇▇ Foundation, a dedicated outpatient clinic for patients with Systemic Sclerosis (Ssc). She has a lab backgroud which she has consolidated in numerous experiences abroad (University of Glasgow, Amsterdam and Paris) and she has ongoing collaborations with European experts in the field (Prof Y Allanore, Paris, France, Prof J Distelr, Erlangen, Germany)

* Position: F=First L=Last C=Correspondent O=Other N=Not applicable

** Autocertificated

di ▇▇▇▇▇ ▇▇▇▇▇▇▇

Birth date:

10/08/1975

Institution: University of Messina Department/Unit: Department of Pharmacology Position Title: collaborator

Biographical Sketch Contributors. N. 2

Personal Statement:

Prof ▇▇ ▇▇▇▇▇ will keep on with XHALIP development planning all toxicology and all animal model of LFD. Prof ▇▇ ▇▇▇▇▇, PhD in Experimental Medicine, PhD in Biotechnology Pharmacology and Clinical Pharmacology and Specialized in Clinical Pathology, is now Full Professor of Biochemistry at the Department of Veterinary Sciences of the University of Messina, Italy. Author of over 335 publications in renowned journals and 11,692 citations. His areas of scientific interest are autoimmunity, acute and chronic inflammation and modulation of the signal pathway. She has various skills in analytical methodologies including experimental animal model, histology, immunohistochemistry and molecular biology techniques for the study of protein expression.

Positions and honors

Official H index: 56.0

( autocertificated )

Scopus Author Id:57218288397 ORCID ID:0000-0001-6725-8581 RESEARCH ID:K-5175-2016

Other awards and honors

Dr. ▇▇ ▇▇▇▇▇ won in 2009 the "Young Researcher" for the scientific production for the macro-area drug Biomedical conferred by the University of Messina. Additionally, she is member of several scientific society including: the Italian Society of Pharmacology (SIF),

the Italian Biochemistry Society (SIB), the American Society Pharmacology and experimental Therapeutics (ASPET)

▇▇▇▇▇▇ ▇▇▇▇▇

Birth date: 07/01/1970

Institution: IRCCS ▇▇▇▇▇ ▇▇▇▇▇

Department/Unit: Department biochemical research and molecular

pharmacology

Position Title: Collaborator

Biographical Sketch Contributors. N. 3

Personal Statement:

Dr ▇▇▇▇▇▇ will be involved in the supervision of all animal tests and will be designing and supervising biodistribution and pharmacokinetic studies in animals.

His experience in the field of pharmacology and pharmacokinetics is large and longstanding, this will guarantee the completion of Aim 1 and 2

From 2004 to 2014 he was mainly involved, first as post-doc and then as permanent researcher, in studies focused on stem cell therapy and preclinical imaging. Since 2014 he is the leader group of the Nanobiology Unit at the ▇▇▇▇▇ ▇▇▇▇▇. During his activity he published and revised more than 150 manuscripts and coordinated Italian and European projects. He belongs to preclinical Ethical Committees

Positions and honors

Official H index: 24.0

( autocertificated )

Scopus Author Id:55911359200 ORCID ID:0000-0002-0239-9532 RESEARCH ID:AAB-2901-2020

Other awards and honors

No award to be reported

▇▇▇▇▇ ▇▇▇▇▇▇▇

Birth date:

21/01/1991

Institution: University of Messina Department/Unit: Department of Pharmacology Position Title: collaborator

Biographical Sketch Contributors. N. 5

Personal Statement:

Dr. ▇▇▇▇▇▇▇ ▇▇▇▇▇, Researcher in Biochemistry at the University of Messina, completed her PhD in Applied Biology and Experimental Medicine at the University of Messina and the Yale University School of Medicine and postdoctoral studies at the University of Messina. She has worked as a researcher for preclinical pharmacology studies and biochemical activities (primary cultures in vitro and experimental models in vivo). You have published more than 80 articles in renowned journals on biochemistry and oxidative stress.

