IT202100025976A1 - Combination of active substances for the treatment of acute myeloid leukemia (AML) with nucleophosmin (NPM1) mutation - Google Patents
Combination of active substances for the treatment of acute myeloid leukemia (AML) with nucleophosmin (NPM1) mutation Download PDFInfo
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- IT202100025976A1 IT202100025976A1 IT102021000025976A IT202100025976A IT202100025976A1 IT 202100025976 A1 IT202100025976 A1 IT 202100025976A1 IT 102021000025976 A IT102021000025976 A IT 102021000025976A IT 202100025976 A IT202100025976 A IT 202100025976A IT 202100025976 A1 IT202100025976 A1 IT 202100025976A1
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/63—Compounds containing para-N-benzenesulfonyl-N-groups, e.g. sulfanilamide, p-nitrobenzenesulfonyl hydrazide
- A61K31/635—Compounds containing para-N-benzenesulfonyl-N-groups, e.g. sulfanilamide, p-nitrobenzenesulfonyl hydrazide having a heterocyclic ring, e.g. sulfadiazine
Description
COMBINAZIONE DI PRINCIPI ATTIVI PER IL TRATTAMENTO DELLA LEUCEMIA ACUTA MIELOIDE (LAM) CON MUTAZIONE DELLA COMBINATION OF ACTIVE INGREDIENTS FOR THE TREATMENT OF ACUTE MYELOID LEUKEMIA (AML) WITH MUTATION OF
NUCLEOFOSMINA (NPM1) NUCLEOPHOSMIN (NPM1)
La presente invenzione riguarda una combinazione di principi attivi per il trattamento della leucemia acuta mieloide (LAM) con mutazione della nucleofosmina (NPM1). In particolare l?invenzione riguarda una combinazione di omacetaxina mepesuccinato e venetoclax per il trattamento della leucemia acuta mieloide (LAM) con mutazione della nucleofosmina (NPM1). The present invention concerns a combination of active ingredients for the treatment of acute myeloid leukemia (AML) with nucleophosmin (NPM1) mutation. In particular, the invention concerns a combination of omacetaxine mepesuccinate and venetoclax for the treatment of acute myeloid leukemia (AML) with nucleophosmin (NPM1) mutation.
? noto che la leucemia acuta mieloide (LAM) ? una malattia eterogenea dal punto di vista clinico e molecolare. Nonostante i continui progressi nella conoscenza della biologia della LAM, ad oggi solo il 40-45% dei pazienti pi? giovani e il 10-15 % dei pazienti pi? anziani (et? ?60 anni) pu? essere curato con terapia standard ed eventualmente sottoposto a trapianto allogenico di cellule staminali. Questa ? una sfida importante, soprattutto perch? i pazienti anziani costituiscono oltre il 50% della popolazione con LAM. Le prospettive sono particolarmente sfavorevoli per i pazienti di qualsiasi et? affetti da malattia recidivante e/o refrattaria (R/R) (con tassi di guarigione non superiori al 10%). Inoltre, poich? l'et? media dei soggetti malati si avvicina ai 70 anni, i pazienti hanno comunemente un cattivo performance status o altre comorbidit?, con un conseguente aumento della mortalit? correlata al trattamento, che impedisce di sottoporre tali pazienti a trattamenti intensivi. ? known that acute myeloid leukemia (AML) is a heterogeneous disease from a clinical and molecular point of view. Despite continuous progress in understanding the biology of LAM, to date only 40-45% of patients are affected. young and 10-15% of patients are younger elderly (age? 60 years) can? be treated with standard therapy and possibly subjected to an allogeneic stem cell transplant. This ? an important challenge, especially because? Elderly patients make up more than 50% of the LAM population. Are the prospects particularly unfavorable for patients of any age? suffering from relapsed and/or refractory (R/R) disease (with cure rates not exceeding 10%). Furthermore, since? the age? average of sick subjects is approaching 70 years of age, patients commonly have a poor performance status or other comorbidities, with a consequent increase in mortality? related to treatment, which prevents these patients from being subjected to intensive treatments.
