IT202100005054A1 - PHARMACEUTICAL COMPOUND FOR USE IN THERAPEUTIC TREATMENT OF CHRONIC HBV INFECTION AND METHOD FOR IDENTIFICATION OF DEPLETED LYMPHOCYTES - Google Patents
PHARMACEUTICAL COMPOUND FOR USE IN THERAPEUTIC TREATMENT OF CHRONIC HBV INFECTION AND METHOD FOR IDENTIFICATION OF DEPLETED LYMPHOCYTES Download PDFInfo
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- IT202100005054A1 IT202100005054A1 IT102021000005054A IT202100005054A IT202100005054A1 IT 202100005054 A1 IT202100005054 A1 IT 202100005054A1 IT 102021000005054 A IT102021000005054 A IT 102021000005054A IT 202100005054 A IT202100005054 A IT 202100005054A IT 202100005054 A1 IT202100005054 A1 IT 202100005054A1
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- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
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Description
Descrizione del trovato avente per titolo: Description of the invention entitled:
"COMPOSTO FARMACEUTICO PER L?USO IN UN TRATTAMENTO TERAPEUTICO DELL?INFEZIONE CRONICA DA HBV E METODO PER L?IDENTIFICAZIONE DI LINFOCITI ESAURITI" "PHARMACEUTICAL COMPOUND FOR USE IN THERAPEUTIC TREATMENT OF CHRONIC HBV INFECTION AND METHOD FOR IDENTIFICATION OF DEPLETED LYMPHOCYTES"
CAMPO DI APPLICAZIONE FIELD OF APPLICATION
Le forme di realizzazione qui descritte si riferiscono a un composto farmacologicamente accettabile per l?uso in un trattamento terapeutico dell?infezione virale cronica da HBV. The embodiments described herein relate to a pharmacologically acceptable compound for use in a therapeutic treatment of chronic HBV viral infection.
Il presente trovato si riferisce, inoltre, anche ad un metodo per l?identificazione di linfociti esauriti che permetta di effettuare una valutazione quali-quantitativa della disfunzione linfocitaria. Furthermore, the present invention also relates to a method for identifying depleted lymphocytes which allows to carry out a qualitative-quantitative evaluation of the lymphocyte dysfunction.
STATO DELLA TECNICA STATE OF THE ART
? noto che il virus dell?epatite B (HBV) ? la causa frequente al mondo di infezioni croniche epatiche. In corso di infezione cronica da HBV i linfociti T virus-specifici sono scarsamente responsivi alla stimolazione antigenica e funzionalmente compromessi. Si ritiene che l esaurimento funzionale (? exhaustion ?) linfocitario sia primariamente causato dalla persistente esposizione ad alte cariche antigeniche virali e che tale inibizione contribuisca alla persistenza virale. La ricostituzione di una risposta antivirale efficiente pu? pertanto rappresentare una strategia razionale per il trattamento dei pazienti con infezione cronica da HBV e per il potenziamento delle attuali terapie antivirali. Varie strategie sono state tentate per il ripristino della funzionalit? linfocitaria, tra cui il blocco dei co-recettori inibitori. E? stato ad esempio dimostrato che il blocco di PD-1 pu? in parte ripristinare le risposte antivirali delle cellule T CD4+ e CD8+ nell'infezione cronica da HBV. Tuttavia, il recupero della funzione linfocitaria ? stato osservato soltanto in una parte dei pazienti ed ? sempre risultato parziale, anche nei pazienti responsivi al trattamento. Attualmente il trattamento dell?infezione cronica da HBV ? principalmente basato sulla terapia con analoghi nucleosidici o nucleotidici (NUC), che nella maggior parte dei pazienti deve, tuttavia, essere continuata per tutta la vita, per non incorrere nel rischio di pericolose riattivazioni virali a seguito della sua sospensione. Esiste pertanto un?urgente necessit? di approcci terapeutici alternativi, che accelerino il controllo virale completo, al fine di abbreviare la durata della terapia con NUC. ? known that the hepatitis B virus (HBV) ? the world's frequent cause of chronic liver infections. During chronic HBV infection, virus-specific T lymphocytes are poorly responsive to antigenic stimulation and functionally compromised. Lymphocyte exhaustion is thought to be primarily caused by persistent exposure to high viral antigenic loads and that such inhibition contributes to viral persistence. Reconstitution of an efficient antiviral response can therefore represent a rational strategy for the treatment of patients with chronic HBV infection and for the enhancement of current antiviral therapies. Have various strategies been attempted to restore functionality? lymphocyte, including blockade of inhibitory co-receptors. AND? been for example demonstrated that the blockade of PD-1 pu? partially restore the antiviral responses of CD4+ and CD8+ T cells in chronic HBV infection. However, the recovery of lymphocyte function ? been observed in only a part of the patients and ? always partial result, even in patients who respond to treatment. Is the treatment of chronic HBV infection currently? mainly based on therapy with nucleoside or nucleotide analogues (NUC), which in most patients must, however, be continued for life, in order not to run the risk of dangerous viral reactivations following its suspension. There is therefore an? Urgent need? of alternative therapeutic approaches, which accelerate complete viral control, in order to shorten the duration of NUC therapy.
? noto che il virus dell?epatite B (HBV) ? la causa frequente al mondo di infezioni croniche epatiche. In corso di infezione cronica da HBV i linfociti T virus-specifici sono scarsamente responsivi alla stimolazione antigenica e funzionalmente compromessi. Si ritiene che l?esaurimento funzionale (? exhaustion ?) linfocitario sia primariamente causato dalla persistente esposizione ad alte cariche antigeniche virali e che tale inibizione contribuisca alla persistenza virale. La ricostituzione di una risposta antivirale efficiente pu? pertanto rappresentare una strategia razionale per il trattamento dei pazienti con infezione cronica da HBV e per il potenziamento delle attuali terapie antivirali. Varie strategie sono state tentate per il ripristino della funzionalit? linfocitaria, tra cui il blocco dei co-recettori inibitori. E? stato ad esempio dimostrato che il blocco di PD-1 pu? in parte ripristinare le risposte antivirali delle cellule T CD4+ e CD8+ nell'infezione cronica da HBV. Tuttavia, il recupero della funzione linfocitaria ? stato osservato soltanto in una parte dei pazienti ed ? sempre risultato parziale, anche nei pazienti responsivi al trattamento. Attualmente il trattamento dell?infezione cronica da HBV ? principalmente basato sulla terapia con analoghi nucleosidici o nucleotidici (NUC), che nella maggior parte dei pazienti deve, tuttavia, essere continuata per tutta la vita, per non incorrere nel rischio di pericolose riattivazioni virali a seguito della sua sospensione. Esiste pertanto un?urgente necessit? di approcci terapeutici alternativi, che accelerino il controllo virale completo, al fine di abbreviare la durata della terapia con NUC. ? known that the hepatitis B virus (HBV) ? the world's frequent cause of chronic liver infections. During chronic HBV infection, virus-specific T lymphocytes are poorly responsive to antigenic stimulation and functionally compromised. Lymphocyte exhaustion is thought to be primarily caused by persistent exposure to high viral antigenic loads and that such inhibition contributes to viral persistence. Reconstitution of an efficient antiviral response can therefore represent a rational strategy for the treatment of patients with chronic HBV infection and for the enhancement of current antiviral therapies. Have various strategies been attempted to restore functionality? lymphocyte, including blockade of inhibitory co-receptors. AND? been for example demonstrated that the blockade of PD-1 pu? partially restore the antiviral responses of CD4+ and CD8+ T cells in chronic HBV infection. However, the recovery of lymphocyte function ? been observed in only a part of the patients and ? always partial result, even in patients who respond to treatment. Is the treatment of chronic HBV infection currently? mainly based on therapy with nucleoside or nucleotide analogues (NUC), which in most patients must, however, be continued for life, in order not to run the risk of dangerous viral reactivations following its suspension. There is therefore an? Urgent need? of alternative therapeutic approaches, which accelerate complete viral control, in order to shorten the duration of NUC therapy.
Per ovviare agli inconvenienti della tecnica nota e per ottenere questi Per ovviare agli inconvenienti della tecnica nota e per ottenere questi ed ulteriori scopi e vantaggi, la Richiedente ha studiato, sperimentato e realizzato il presente trovato. To overcome the drawbacks of the known art and to obtain these To overcome the drawbacks of the known art and to obtain these and further objects and advantages, the Applicant has studied, tested and implemented the present invention.
ESPOSIZIONE DEL TROVATO DISCOVERY DISPLAY
Il presente trovato ? espresso e caratterizzato nelle rivendicazioni indipendenti. Le rivendicazioni dipendenti espongono altre caratteristiche del presente trovato o varianti dell?idea di soluzione principale. The present found ? expressed and characterized in the independent claims. The dependent claims disclose other characteristics of the present invention or variants of the idea of the main solution.