Her role in this project will be to collaborate in the generation and analysis of all models of disease

Positions and honors

Official H index: 27.0

( autocertificated )

Scopus Author Id:57192308076 ORCID ID:0000-0003-0223-1403 RESEARCH ID:AAC-1745-2019

Other awards and honors

Dr. ▇▇▇▇▇ won in 2018 the SIF Research Scholarship for Short Periods Abroad for a study stay at Yale University, School of Medicine, Department of Pharmacology, USA. Additionally, she won the travel grant for the participation in the 45th FEBS Congress for the Italian Society of Biochemistry and Molecular Biology (SIB). she is member of several scientific society including: the Italian Society of Pharmacology (SIF), SIB, the American Society Pharmacology and experimental Therapeutics (ASPET)

Expertise Research Collaborators

* Position: F=First L=Last C=Correspondent O=Other N=Not applicable

** Autocertificated

* percentage calculated as average value between all the Operating Units.

Report the Co-Funding Contributor:

salaries of Meloni (2pm/yeart), Codullo (2pm/year), Bigini (2pm/year) and Di Paola (1 pm/year) are included. Salaries are provided by IRCCS San ▇▇▇▇▇▇, IRCCS ▇▇▇▇▇ ▇▇▇▇▇ and University of Messina, respectively.

Proposed total budget UO1 Institution: Fondazione Policlinico San ▇▇▇▇▇▇ (Euro)

Report the Co-Funding Contributor:

Proposed total budget UO2 Institution: University of Messina (Euro)

Report the Co-Funding Contributor:

Proposed total budget UO3 Institution: IRCCS ▇▇▇▇▇ ▇▇▇▇▇ (Euro)

Report the Co-Funding Contributor:

Principal Investigator Data

Cognome: MELONI Nome: FEDERICA

Codice fiscale: ▇▇▇▇▇▇▇▇▇▇▇▇▇▇▇▇ Documento: Passaporto, Numero: ▇▇▇▇▇▇▇▇▇ Data di nascita: 30/09/1961

Luogo di nascita: pavia Provincia di nascita: PV

Indirizzo lavorativo: ▇▇▇▇▇▇▇▇ ▇▇▇▇▇ ▇▇ ▇▇▇▇▇: ▇▇▇▇▇

CAP: 27100

Provincia: PV

Email: ▇▇▇▇▇▇▇▇▇▇.▇▇▇▇▇▇@▇▇▇▇▇▇▇.▇▇ Altra email: ▇.▇▇▇▇▇▇@▇▇▇▇▇▇▇.▇▇.▇▇ Telefono: ▇▇▇▇▇▇▇▇▇▇▇▇▇

Altro telefono: ▇▇▇▇▇▇▇▇▇▇

Fax: ▇▇▇▇▇▇▇▇▇▇

Qualifica: Universitario covenzionato Struttura: uos transplant center

Istituzione: fondaz irccs policlinico san ▇▇▇▇▇▇ Datore/ente di lavoro? Si

Datore/ente di lavoro SSN? No

Nome datore/ente di lavoro non SSN: Università di Pavia Nome istituzione SSN: Fondazione irccs policlinico san ▇▇▇▇▇▇

Tipo contratto: Distaccato presso IRCCS/Ente SSN a seguito Convenzione Università/Altro Ente Ricerca

Con l'invio della presente proposta si dichiara che la stessa o parti significative di essa non sono oggetto di altri finanziamenti pubblici o privati e che di conseguenza vi è assenza del c.d. doppio finanziamento ai sensi dell'art. 9 del Regolamento (UE) 2021/241, ossia che non ci sia una duplicazione del finanziamento degli stessi costi da parte di altri programmi dell'Unione, nonché con risorse ordinarie da Bilancio statale.