Le opzioni terapeutiche standard disponibili per i pazienti anziani non idonei a regimi di chemioterapia intensiva sono agenti ipometilanti (HMA), in particolare azacitidina o decitabina, o citarabina a basso dosaggio (LDAC). Ad oggi, i suddetti agenti ipometilanti o la citarabina sono preferibilmente somministrati in associazione con l?agente proapoptotico venetoclax, inibitore della proteina antiapoptotica B-cell leucemia/linfoma-2 (Bcl-2), di recente introduzione nella terapia delle LAM. Alternativamente, sempre nei pazienti anziani, sono adottate misure puramente di supporto. Standard treatment options available for elderly patients ineligible for intensive chemotherapy regimens are hypomethylating agents (HMA), particularly azacitidine or decitabine, or low-dose cytarabine (LDAC). To date, the aforementioned hypomethylating agents or cytarabine are preferably administered in association with the proapoptotic agent venetoclax, inhibitor of the antiapoptotic protein B-cell leukemia/lymphoma-2 (Bcl-2), recently introduced in the therapy of AML. Alternatively, again in elderly patients, purely supportive measures are adopted.
Venetoclax ? un inibitore selettivo della proteina Bcl-2, che ? sovraespressa in pi? tipi di cancro. Il gene BCL-2 ? un oncogene anti-apoptotico, il che significa che pu? prevenire l'apoptosi di cellule maligne. Con l'inibizione della proteina Bcl-2 pu? essere attivata l'apoptosi di cellule tumorali. I primi brevetti per venetoclax sono stati concessi negli Stati Uniti tra il 2013 e il 2015 [US8580794B2; US8546399B2; US9045475B2; US9174982B2;]. Venetoclax ha mostrato un?alta affinit? di legame verso la proteina Bcl-2 ed efficacia in un contesto cellulare. Inoltre, ? stato mostrato il legame selettivo di venetoclax a Bcl-2. Il percorso di sintesi originale e il percorso di produzione sono stati descritti nelle domande di brevetto di AbbVie [WO2010/138588A2; WO2011/149492A1; WO2012/071374A1; WO2012/071336A1; WO2014/165044A1]. Un percorso alternativo di sintesi pu? essere trovato nel brevetto CN104370905B. Venetoclax? a selective inhibitor of the Bcl-2 protein, which is overexpressed more? types of cancer. The BCL-2 gene? an anti-apoptotic oncogene, which means it can? prevent apoptosis of malignant cells. With the inhibition of the Bcl-2 protein it can? apoptosis of tumor cells be activated. The first patents for venetoclax were granted in the United States between 2013 and 2015 [US8580794B2; US8546399B2; US9045475B2; US9174982B2;]. Venetoclax showed a high affinity? binding to the Bcl-2 protein and efficacy in a cellular context. Furthermore, ? Selective binding of venetoclax to Bcl-2 was shown. The original synthesis route and manufacturing route were described in AbbVie's patent applications [WO2010/138588A2; WO2011/149492A1; WO2012/071374A1; WO2012/071336A1; WO2014/165044A1]. An alternative synthesis path can? be found in patent CN104370905B.
Venetoclax pu? essere utilizzato per il trattamento di una variet? di tumori. Can Venetoclax? be used for the treatment of a variety? of tumors.
In particolare, venetoclax ? indicato: i) per il trattamento di pazienti adulti con leucemia linfatica cronica (LLC) o linfoma a piccoli linfociti (SLL), con o senza delezione 17p, che hanno ricevuto almeno una precedente terapia; e, come detto sopra, ii) in combinazione con azacitidina o decitabina o citarabina a basso dosaggio per il trattamento della leucemia acuta mieloide (LAM) di nuova diagnosi negli adulti di et? pari o superiore a 75 anni o con comorbilit? che ne precludono l'uso della chemioterapia di induzione intensiva. In particular, venetoclax ? indicated: i) for the treatment of adult patients with chronic lymphocytic leukemia (CLL) or small lymphocytic lymphoma (SLL), with or without 17p deletion, who have received at least one previous therapy; and, as mentioned above, ii) in combination with azacitidine or decitabine or low-dose cytarabine for the treatment of newly diagnosed acute myeloid leukemia (AML) in older adults. 75 years of age or older or with comorbidities? which preclude the use of intensive induction chemotherapy.
Inoltre, venetoclax ? risultato essere efficace nei tumori resistenti. Furthermore, venetoclax ? found to be effective in resistant tumors.
Tuttavia, nonostante i risultati promettenti delle recenti terapie di associazione con venetoclax sopra descritte per il trattamento della LAM in soggetti non idonei a regimi di chemioterapia intensiva, la maggior parte dei pazienti va comunque incontro a recidiva e soccombe alla propria malattia. However, despite the promising results of the recent combination therapies with venetoclax described above for the treatment of AML in subjects unsuitable for intensive chemotherapy regimens, the majority of patients still experience relapse and succumb to their disease.