In accordo con forme di realizzazione, si descrive un composto farmacologicamente accettabile selezionato all?interno di un vasto gruppo di composti comprendete inibitori delle deacetilasi istoniche e coenzimi ossidoriduttivi o loro precursori per l?uso in un metodo per il trattamento terapeutico di una infezione virale cronica da HBY. In accordance with embodiments, there is disclosed a pharmacologically acceptable compound selected from a broad group of compounds including histone deacetylase inhibitors and redox coenzymes or precursors thereof for use in a method for the therapeutic treatment of a chronic viral infection from HBY.
Il trovato fornisce una strategia innovativa ed efficiente per il ripristino delle funzioni antivirali dei linfociti T virus-specifici esauriti, basata sulla fine selezione e caratterizzazione dei processi cellulari disfunzionali su cui agire per indurre un efficace ripristino complessivo delle funzioni dei linfociti T e in tal modo ottenere un pieno controllo della malattia e conseguentemente la risoluzione dell?infezione. The invention provides an innovative and efficient strategy for the restoration of the antiviral functions of depleted virus-specific T lymphocytes, based on the fine selection and characterization of the dysfunctional cellular processes on which to act in order to induce an effective overall restoration of the T lymphocyte functions and thus obtain full control of the disease and consequently the resolution of the infection.
In particolare, i pathways risultati maggiormente compromessi nei linfociti T esauriti, sono quelli coinvolti nelle funzioni mitocondriali, del proteasoma, del sistema di risposta al danno del DNA e di regolazione della trascrizione genica. In particular, the pathways most compromised in depleted T lymphocytes are those involved in mitochondrial, proteasome, DNA damage response system and gene transcription regulation functions.
Il trovato offre una strategia di intervento che agisce su uno o pi? dei suddetti pathways compromessi per ridurre i sintomi dell?infezione cronica e/o portare ad una sua risoluzione. The invention offers an intervention strategy that acts on one or more? of the aforementioned compromised pathways to reduce the symptoms of the chronic infection and/or lead to its resolution.
Secondo le forme di realizzazione, viene restituita ai linfociti esauriti, almeno parzialmente, la funzione anti-virale rendendoli nuovamente responsivi ad un?idonea stimolazione, ripristinandone, quindi, la loro normale funzione citotossica. According to the embodiments, the anti-viral function is restored to the depleted lymphocytes, at least partially, making them again responsive to a suitable stimulation, thus restoring their normal cytotoxic function.
In particolare, il trovato, correggendo la funzione di almeno uno dei pathway sopra menzionati, induce i linfociti T CD8 esauriti a potenziare la funzionalit? antivirale, sia in termini di produzione di citochine antivirali quali, ad esempio, IFN-?, TNF-?, IL-2 sia di attivit? citotossica, espressa dalla up-regolazione del marcatore CD107a. In particular, the invention, by correcting the function of at least one of the aforementioned pathways, induces the depleted CD8 T lymphocytes to enhance the functionality antiviral, both in terms of production of antiviral cytokines such as, for example, IFN-?, TNF-?, IL-2 and of activity? cytotoxic, expressed by up-regulation of the CD107a marker.
Il trovato pu? essere utilizzato sia come trattamento di prima linea, cio? su pazienti non ancora trattati, sia in pazienti gi? in trattamento con altri farmaci comunemente utilizzati per il trattamento dell?infezione cronica da HBV. The found can? be used both as a first-line treatment, cio? on patients not yet treated, both in patients already? being treated with other medicines commonly used to treat chronic HBV infection.
Secondo le forme di realizzazione, il trovato pu? essere usato in combinazione con altri farmaci per creare un effetto sinergico o quanto meno additivo. According to the embodiments, the invention can be used in combination with other drugs to create a synergistic or at least additive effect.
ILLUSTRAZIONE DEI DISEGNI ILLUSTRATION OF THE DESIGNS
Questi ed altri aspetti, caratteristiche e vantaggi del presente trovato appariranno chiari dalla seguente descrizione di forme di realizzazione, fomite a titolo esemplificativo, non limitativo, con riferimento agli annessi disegni in cui: These and other aspects, characteristics and advantages of the present invention will become clear from the following description of embodiments, provided by way of non-limiting example, with reference to the accompanying drawings in which:
- la figura 1 ? una sequenza di pannelli heat-map ciascuno raffigurante l?espressione di geni coinvolti in rispettivi pathways nei linfociti CD8 di pazienti con infezione cronica da HBV (CHR) rispetto a soggetti guariti spontaneamente da un?epatite B acuta (RES); - figure 1 ? a sequence of heat-map panels each depicting the expression of genes involved in respective pathways in CD8 lymphocytes of patients with chronic HBV infection (CHR) compared to subjects who spontaneously recovered from acute hepatitis B (RES);
- le figure 2a - 2c sono grafici che illustrano la disfunzione mitocondriale nei linfociti CD8 isolati da pazienti con epatite B cronica rispetto a soggetti guariti spontaneamente e a soggetti sani; - Figures 2a - 2c are graphs illustrating the mitochondrial dysfunction in CD8 lymphocytes isolated from patients with chronic hepatitis B compared to spontaneously recovered subjects and healthy subjects;
- la figura 3 ? un grafico che mostra l accumulo di aggregati proteici intracellulari come conseguenza della disfunzione della proteostasi (digestione e smaltimento di proteine o organuli intracellulari alterati); - le figure 4a - 4c sono grafici che riportano la disfunzione della risposta al danno al DNA; - figure 3 ? a graph showing the accumulation of intracellular protein aggregates as a result of proteostasis dysfunction (digestion and disposal of impaired intracellular proteins or organelles); Figures 4a - 4c are graphs reporting the dysfunction of the DNA damage response;
- la figura 5 ? un grafico che riporta l alterazione di un meccanismo regolatore della trascrizione del DNA, in particolare della acetilazione istonica; - figure 5 ? a graph showing the alteration of a regulatory mechanism of DNA transcription, in particular of histone acetylation;
- le figure 6a e 6b sono grafici che mostrano rispettivamente il deficit nella formazione di catene di poli(ADP-riboso) o ?parilazione? (relativa principalmente ai meccanismi di attivazione della risposta al danno del DNA) e sull?espressione di superficie di CD38; - Figures 6a and 6b are graphs showing respectively the deficit in the formation of poly(ADP-ribose) or ?parilation? (mainly related to the activation mechanisms of the DNA damage response) and on the surface expression of CD38;
- le figure 7a - 7e sono grafici di confronto nella correzione di pathways secondo forme di realizzazione del trovato; - figures 7a - 7e are comparison graphs in the correction of pathways according to embodiments of the invention;
- le figure 8a - 8b sono grafici di confronto sulla produzione di citochine secondo forme di realizzazione del trovato; - figures 8a - 8b are comparison graphs on the production of cytokines according to embodiments of the invention;
- le figure 9a - 9c sono grafici di confronto sulla produzione di citochine secondo ulteriori forme di realizzazione del trovato; - figures 9a - 9c are comparison graphs on the production of cytokines according to further embodiments of the invention;
- la figura 10 mostra grafici sulla proliferazione cellulare secondo forme di realizzazione del trovato. - figure 10 shows graphs on cell proliferation according to embodiments of the invention.
Per facilitare la comprensione, numeri di riferimento identici sono stati utilizzati, ove possibile, per identificare elementi comuni identici nelle figure. Va inteso che elementi e caratteristiche di una forma di realizzazione possono essere convenientemente combinati o incorporati in altre forme di realizzazione senza ulteriori precisazioni. For ease of understanding, identical reference numerals have been used wherever possible to identify identical commonalities in the figures. It should be understood that elements and features of one embodiment may be conveniently combined or incorporated into other embodiments without further specification.
DESCRIZIONE DI FORME DI REALIZZAZIONE DESCRIPTION OF EMBODIMENTS
Si far? ora riferimento nel dettaglio alle possibili forme di realizzazione del trovato, delle quali uno o pi? esempi sono illustrati nelle figure allegate a titolo esemplificativo non limitativo. Anche la fraseologia e terminologia qui utilizzata ? a fini esemplificativi non limitativi. Will it be done? now reference in detail to the possible embodiments of the invention, of which one or more? examples are illustrated in the attached figures by way of non-limiting example. Even the phraseology and terminology used here? for non-limiting example purposes.
Il presente trovato si riferisce ad un composto farmacologicamente accettabile selezionato in un gruppo che consiste di inibitori delle deacetilasi istoniche e di coenzimi ossidoriduttivi o loro precursori in un metodo per l?uso in un trattamento terapeutico dell? infezione virale cronica da HBV. The present invention relates to a pharmacologically acceptable compound selected from a group consisting of histone deacetylase inhibitors and redox coenzymes or their precursors in a method for use in a therapeutic treatment of chronic HBV viral infection.