By submitting this proposal, I declare that no significant part or parts of it are recipient of any other public or private funding and that consequently there isn't any so-called double financing pursuant to art. 9 of Regulation (EU) 2021/241, i.e. that there is no duplication in the financing of the same costs by other Euopean Union programs or any other ordinary resources from the State budget.

Project validation result

Message:

Success

First Year

Second Year

Third Year

12

11

10

9

8

7

6

5

4

3

2

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12

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Unit 3 Univ of Messina Unit 2 IRCCS ▇. ▇▇▇▇▇

Unit 1 IRCCS San ▇▇▇▇▇▇

WorkPackages

WP1 liposome preparation C

task 1.1 preparationof fluorescent HA coatedandUn-coated LIPOSOMES X

task 1.2 preparationof Imatinibloaded HA coatedand UN-coatedLiposomes X

task 1.3 preparationof nintedanibcloadedHA coatedandun-coatedliposomes X

task 1.4 preparationof ruxolitinib loadedHA coatedand Un-coatedLiposomes X

WP2 invitro a se sment of uptake andactivity of different XHALIPpreparations C P

task 1.1 Uptake by MC, Epithelial cells, Endothelial cellsmacrophagesand other inflammatory cells X task 2.2 distributionacrossalveolo-capillarybarrier invitro X

task 2.3 invitro activityondifferent cell types(prioliferation , colagen production, migration,viability, cytokinerelease) X

task 2.4 distruibutionin PCLScultures X X

WP3 pharmacokinetics studiesin Animals P C task 3.1 Combined Maldi-Confocal Imagingoptimization for thetrackingof XHALIP inlungs task 3,2 Biodistribution, nanosafety assessment inanimals

task 3.3 Pharmacokinetics

task 4.1 acute toxicity studies task4.2 chronictoxicitystudies Task 4.3analysisof cell toxicity

Task 5.1animal model of fibrosis Task 4.5animal model of BOS

Task 6.1coordination Task 6.2dissemination

C: coordinator / P participant

WP4 Animal Toxicology

WP5: Activity in animal models

WP6 coordination and di semination

P P C

x x

x x x

P P C

X X X

X X X

C P P X

X X X

PR2

CTR

LIP

LIP-HA

Fig. 1: Uptake of liposomes coated with HA (LIP-HA) and uncoated (LIP) by MC isolated from ILD-CTD

PR2

A

% di vitalità cellulare (% vs CTR 100%)

120

100

BOS derived MC

B

% di vitalità cellulare (% vs CTR 100%)

120

100

ILD-CTD derived MC

80

60

40

20

0

24h

48h 72h

LIP-Im

LIP-HA-Im

Im

80

60

40

20

0

24h 48h 72h

LIP-Im

LIP-HA-Im

Im

C

% di vitalità cellulare (% vs CTR 100%)

160

140

120

100

80

60

40

A549 cell line (CD44 high)

LIP-Im

LIP-HA-Im

Im

Fig. 2: Cell viability

MC from BOS patients (A), MC from ILD-CTD patients (B), A549 cell line (C) after treatment with Imatinib Loaded Liposomes (LIP), Imatinib Loaded HA coated Liposomes (LIP-HA) and an analogous dose, compared to the one used for liposome, of free Imatinib (Im).

20

0

24h 48h 72h

PR2

A

p-c-Abl (Y393)/β-actin (normalized to CTR)

1.0

0.5

Phospho-c-Abl expression

CTR LIPIm

LIPHAIm

Im

B

Col1a1/β-actina (normalized to CTR)

1.0

0.5

Collagen 1a1 expression

CTR LIPIm

LIPHAIm

Im

0.0

BOS

ILD-CTD

0.0

BOS ILD-CTD

Fig. 3: (A) Expression of Phospho-c-Abl by cells treated with Imatinib Loadesd Liposomes coated with HA and uncoated. Free drug (at the same concentration of liposome) is used as control. (B) Collagen 1a1 expression in cells treated under the same conditions as in Fig. 3A.

PR5