Un linfocita T viene considerato avere una funzione ridotta o assente, altrimenti detto funzionalmente esaurito, quando, anche in presenza di un?idonea stimolazione antigenica l?azione effettrice antivirale che fornisce ? ridotta o assente. A T lymphocyte is considered to have a reduced or absent function, otherwise called functionally exhausted, when, even in the presence of a suitable antigenic stimulation, the antiviral effector action it provides is? reduced or absent.
In forme di realizzazione, l?infezione virale cronica pu? essere concomitante con un tumore, che pu? essere occulto. In embodiments, chronic viral infection can be concomitant with a tumor, which can? be occult.
In accordo con forme di realizzazione il tumore pu? essere un tumore al fegato. In accordance with embodiments the tumor can? be liver cancer.
In forme di realizzazione, il metodo pu? comprendere la somministrazione di altri farmaci. In accordo con forme di realizzazione, il metodo pu? comprendere la somministrazione di antivirali. In forme di realizzazione il metodo pu? comprendere la somministrazione di chemioterapici o farmaci antitumorali. In embodiments, the method can understand the administration of other drugs. According to embodiments, the method can include the administration of antivirals. In embodiments the method can? include the administration of chemotherapy or anticancer drugs.
In forme di realizzazione, il metodo pu? prevedere la somministrazione di farmaci analoghi nucleot(s)idici (NUC), per esempio nel caso di malattia cronica da HBV. In embodiments, the method can provide for the administration of nucleot(s)ide analogues (NUC) drugs, for example in the case of chronic HBV disease.
Secondo forme di realizzazione, i coenzimi ossidoriduttivi o i loro precursori possono essere somministrati in combinazione con un inibitore di CD38 selezionato da un gruppo, non limitativo, comprendente, anticorpi, analoghi del NAD, flavonoidi, 4-amminochinolina. According to embodiments, the redox coenzymes or their precursors can be administered in combination with a CD38 inhibitor selected from a non-limiting group including, antibodies, NAD analogs, flavonoids, 4-aminoquinoline.
Secondo forme di realizzazione gli anticorpi inibitori di CD38 possono essere selezionati da un gruppo, non limitativo, comprendente, daratumumab, isatuximab, MOR202 e TAK079. According to embodiments, the CD38 inhibitory antibodies can be selected from a non-limiting group including, daratumumab, isatuximab, MOR202 and TAK079.
Secondo forme di realizzazione gli analoghi del NAD inibitori di CD38 possono essere selezionati da un gruppo, non limitativo, comprendente, ARA-F-NAD, ARA-F-NFM, Fosfoestere/C48, Carba-NAD e Pseudo-Carba-NAD. According to embodiments, the CD38 inhibitory NAD analogs can be selected from a non-limiting group including, ARA-F-NAD, ARA-F-NFM, Phosphoester/C48, Carba-NAD and Pseudo-Carba-NAD.
Secondo forme di realizzazione i flavonoidi inibitori di CD38 possono essere selezionati da un gruppo, non limitativo, comprendente, apigenina, luteolinidina kuromanina e rhein/K-rhein. According to embodiments, the flavonoid inhibitors of CD38 can be selected from a non-limiting group including apigenin, luteolinidine kuromanin and rhein/K-rhein.
Secondo forme di realizzazione le 4-ammino-chinoline inibitori di CD38 possono essere selezionate da un gruppo, non limitativo, comprendente, 78c, lah e lai. According to embodiments, the 4-amino-quinolines inhibitors of CD38 can be selected from a non-limiting group comprising 78c, lah and lai.
Gli acidi idrossamici possono essere selezionati da un gruppo, non limitativo, comprendente, Tricostatina A, SAHA, Belinostat, Panabiostat, Givinostat, Resminostat, Abexinostat, Quisinostat, Rocilinostat, Practinostat e CHR-3996. Hydroxamic acids can be selected from a non-limiting group including, Trichostatin A, SAHA, Belinostat, Panabiostat, Givinostat, Resminostat, Abexinostat, Quisinostat, Rocilinostat, Practinostat and CHR-3996.
Gli acidi grassi a catena corta possono essere selezionati da un gruppo, non limitativo, comprendente, acido valproico, acido butirrico e acido fenilbutirrico. The short-chain fatty acids can be selected from a non-limiting group including valproic acid, butyric acid and phenylbutyric acid.
Le benzamidi possono essere selezionate da un gruppo, non limitativo, comprendente, Entinostat, Tacedinaline, 4SC202 e Mocetinostat. Benzamides can be selected from a non-limiting group including, Entinostat, Tacedinaline, 4SC202 and Mocetinostat.
I tetrapeptidi ciclici possono essere selezionati da un gruppo, non limitativo, comprendente, Romidepsina. The cyclic tetrapeptides can be selected from a non-limiting group including Romidepsin.
Gli inibitori delle sirtuine possono essere selezionati da un gruppo, non limitativo, comprendente, nicotinammide, sirtinolo, cambinolo e MX-527. Sirtuin inhibitors can be selected from a non-limiting group including nicotinamide, sirtinol, cambinol and MX-527.
Secondo forme di realizzazione, i coenzimi ossidoriduttivi sono selezionati da un gruppo comprendente Nicotinammideadenindinucleotide (NAD<+>/NADH,H<+>), Nicotinammideadenindinucleotidefosfato (NADP<+>/NADPH,H<+>), Flavin adenina dinucleotide (FAD/FADH2), Flavina mononucleotide (FMN/FMNFH2), Coenzima Q (CoQ/CoQH2); il gruppo prostetico eme contenuto nei citocromi (Cyt[Fe<2+>]/Cyt[Fe<3+>]) o loro precursori. According to embodiments, the redox coenzymes are selected from a group including Nicotinamide Adenine Dinucleotide (NAD<+>/NADH,H<+>), Nicotinamide Adenine Dinucleotide Phosphate (NADP<+>/NADPH,H<+>), Flavin Adenine Dinucleotide (FAD/ FADH2), Flavin mononucleotide (FMN/FMNFH2), Coenzyme Q (CoQ/CoQH2); the heme prosthetic group contained in cytochromes (Cyt[Fe<2+>]/Cyt[Fe<3+>]) or their precursors.
In una forma di realizzazione preferita, l?inibitore delle deacetilasi istoniche ? Entinostat (MS-275). In a preferred embodiment, the histone deacetylase inhibitor ? Entinostat (MS-275).
In un?altra forma di realizzazione preferita, il coenzima riduttivo ? Nicotinamide mononucleotide (NMN). In another preferred embodiment, the reducing coenzyme is Nicotinamide mononucleotide (NMN).
Il composto farmacologicamente accettabile pu? essere somministrato al paziente in una o pi? delle seguenti vie di somministrazione variamente combinate, come orale, intravenosa, trans-mucosale, polmonare, transdermale, oculare, buccale, sottolinguale, intraperitoneale, intratecale, intramuscolare. The pharmacologically acceptable compound can? be administered to the patient in one or more? of the following variously combined routes of administration, such as oral, intravenous, trans-mucosal, pulmonary, transdermal, ocular, buccal, sublingual, intraperitoneal, intrathecal, intramuscular.
Il composto farmacologicamente accettabile pu? essere in composizione solida, per esempio, pastiglie, compresse, tavolette, polveri. Opzionalmente, la composizione solida per somministrazione orale pu? contenere idonei carrier o eccipienti o ingredienti per il rilascio controllato. The pharmacologically acceptable compound can? be in solid composition, for example, lozenges, tablets, tablets, powders. Optionally, the solid composition for oral administration can contain suitable carriers or excipients or ingredients for controlled release.
Il composto farmacologicamente accettabile pu? essere liquido preparato come soluzione acquosa o emulsione. Il composto farmacologicamente accettabile liquido pu? essere, per esempio, una soluzione iniettabile o una soluzione somministrabile per via orale. The pharmacologically acceptable compound can? be liquid prepared as an aqueous solution or emulsion. The liquid pharmacologically acceptable compound can? be, for example, an injectable solution or an orally administered solution.
Il trovato si riferisce, inoltre, ad un metodo per l?identificazione di linfociti esauriti che comprende valutare almeno una tra le funzioni cellulari a scelta: funzione mitocondriale, funzione del proteasoma, risposta al danno del DNA e regolazione della trascrizione, in associazione alla rilevazione della produzione di citochine antivirali in risposta ad uno stimolo antigene-specifico. The invention also refers to a method for identifying depleted lymphocytes which comprises evaluating at least one of the cellular functions of your choice: mitochondrial function, proteasome function, response to DNA damage and regulation of transcription, in association with the detection of the production of antiviral cytokines in response to an antigen-specific stimulus.
Forme di realizzazione possono prevedere che la valutazione della funzione mitocondriale preveda determinare il potenziale di membrana mitocondriale e/o la quantit? di specie reattive dell?ossigeno. Embodiments may provide that the assessment of mitochondrial function involves determining the mitochondrial membrane potential and/or the amount of reactive oxygen species.
Forme di realizzazione possono prevedere che la valutazione della funzione del proteosoma prevede rilevare aggregati proteici intracellulari. Embodiments may provide that the assessment of proteasome function involves detecting intracellular protein aggregates.
Forme di realizzazione possono prevedere che la valutazione della risposta al danno del DNA prevede rilevare la fosforilazione di almeno uno tra ATM, H2AX, 53BP1 e della formazione di catene di poli(ADP)riboso (PAR), indicativa dell? attivit? degli enzimi della famiglia PARP (poli(ADP-riboso)polimerasi). Embodiments may provide that the assessment of the DNA damage response involves detecting phosphorylation of at least one of ATM, H2AX, 53BP1 and the formation of poly(ADP)ribose (PAR) chains, indicative of the activity? of PARP (poly(ADP-riboso)polymerase) family enzymes.
In accordo con forme di realizzazione, il rilevamento della formazione di catene di poli(ADP)riboso pu? avvenire in presenza o assenza di NAD esogeno. According to embodiments, the detection of the formation of poly(ADP)ribose chains can occur in the presence or absence of exogenous NAD.
Forme di realizzazione possono prevedere che la valutazione della regolazione della trascrizione preveda il rilevamento dell?acetilazione dell?istone H3. Embodiments may have the assessment of transcription regulation include the detection of histone H3 acetylation.
Sebbene il metodo sia utilizzato per la rilevazione di linfociti esauriti nell?infezione da HBV, ? plausibile pensare che tale metodo consenta di rilevare linfociti esauriti anche in altre patologie, in particolare in patologie virali croniche. Although the method is used for the detection of depleted lymphocytes in HBV infection, it is ? plausible to think that this method allows to detect depleted lymphocytes also in other pathologies, in particular in chronic viral pathologies.
Nel seguito il principio del trovato ? descritto con riferimento a particolari composti di inibitori delle deacetilasi istoniche e di coenzimi ossidoriduttivi o loro precursori, tuttavia ci? non esclude dall?applicazione del trovato altri composti con effetto analogo o simile. Allo scopo di identificare bersagli molecolari idonei per strategie di ripristino della funzionalit? linfocitaria, ? stato eseguita un?analisi di espressione genica dei linfociti T HBV-specifici isolati da pazienti con epatite cronica B (Chr) e quindi ? esauriti I profili trascrizionali ottenuti da linfociti T HBV-specifici esauriti o in breve, T esauriti, sono stati confrontati con i profili ottenuti da: In the following, the principle of the invention ? described with reference to particular compounds of histone deacetylase inhibitors and redox coenzymes or their precursors, however this? does not exclude from the application of the invention other compounds with analogous or similar effect. In order to identify suitable molecular targets for functional restoration strategies lymphocyte, ? was an analysis of gene expression of HBV-specific T lymphocytes isolated from patients with chronic hepatitis B (Chr) performed and therefore ? depleted Transcriptional profiles obtained from depleted HBV-specific T lymphocytes or depleted T for short, were compared to profiles obtained from:
- linfociti T HBV-specifici funzionalmente integri isolati da pazienti in fase acuta di un?epatite B (Ac), - functionally intact HBV-specific T lymphocytes isolated from patients in the acute phase of hepatitis B (Ac),
- linfociti T di pazienti che hanno spontaneamente risolto l?infezione da HBV (Res) e, - T lymphocytes from patients who have spontaneously resolved their HBV infection (Res) and,
- linfociti T influenza-specifici (FLU) prelevati da soggetti sani (He). Le analisi effettuate tramite tecniche di Gene Set Enrichment Analysis (GSEA), hanno evidenziato la tendenza alla down-regolazione di molti pathways cellulari nei linfociti T HBV-specifici prelevati da pazienti con infezione da HBV cronica. I pathways risultati maggiormente compromessi nei linfociti T esauriti risultano essere quelli coinvolti nelle funzioni mitocondriali, del proteasoma, del sistema di risposta al danno del DNA e di regolazione della trascrizione genica (Figura 1). - influenza-specific T lymphocytes (FLU) taken from healthy subjects (He). Analyzes performed using Gene Set Enrichment Analysis (GSEA) techniques have highlighted the down-regulation tendency of many cellular pathways in HBV-specific T lymphocytes taken from patients with chronic HBV infection. The pathways most compromised in depleted T lymphocytes are those involved in mitochondrial, proteasome, DNA damage response system and gene transcription regulation functions (Figure 1).
Sono stati utilizzati adeguati test per lo studio dei pahways compromessi ed ? stato inoltre valutato l?effetto di diverse sostanze sulla correzione di tali funzioni e di conseguenza sul ripristino della funzionalit? linfocitaria anti- virale. Have adequate tests been used to study compromised pahways and ? was also evaluated the effect of various substances on the correction of these functions and consequently on the restoration of functionality? lymphocytic anti-viral.
1- Test utilizzati per la valutazione quali-quantitativa della disfunzione linfocitaria in citofluorimetria. 1- Tests used for the qualitative-quantitative evaluation of lymphocyte dysfunction in cytofluorimetry.
a- Funzione mitocondriale: sonde JC-1 e D?OC6 per la determinazione del potenziale di membrana mitocondriale, sonda MitoSox, per il rilevamento dei livelli di specie reattive dell?Ossigeno (ROS) a livello mitocondriale rispettivamente (Figure 2a - 2c); a- Mitochondrial function: JC-1 and D?OC6 probes for determining the mitochondrial membrane potential, MitoSox probe, for detecting the levels of reactive oxygen species (ROS) at the mitochondrial level respectively (Figures 2a - 2c);
b- Funzionalit? della proteostasi cellulare: sonda Proteostat per il rilevamento degli aggregati proteici intracellulari (Figura 3); c- Risposta al danno del DNA: marcatura intracellulare con anticorpo anti-phosphoATM, anti-phosphoH2AX, anti-53BPl, anti-poli(ADP-riboso) (PAR) (in assenza e in presenza di NAD esogeno) (Figure 4a - 4c e 6a); b- Functionality? of cellular proteostasis: Proteostat probe for the detection of intracellular protein aggregates (Figure 3); c- Response to DNA damage: intracellular labeling with anti-phosphoATM, anti-phosphoH2AX, anti-53BPl, anti-poly(ADP-ribose) (PAR) antibody (in the absence and in the presence of exogenous NAD) (Figures 4a - 4c and 6a);
d- Regolazione della trascrizione: marcatura intracellulare con anticorpo anti-acetyl-istone H3 (Figura 5). d- Regulation of transcription: intracellular labeling with anti-acetyl-histone H3 antibody (Figure 5).
2- Strategie di correzione delle funzioni intracellulari alterate nei linfociti esauriti. 2- Strategies for correcting impaired intracellular functions in depleted lymphocytes.
a- Correzione della funzione mitocondriale, del proteasoma, dei meccanismi di risposta al danno del DNA. a- Correction of mitochondrial function, proteasome, response mechanisms to DNA damage.
Poich? enzimi importanti per l?attivit? mitocondriale e metabolica e la capacit? di efficiente risposta al danno del DNA, soprattutto per quanto riguarda gli enzimi della famiglia PARP, si basano sul consumo del cofattore NAD+ (Figura 6a), cos? come l?attivit? dell?enzima CD38, che ha un ruolo importante nell?attivazione T linfocitaria, i dati di disfunzione di tali processi suggeriscono una possibile carenza di NAD intracellulare. Una strategia di correzione pu? essere rappresentata dalla somministrazione di precursori del NAD stesso, singolarmente o in associazione all?inibizione del CD38, in quanto quest?ultimo ? risultato fortemente espresso nei linfociti dei pazienti con infezione cronica da HBV (Figura 6b). Sulla base di queste valutazioni ? stato testato in vitro un precursore del NAD, Nicotinamide mononucleotide (NMN), da solo o in associazione con un inibitore di CD38. because important enzymes for? activity? mitochondrial and metabolic and the ability? of efficient response to DNA damage, especially as regards the enzymes of the PARP family, are based on the consumption of the cofactor NAD+ (Figure 6a), so? how the? activity? of the enzyme CD38, which has an important role in T lymphocyte activation, the data of dysfunction of these processes suggest a possible deficiency of intracellular NAD. A correction strategy can be represented by the administration of precursors of NAD itself, individually or in association with the inhibition of CD38, as the latter ? result strongly expressed in lymphocytes of patients with chronic HBV infection (Figure 6b). Based on these assessments ? A precursor of NAD, Nicotinamide mononucleotide (NMN), alone or in combination with a CD38 inhibitor was tested in vitro.
b- Correzione di meccanismi di regolazione epigenetica. b- Correction of epigenetic regulation mechanisms.
Infine, sulla base dell?estesa down-regolazione di numerosi processi biologici, associata allaumentata espressione di geni ad attivit? regolatoria negativa tra cui anche HDACl, e alla scarsa capacit? di acetilazione osservata in esperimenti ex vivo (Figura 5), si ? ritenuto opportuno testare l?effetto di un inibitore delle deacetilasi (HDAC inhibitor ), in particolare di Entinostat. E? noto infatti che l acetilazione delle proteine istoniche associate al DNA ? uno dei meccanismi di regolazione epigenetici che rendono permissiva la trascrizione della cromatina. Finally, on the basis of the extensive down-regulation of numerous biological processes, associated with the increased expression of genes with negative regulatory including HDACl, and the low capacity? of acetylation observed in ex vivo experiments (Figure 5), yes? considered appropriate to test the effect of a deacetylase inhibitor (HDAC inhibitor), in particular Entinostat. AND? in fact, it is known that the acetylation of histone proteins associated with DNA is one of the epigenetic regulatory mechanisms that make chromatin transcription permissive.
In considerazione delle indicazioni ottenute mediante l?applicazione dei test sopra citati e i risultati di ripristino linfocitario ottenuti in vitro con alcuni dei composti testati, ? realistico pensare che le informazioni derivate da questi risultati possano essere traslate in nuove ed efficienti terapie anti-HBV. Tali strategie di ricostituzione funzionale linfocitaria potrebbero essere applicabili a pazienti con infezione cronica non ancora in trattamento, ma anche in associazione ai farmaci antivirali attualmente utilizzati nella pratica clinica, allo scopo di abbreviarne il periodo di somministrazione, accelerando il processo di risoluzione dell?infezione. In consideration of the indications obtained through the application of the tests mentioned above and the results of lymphocyte recovery obtained in vitro with some of the compounds tested, ? realistic to think that the information derived from these results can be translated into new and efficient anti-HBV therapies. These lymphocyte functional reconstitution strategies could be applicable to patients with chronic infection not yet undergoing treatment, but also in association with antiviral drugs currently used in clinical practice, in order to shorten the period of administration, accelerating the process of resolution of the infection.
MATERIALI E METODI MATERIALS AND METHODS
Con l?intento di studiare le funzioni intracellulari alterate e il loro eventuale ripristino e conseguentemente la funzionalit? linfocitaria antivirale sono stati testati in vitro i seguenti composti: With the intention of studying the altered intracellular functions and their eventual restoration and consequently the functionality? lymphocyte antiviral the following compounds were tested in vitro:
- Nicptinamide mononucleotide (NMN) - Nicptinamide mononucleotide (NMN)
- Inibitore di CD38 - CD38 inhibitor
- Entinostat (MS-275) - Entinostat (MS-275)
E stato valutato l?effetto di tali sostanze su alcune funzioni cellulari alterate, a livello di mitocondrio, di risposta al danno del DNA, di controllo della trascrizione e la loro capacit? di migliorare le funzioni antivirali dei linfociti T CD8+ HBV-specifici, misurate come produzione di citochine e capacit? proliferativa. Per quanto riguarda la funzionalit? mitocondriale ? stata analizzata la percentuale di linfociti depolarizzati nella coltura linfocitaria non trattata, rispetto a quella trattata con i diversi composti. Per quanto riguarda la risposta al danno del DNA ? stata valutata la fosforilazione della variante istonica H2AX. Infine, ? stata studiata l?acetilazione dell?istone H3 e dell?istone H4 per valutare un aspetto del controllo della trascrizione genica. Per lo studio delle risposte anti- virali, le cellule sono state stimolate per 10 giorni con peptidi HBV-specifici, corrispondenti alla proteina virale CORE, in presenza o in assenza dei diversi composti. Attraverso marcatura intracellulare e successiva analisi citofluorimetrica ? stata valutata la produzione delle principali citochine antivirali. The effect of these substances on some altered cellular functions, at the level of the mitochondria, response to DNA damage, transcription control and their capacity has been evaluated. to improve the antiviral functions of HBV-specific CD8+ T lymphocytes, measured as cytokine production and ability? proliferative. As for functionality? mitochondrial ? The percentage of depolarized lymphocytes in the untreated lymphocyte culture compared to that treated with the different compounds was analysed. As for the DNA damage response? phosphorylation of the histone variant H2AX was evaluated. In the end, ? Histone H3 and histone H4 acetylation was studied to evaluate one aspect of gene transcription control. To study the antiviral responses, the cells were stimulated for 10 days with HBV-specific peptides, corresponding to the viral CORE protein, in the presence or absence of the different compounds. Through intracellular labeling and subsequent cytofluorimetric analysis ? the production of the main antiviral cytokines was evaluated.
1 - Pazienti e controlli 1 - Patients and controls
Sono stati reclutati 70 pazienti volontari (44 di sesso maschile e 26 femminile) con epatite cronica attiva da HBV HBeAg-negativa, nessuno dei quali era mai stato sottoposto a terapia. La diagnosi si ? basata sull?evidenza di livelli elevati di alanina aminotransferasi (ALT) mantenuti per pi? di sei mesi e positivit? per HBsAg, anti-HBc, anti-HBe e HBV-DNA. We recruited 70 volunteer patients (44 male and 26 female) with HBeAg-negative HBV chronic active hepatitis, none of whom had ever undergone therapy. Is the diagnosis? based on evidence of elevated alanine aminotransferase (ALT) levels maintained for longer than of six months and positivit? for HBsAg, anti-HBc, anti-HBe and HBV-DNA.
Tali pazienti volontari, al momento del prelievo, avevano un?et? compresa tra 24 e 70 anni; HBV-DNA variabile tra 3x10<3 >e 9,6x10<8 >IU/ml; livelli di ALT tra 14 e 379 U/l; 50 pazienti erano infettati da virus dell?epatite B di genotipo D, 3 di genotipo A, 2 di genotipo C e uno di genotipo E, mentre per i rimanenti pazienti il genotipo virale non ? stato rilevato. Questi pazienti sono stati sottoposti a screening per HLA-A2, realizzato mediante marcatura delle cellule mononucleate del sangue periferico (PBMC) con l? anticorpo fluorescente anti-HLA-A2.01 (BD Biosciences, San Jose, CA) e successiva valutazione mediante analisi citofluorimetrica. These voluntary patients, at the time of sampling, had an age? aged between 24 and 70; HBV-DNA variable between 3x10<3 > and 9.6x10<8 >IU/ml; ALT levels between 14 and 379 U/l; 50 patients were infected with hepatitis B virus of genotype D, 3 of genotype A, 2 of genotype C and one of genotype E, while for the remaining patients the viral genotype was not? been detected. These patients were screened for HLA-A2 by labeling peripheral blood mononuclear cells (PBMCs) with the? anti-HLA-A2.01 fluorescent antibody (BD Biosciences, San Jose, CA) and subsequent evaluation by flow cytometric analysis.
Tutti i pazienti volontari reclutati sono risultati negativi per la ricerca di anticorpi anti-HCV (virus dell?epatite C), virus delta, HIV-1 e HIV-2 (virus dell?immunodeficienza umana di tipo 1 e di tipo 2) e per altri marcatori di epatite virale o autoimmune. All recruited patient volunteers tested negative for antibodies to HCV (hepatitis C virus), delta virus, HIV-1 and HIV-2 (human immunodeficiency virus type 1 and type 2) and for other markers of viral or autoimmune hepatitis.
2 - Isolamento dei linfociti del sangue periferico 2 - Isolation of peripheral blood lymphocytes
Le cellule linfomononucleate del sangue periferico ( Peripheral Blood Mononuclear Cells, PBMCs) sono state ottenute a partire da sangue fresco eparinato mediante centrifugazione su gradiente di densit? Ficoll-Hypaque. Le PBMCs sono state quindi congelate in medium di congelamento (Siero Fetale Bovino -FBS 90% e Dimetilsolfossido -DMSO 10%) e crioconservate in azoto liquido fino al momento del loro utilizzo per gli esperimenti. Peripheral blood mononuclear cells (PBMCs) were obtained from fresh heparinized blood by density gradient centrifugation. Ficoll-Hypaque. The PBMCs were then frozen in freezing medium (Fetal Bovine Serum -FBS 90% and Dimethyl sulfoxide -DMSO 10%) and cryopreserved in liquid nitrogen until their use for the experiments.
3 - Composti utilizzati per il trattamento delle cellule in vitro Sono stati studiati in vitro diversi composti, per ognuno dei quali ? inizialmente stato testato un ampio range di concentrazioni; successivamente sono state scelte le concentrazioni risultate pi? efficaci e indicate di seguito per ciascuna sostanza. 3 - Compounds used for the treatment of cells in vitro Different compounds have been studied in vitro, for each of which ? a wide range of concentrations was initially tested; were subsequently chosen the concentrations that resulted pi? effective and listed below for each substance.
- Nicotinamide mononucleotide (NMN, Sigma- Aldrich) alle concentrazioni di 10, 100 e 500 ??. - Nicotinamide mononucleotide (NMN, Sigma-Aldrich) at concentrations of 10, 100 and 500 ??.
- Inibitore di CD38 (#538763 | CD38 Inhibitor - Calbiochem) alla concentrazione di 1 e 5 nM, in combinazione con NMN. - CD38 inhibitor (#538763 | CD38 Inhibitor - Calbiochem) at concentrations of 1 and 5 nM, in combination with NMN.
- Entinostat (MS-275, Sigma- Aldrich) alle concentrazioni di 0,1 e 0,05 ??. - Entinostat (MS-275, Sigma-Aldrich) at concentrations of 0.1 and 0.05??.
4 - Espansione in vitro dei linfociti T 4 - In vitro expansion of T lymphocytes
Le PBMC isolate da sangue periferico e crioconservate in azoto liquido, sono state scongelate, sottoposte a lavaggio in HBSS e risospese in terreno completo (RPMI 1640 addizionato con 40 ?g/ml di Gentamicina, 2,5 ?g/ml di Fungizone, 0,05 mM di 2-Mercaptoetanolo, IX NE A e 8% siero umano), distribuite in piastre da 96 pozzetti con fondo tondo e stimolate con peptidi virali. PBMCs isolated from peripheral blood and cryopreserved in liquid nitrogen were thawed, washed in HBSS and resuspended in complete medium (RPMI 1640 supplemented with 40 ?g/ml of Gentamicin, 2.5 ?g/ml of Fungizone, 0 05 mM 2-Mercaptoethanol, IX NE A and 8% human serum), distributed in 96-well round bottom plates and stimulated with viral peptides.
Per stimolare la popolazione dei linfociti T HBV-specif?ci in vitro ? stata utilizzata una miscela di peptidi sintetici, della lunghezza di 15 aminoacidi ciascuno, con sequenze sovrapposte di 10 residui e corrispondenti alla sequenza della proteina core di HBV di genotipo D (sintetizzati da Chiron Mimotopes, Victoria, Australia). To stimulate the HBV-specific T lymphocyte population in vitro ? A mixture of synthetic peptides, each 15 aminoacids long, with overlapping sequences of 10 residues and corresponding to the core protein sequence of HBV genotype D (synthesized by Chiron Mimotopes, Victoria, Australia) was used.
Per lo studio della risposta dei linfociti T nei pazienti HLA-A2 positivi sono stati impiegati peptidi sintetici corrispondenti all?epitopo HLA-A2 ristretto della proteina core (aa. 18-27: FLPSDFFPSV) di HBV di genotipo D. Le cellule sono state incubate per 10 giorni in presenza o assenza dei composti da testare. Al terzo giorno di coltura cellulare ? stata aggiunta 1L-2 alla concentrazione di 50 U/ml. To study the response of T lymphocytes in HLA-A2 positive patients, synthetic peptides corresponding to the HLA-A2 restricted epitope of the core protein (aa. 18-27: FLPSDFFFPSV) of HBV genotype D were used. The cells were incubated for 10 days in the presence or absence of the compounds to be tested. On the third day of cell culture ? 1L-2 was added at a concentration of 50 U/ml.
5 - Valutazione del potenziale di membrana mitocondriale L?analisi citofluorimetrica condotta sulle colture linfocitarie utilizzando la sonda JC-1 (Molecular Probes, ) permette di quantificare la percentuale di cellule caratterizzate da depolarizzazione della membrana mitocondriale (MMP). Infatti la sonda JC-1 ? un colorante cationico fluorescente ad alta affinit? per i mitocondri, che in forma monomerica emette fluorescenza nella lunghezza d?onda del verde (picco di emissione a circa 527 nm). In presenza di un elevato potenziale di membrana mitocondriale (MMP), si accumula proporzionalmente ad esso all?interno del mitocondrio, dove raggiungendo alte concentrazioni, forma aggregati che emettono fluorescenza nella lunghezza d?onda del rosso (circa 590 nm). In caso di calo del potenziale di membrana mitocondriale (depolarizzazione) si ha la diffusione del colorante nel citoplasma, di conseguenza il picco di emissione del JC-1 subisce uno shift, con passaggio da una fluorescenza rossa ad una verde. Le colture linfocitarie, dopo stimolazione con i peptidi di HBV, sono state incubate alla temperatura di 37?C per 10 giorni in presenza o assenza dei diversi composti in esame. Dopo aver eseguito la marcatura di superficie con gli anticorpi anti-CD3 e anti-CD8 per 15 minuti a temperatura ambiente, le cellule sono state risospese in terreno RPMI con 10% di FCS preriscaldato a 37?C e incubate con la sonda JC-1 (2,5 ?g/ml) o DiOC6 (20nM) o MitoSox Red (5 ??) per 15 minuti alla temperatura di 37?C, protette dalla luce. In seguito all?aggiunta della sonda di vitalit? 7AAD, i campioni sono stati acquisiti ed analizzati mediante citofluorimetro a flusso a otto colori (FACS-Canto II, Becton-Dickinson, CA, USA), utilizzando il software FACS-DIVA. Come controllo positivo, per individuare i linfociti depolarizzati, le cellule sono state trattate con il protonoforo valinomicina alla concentrazione di 100 ?? per 10 minuti a 37?C, prima dell?aggiunta della sonda JC-1 o D?OC6. 5 - Evaluation of the mitochondrial membrane potential The cytofluorimetric analysis conducted on lymphocyte cultures using the JC-1 probe (Molecular Probes, ) allows to quantify the percentage of cells characterized by mitochondrial membrane depolarization (MMP). In fact, the JC-1 probe? a high affinity cationic fluorescent dye for mitochondria, which in monomeric form emits fluorescence in the green wavelength (emission peak at about 527 nm). In the presence of a high mitochondrial membrane potential (MMP), it accumulates proportionally to it inside the mitochondrion, where, reaching high concentrations, it forms aggregates that emit fluorescence in the red wavelength (about 590 nm). In the event of a drop in the mitochondrial membrane potential (depolarization), there is diffusion of the dye in the cytoplasm, consequently the emission peak of JC-1 undergoes a shift, with a transition from red to green fluorescence. The lymphocyte cultures, after stimulation with HBV peptides, were incubated at a temperature of 37°C for 10 days in the presence or absence of the different compounds under test. After surface labeling with anti-CD3 and anti-CD8 antibodies for 15 min at room temperature, cells were resuspended in RPMI medium with 10% FCS prewarmed to 37°C and incubated with probe JC-1 (2.5 ?g/ml) or DiOC6 (20nM) or MitoSox Red (5 ??) for 15 minutes at 37?C, protected from light. Following the addition of the vitality probe? 7AAD, samples were acquired and analyzed by eight-color flow cytometry (FACS-Canto II, Becton-Dickinson, CA, USA), using the FACS-DIVA software. As a positive control, to detect depolarized lymphocytes, cells were treated with the protonophore valinomycin at a concentration of 100?? for 10 minutes at 37?C, before adding JC-1 or D?OC6 probe.
6 - Rilevazione degli aggregati proteici 6 - Detection of protein aggregates
L?analisi citofluorimetrica condotta sui linfociti T utilizzando il kit ?ProteoStat? Aggresome Detection? (Enzo Life Sciences, New York, NY) ha permesso la quantificazione degli aggregati proteici cellulari. Tale kit contiene una sonda fluorescente (Proteostat) che si intercala nella struttura quaternaria originata in seguito ad aggregazione proteica e ne permette la rilevazione. Le colture linfocitarie in presenza o assenza dei trattamenti sono state sottoposte alla marcatura di superficie (anticorpi anti-CD3, anti-CD8) per 15 minuti a temperatura ambiente, a fissazione (Citofix, BD) e a permeabilizzazione (Triton X 0,5%) per permettere la marcatura intracellulare con la sonda Proteostat. The cytofluorimetric analysis conducted on T lymphocytes using the ?ProteoStat? Aggressive Detection? (Enzo Life Sciences, New York, NY) has enabled the quantification of cellular protein aggregates. This kit contains a fluorescent probe (Proteostat) which intercalates in the quaternary structure originating following protein aggregation and allows its detection. Lymphocyte cultures in the presence or absence of treatments were subjected to surface labeling (anti-CD3, anti-CD8 antibodies) for 15 minutes at room temperature, fixation (Citofix, BD) and permeabilization (Triton X 0.5%) to allow intracellular labeling with the Proteostat probe.
La quantit? di aggregati proteici presenti nei campioni analizzati ? stata determinata confrontando la mediana dell?intensit? di fluorescenza (MFI) rilevata nei campioni trattati con i diversi composti e quella rilevata nei corrispondenti campioni non trattati. The quantity? of protein aggregates present in the analyzed samples ? been determined by comparing the median of? intensity? of fluorescence (MFI) detected in the samples treated with the different compounds and that detected in the corresponding untreated samples.
7 - Fosforilazione dell?istone H2AX e acetilazione dell?istone H3 e H4 7 - Phosphorylation of histone H2AX and acetylation of histone H3 and H4
Per studiare la capacit? di risposta al danno del DNA e quindi l?attivazione dei sensori del danno, cos? come l? acetilazione degli istoni come meccanismo epigenetico che attiva la trascrizione del DNA in risposta ad uno stimolo antigenico, le colture linfocitarie trattate con le diverse sostanze in studio, al nono/decimo giorno di coltura sono state divise a met?, per poter ri-stimolare ciascuna coltura per due ore con gli stessi peptidi HBV, mantenendone una met? non stimolata, per controllo. Questo per poter calcolare l?up-regolazione, in termini di incremento rispetto al valore basale, sia della fosforilazione di H2AX, che dell?acetilazione degli istoni H3 o H4 in presenza o in assenza dei diversi trattamenti. Brevemente, le cellule sono state fissate con il reagente BD Phosflow Fix Buffer I (Becton Dickinson, BD) pre-riscaldato a 37?C, per 10 minuti a 37?C. Quindi dopo gli opportuni lavaggi, le cellule sono state risospese nel reagente permeabilizzante BD Phosflow Perm/Wash Buffer I e sono state incubate a 4?C per 30? con gli anticorpi anti-CD3, CD8, e anti-phosphoH2AX (BD) oppure anti-acetyl H3 o anti-acetyl H4 (Merck, Millipore). I campioni sono stati quindi acquisiti mediante citofluorimetro a flusso a otto colori (FACS-Canto II, Becton-Dickinson, CA, USA), utilizzando il software FACS-DIVA sia per l?acquisizione che per l?analisi. To study the ability of response to the damage of the DNA and therefore the activation of the sensors of the damage, cos? how the? histone acetylation as an epigenetic mechanism that activates DNA transcription in response to an antigenic stimulus, the lymphocyte cultures treated with the different substances under study, on the ninth/tenth day of culture were divided in half, in order to be able to re-stimulate each culture for two hours with the same HBV peptides, keeping one half? unstimulated, for control. This is to be able to calculate the up-regulation, in terms of increase with respect to the baseline value, of both the phosphorylation of H2AX and the acetylation of histones H3 or H4 in the presence or absence of the different treatments. Briefly, cells were fixed with BD Phosflow Fix Buffer I reagent (Becton Dickinson, BD) pre-warmed to 37°C, for 10 minutes at 37°C. Then after appropriate washings, the cells were resuspended in BD Phosflow Perm/Wash Buffer I permeabilization reagent and incubated at 4?C for 30? with anti-CD3, CD8, and anti-phosphoH2AX (BD) or anti-acetyl H3 or anti-acetyl H4 antibodies (Merck, Millipore). Samples were then acquired by eight-color flow cytometry (FACS-Canto II, Becton-Dickinson, CA, USA), using FACS-DIVA software for both acquisition and analysis.
8 - Valutazione della produzione di citochine intracellulari (ICS) Allo scopo di valutare la funzionalit? anti-virale dei linfociti T ? stata studiata la produzione di citochine intracellulari e l?up-regolazione di CD107a, quale misura indiretta di attivit? citotossica, in seguito a stimolo virus-specifico. Le cellule sono state incubate in presenza di peptidi virali (alla concentrazione finale di 1 ??) e anticorpo anti-CD107a per un?ora a 37?C. Successivamente sono stati aggiunti 10 pg/ml di Brefeldina-A per ulteriori 3 ore di incubazione, al fine di bloccare la secrezione delle citochine prodotte da parte delle cellule. Al termine dell?incubazione, le cellule sono state lavate con tampone PBS IX ( Phosphate buffered saline)+ 0,5% FCS ( Fetal Calf Serum ) e colorate con gli opportuni marcatori di superficie (Destramero MHC di Classe I, anticorpo anti-CD3, anti-CD8, anti-CD4). 8 - Evaluation of the production of intracellular cytokines (ICS) In order to evaluate the functionality? anti-viral of T lymphocytes ? The production of intracellular cytokines and the up-regulation of CD107a was studied, as an indirect measure of activity cytotoxic, following a virus-specific stimulus. The cells were incubated in the presence of viral peptides (at the final concentration of 1 ??) and anti-CD107a antibody for one hour at 37°C. Subsequently 10 µg/ml of Brefeldin-A were added for a further 3 hours of incubation, in order to block the secretion of the cytokines produced by the cells. At the end of the incubation, the cells were washed with PBS IX buffer ( Phosphate buffered saline) + 0.5% FCS ( Fetal Calf Serum ) and stained with the appropriate surface markers (MHC Class I dextramer, anti-CD3 antibody , anti-CD8, anti-CD4).
Le cellule sono state poi trattate con medium A e medium B, rispettivamente per il fissaggio e la permeabilizzazione, (Nordic MUbio, Susteren Olanda) per consentire la reazione a livello intracellulare degli anticorpi monoclonali anti-IFN-?, anti-TNF-? e anti-IL-2 durante l incubazione di 15 minuti a temperatura ambiente. Dopo opportuno lavaggio, si ? proceduto all?analisi mediante citometria a flusso. The cells were then treated with medium A and medium B, respectively for fixation and permeabilization, (Nordic MUbio, Susteren The Netherlands) to allow the intracellular reaction of the anti-IFN-?, anti-TNF-?, anti-TNF-? and anti-IL-2 during a 15 minute incubation at room temperature. After appropriate washing, yes ? proceeded to the analysis by flow cytometry.
9 - Analisi statistiche 9 - Statistical analysis
L?effetto sulle funzioni intracellulari e i livelli di citochine prodotte dai linfociti T nelle differenti condizioni sperimentali sono stati confrontati utilizzando il test di Wilcoxon per ranghi o per dati appaiati. L?entit? dell?aumento, o fold change (FC), della produzione di citochine da parte delle cellule trattate con le diverse sostanze testate (FC?1) ? stato valutato con il test di Wilcoxon per ranghi. The effect on intracellular functions and levels of cytokines produced by T lymphocytes in the different experimental conditions were compared using the Wilcoxon test for ranks or paired data. The entity? of the increase, or fold change (FC), of the production of cytokines by the cells treated with the different substances tested (FC?1) ? was evaluated with the Wilcoxon test for ranks.
RISULTATI DEL TRATTAMENTO TREATMENT RESULTS
1 - Recupero delle funzionalit? mitocondriali e del proteasoma Per valutare l?effetto di NMN e anche di Entinostat sulle funzionalit? mitocondriali dei linfociti T CD8 HBV-specifici ?esauriti?, la percentuale di cellule depolarizzate presenti nelle colture linfocitarie effettuate in presenza o assenza dei rispettivi composti ? stata valutata utilizzando la sonda fluorescente JC-1, che accumulandosi all?interno del mitocondrio in funzione del potenziale di membrana mitocondriale, permette di evidenziare la percentuale di cellule depolarizzate. Come ? possibile osservare nella Figura 7a, sia nelle colture trattate con NMN, che in quelle trattate con Entinostat si ? osservata una significativa riduzione della percentuale di linfociti CD8 depolarizzati se confrontate con le corrispondenti colture non trattate di controllo. Nelle stesse colture trattate con NMN o con Entinostat si ? potuto osservare una diminuzione di aggregati proteici intracellulari (aggresomi) nelle colture trattate con entrambe le sostanze in esame, rispetto alle corrispondenti colture non trattate, indicando una maggiore efficienza della proteostasi nella degradazione degli aggregati proteici (Figura 7b). 1 - Recovery of functionalities? mitochondrial and proteasome To evaluate the effect of NMN and also of Entinostat on functional? mitochondrial concentrations of HBV-specific CD8 T lymphocytes ?depleted?, the percentage of depolarized cells present in lymphocyte cultures performed in the presence or absence of the respective compounds ? was evaluated using the JC-1 fluorescent probe, which by accumulating inside the mitochondrion as a function of the mitochondrial membrane potential, allows to highlight the percentage of depolarized cells. As ? possible to observe in Figure 7a, both in the cultures treated with NMN and in those treated with Entinostat yes? observed a significant reduction in the percentage of depolarized CD8 lymphocytes when compared to the corresponding untreated control cultures. In the same cultures treated with NMN or with Entinostat yes? could observe a decrease of intracellular protein aggregates (aggresomes) in cultures treated with both test substances, compared to the corresponding untreated cultures, indicating a higher efficiency of proteostasis in the degradation of protein aggregates (Figure 7b).
2- Incremento della risposta al danno del DNA 2- Increased response to DNA damage
Le colture linfocitarie sono state ri-stimolate con i peptidi HBV per due ore, e quindi si ? effettuata la colorazione intracellulare con anticorpo anti-phosphoH2AX della coltura ri-stimolata e non, per valutarne l?incremento della fosforilazione di H2AX rispetto al valore basale, poich? nei dati di validazione sui campioni ex-vivo era stata precedentemente osservata una minore up-regolazione di tale fosforilazione in seguito a stimolo (Figura 4). Come possibile osservare in Figura 7c, sia nelle colture trattate con NMN, che in quelle trattate con Entinostat si ? osservato un significativo incremento del rapporto tra il valore di intensit? di fluorescenza (MFI) di phosphoH2AX nella coltura ristimolata. Lymphocyte cultures were re-stimulated with HBV peptides for two hours, and then ? performed intracellular staining with anti-phosphoH2AX antibody of the re-stimulated and non-stimulated culture, to evaluate the increase in H2AX phosphorylation compared to the baseline value, since? in the validation data on ex-vivo samples, a minor up-regulation of this phosphorylation following stimulation had previously been observed (Figure 4). As it is possible to observe in Figure 7c, both in the cultures treated with NMN and in those treated with Entinostat yes? observed a significant increase in the relationship between the value of intensity? fluorescence analysis (MFI) of phosphoH2AX in the resampled culture.
3- Incremento dell?acetilazione degli istoni H3 e H4 3- Increased acetylation of histones H3 and H4
Le colture linfocitarie sono state ri-stimolate con i peptidi HBV per due ore, e quindi si ? effettuata la colorazione intracellulare con anticorpo anti-acetylH3 o anti-acetylH4 della coltura ri-stimolata e non, per valutarne l?incremento dell?acetilazione rispetto al valore basale, poich? nei dati di validazione sui campioni ex-vivo era stata precedentemente osservata una minore up-regolazione dell?acetilazione istonica in seguito a stimolo (Figura 5). Come ? possibile osservare in Figure 7d e 7e, sia nelle colture trattate con NMN, che in quelle trattate con Entinostat si ? osservato un significativo incremento del rapporto tra i valori di intensit? di fluorescenza (MFI) di acetyl-FI3 o H4 nelle colture ristimolate. Lymphocyte cultures were re-stimulated with HBV peptides for two hours, and then ? intracellular staining with anti-acetylH3 or anti-acetylH4 antibody of the re-stimulated and non-stimulated culture was performed, to evaluate the increase in acetylation compared to the baseline value, since? minor up-regulation of histone acetylation upon stimulation was previously observed in the validation data on ex-vivo samples (Figure 5). As ? possible to observe in Figures 7d and 7e, both in the cultures treated with NMN and in those treated with Entinostat yes? observed a significant increase in the relationship between the intensity values? of fluorescence (MFI) of acetyl-FI3 or H4 in ristimulated cultures.
Recupero delle funzionalit? antivirali Recovery of features? antivirals
Per valutare il ripristino della funzionalit? antivirale dei linfociti ?esauriti? HBV-specifici, sono state prodotte in vitro linee di linfociti a breve termine (10 giorni), in presenza o assenza delle diverse sostanze in studio, generate mediante stimolazione di PBMCs di pazienti con epatite cronica, con peptidi sintetici corrispondenti alla proteina core di HBV. NMN ? stato testato su campioni provenienti da 26 pazienti volontari, F inibitore di CD38 in combinazione con NMN su 22, Entinostat su 22. Al decimo giorno di incubazione, ? stata valutata la produzione di citochine in risposta a stimolazione con i peptidi HBV-specifici (corrispondenti alla proteina core), attraverso marcatura intracellulare delle citochine (ICS) e successiva analisi tramite citometria a flusso. Dai risultati ? emerso che tutti i composti studiati promuovono un aumento statisticamente significativo della produzione di citochine antivirali da parte dei linfociti T CD8, rispetto alle cellule non trattate. L?incremento della produzione di citochine da parte dei linfociti CD8 in seguito a stimolo HBV-specifico ? stato calcolato come rapporto tra la percentuale di cellule producenti citochine nel campione trattato con i diversi composti rispetto al non trattato (?fold change?, FC). To evaluate the restoration of functionality? antiviral of lymphocytes ?exhausted? HBV-specific, short-term (10 days) lymphocyte lines were produced in vitro, in the presence or absence of the different study substances, generated by stimulation of PBMCs of patients with chronic hepatitis, with synthetic peptides corresponding to the HBV core protein . NMN ? been tested on samples from 26 patient volunteers, CD38 F inhibitor in combination with NMN on 22, Entinostat on 22. On the tenth day of incubation, ? The production of cytokines in response to stimulation with HBV-specific peptides (corresponding to the core protein) was evaluated by intracellular labeling of cytokines (ICS) and subsequent analysis by flow cytometry. From the results ? showed that all compounds studied promote a statistically significant increase in the production of antiviral cytokines by CD8 T lymphocytes, compared to untreated cells. The increase in cytokine production by CD8 lymphocytes following an HBV-specific stimulus ? was calculated as the ratio between the percentage of cytokine-producing cells in the sample treated with the different compounds compared to the untreated sample (?fold change?, FC).
Il trattamento con Nicotinamide mononucleotide (NMN) ? stato in grado di indurre un incremento medio nella produzione di IFN-? di 3,396 volte, di 4,04 per il TNF-a, 2,72 per IL2, 1,79 per CD107a e di 3,79 per i linfociti CD8 che producono sia IFN-? che TNF-? (Figura 8a). Treatment with Nicotinamide mononucleotide (NMN) ? been able to induce an average increase in the production of IFN-? by 3.396-fold, 4.04 for TNF-a, 2.72 for IL2, 1.79 for CD107a and 3.79 for CD8 lymphocytes that produce both IFN-? what TNF-? (Figure 8a).
Il trattamento con Entinostat ? stato in grado di indurre un incremento medio nella produzione di IFN-? di 5,5 volte, di 5 volte per il TNF-?, di 2 volte per IL2, 2,7 per CD107a e di 5,7 volte per i linfociti che producono sia IFN-? che TNF-? La produzione di IFN-? e/o TNF-? ? risultata aumenta di almeno 1,5 volte in 18 pazienti su 22, pari all?81,8% (Figura 8b). The treatment with Entinostat ? been able to induce an average increase in the production of IFN-? by 5.5-fold, 5-fold for TNF-?, 2-fold for IL2, 2.7-fold for CD107a and 5.7-fold for lymphocytes that produce both IFN-? what TNF-? The production of IFN-? and/or TNF-? ? result increases by at least 1.5-fold in 18 of 22 patients, equal to 81.8% (Figure 8b).
In presenza di NMN la produzione di IFN-? e/o TNF-? aumenta di almeno 1,5 volte in 13 pazienti su 22, mentre associando a NMN l?inibitore di CD38 si ? potuto osservare un incremento di almeno 1,5 volte di IFN-? e/o TNF-? in 18 campioni su un totale di 22 campioni esaminati (Figure 9a-9c). In the presence of NMN, the production of IFN-? and/or TNF-? increases by at least 1.5 times in 13 out of 22 patients, while associating the CD38 inhibitor with NMN is ? could observe at least a 1.5-fold increase in IFN-? and/or TNF-? in 18 samples out of a total of 22 samples examined (Figures 9a-9c).
Inoltre, si ? osservato un aumento della capacit? proliferativa (Figura 10). Also, yes? observed an increase in capacity? proliferative (Figure 10).
? chiaro che al composto farmacologicamente accettabile per l?uso in un trattamento terapeutico dell?infezione virale cronica da HBY fin qui descritto possono essere apportate modifiche e/o aggiunte di parti o fasi, senza per questo uscire dall?ambito del presente trovato come definito dalle rivendicazioni. ? it is clear that modifications and/or additions of parts or phases can be made to the pharmacologically acceptable compound for use in a therapeutic treatment of chronic HBY viral infection described up to now, without thereby departing from the scope of the present invention as defined by the claims.
Nelle rivendicazioni che seguono, i riferimenti tra parentesi hanno il solo scopo di facilitare la lettura e non devono essere considerati come fattori limitativi per quanto attiene all?ambito di protezione sotteso dalle specifiche rivendicazioni. In the claims that follow, the references in brackets have the sole purpose of facilitating the reading and must not be considered as limiting factors as regards the scope of protection implied by the specific claims.
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