Inoltre, sono noti diversi studi pubblicati che hanno impiegato omoarringtonina (HHT) o omacetaxina mepesuccinato nel trattamento delle LAM (1). Furthermore, there are several published studies that have used homoharringtonine (HHT) or omacetaxine mepesuccinate in the treatment of AML (1).
L'omacetaxina mepesuccinato (nota anche come omacetaxina, omoarringtonina o HHT) ? una forma semisintetica di un estratto alcaloide della pianta Cephalotaxus fortunei. HHT si lega alla fessura del sito A dei ribosomi prevenendo la fase di allungamento iniziale della sintesi proteica e che porta a un'inibizione transitoria ma profonda della sintesi proteica con effetto soprattutto sui livelli di proteine con pi? breve emivita, compresa la proteina antiapoptotica myeloid cell leukemia-1 (Mcl-1)(2). Da sottolineare come Mcl-1 sia coinvolta nei meccanismi di resistenza a seguito dell'inibizione di Bcl-2. Omacetaxine mepesuccinate (also known as omacetaxine, homoharringtonine or HHT) is a semi-synthetic form of an alkaloid extract of the Cephalotaxus fortunei plant. HHT binds to the A-site cleft of the ribosomes preventing the initial elongation phase of protein synthesis and leading to a transient but profound inhibition of protein synthesis with an effect mainly on the levels of proteins with more? short half-life, including the antiapoptotic protein myeloid cell leukemia-1 (Mcl-1) (2). It should be underlined that Mcl-1 is involved in resistance mechanisms following the inhibition of Bcl-2.
Omacetaxina mepesuccinato ? noto per il trattamento di pazienti adulti con leucemia mieloide cronica (LMC) in fase cronica o accelerata con resistenza e/o intolleranza a due o pi? inibitori della tirosin-chinasi (TKI). Omacetaxine mepesuccinate? known for the treatment of adult patients with chronic or accelerated phase chronic myeloid leukemia (CML) with resistance and/or intolerance to two or more tyrosine kinase inhibitors (TKIs).
L?omacetaxina ? stata approvata dalla FDA, con il nome commerciale Synribo, per il trattamento di pazienti adulti con leucemia mieloide cronica (LMC) resistente e/o intollerante a due o pi? inibitori di tirosin chinasi. Synribo ? un farmaco commercializzato da Teva Pharms Intl, ed ? incluso in un NDA (New Drug Application) approvato dalla FDA (NDA: 203585). Synribo non ? in commercio in Europa. C'? un brevetto che protegge questo farmaco: ?Trattamento della leucemia mieloide cronica, resistente o intollerante a ST1571, che coinvolge l'omoarringtonina da sola o in combinazione con altri agenti? [US6987103B2]. L?omacetaxina? has been approved by the FDA, under the trade name Synribo, for the treatment of adult patients with chronic myeloid leukemia (CML) resistant and/or intolerant to two or more tyrosine kinase inhibitors. Synribo ? a drug marketed by Teva Pharms Intl, and it is included in an FDA-approved NDA (New Drug Application) (NDA: 203585). Synribo isn't it? on the market in Europe. Is there? a patent protecting this drug: ?Treatment of chronic myeloid leukemia, resistant or intolerant to ST1571, involving homoharringtonine alone or in combination with other agents? [US6987103B2].
Inoltre, ? nota una recente pubblicazione concernente la sperimentazione di laboratorio in modelli cellulari di LAM e anche nel modello animale (murino) della associazione venetoclax e omoarringtonina (3). Furthermore, ? notes a recent publication concerning laboratory testing in cellular models of AML and also in the animal (murine) model of the combination of venetoclax and homoharringtonine (3).
Da una ricerca su clinicaltrial.gov si rileva che sono stati appena attivati (not yet recruiting) nel setting terapia delle LAM i seguenti trial clinici, che prevedono l?associazione di omacetaxina mepesuccinato e venetoclax per il trattamento di pazienti con LAM: A search on clinicaltrial.gov shows that the following clinical trials have just been activated (not yet recruiting) in the AML therapy setting, which involve the association of omacetaxine mepesuccinate and venetoclax for the treatment of patients with AML:
- ?Omacetaxine and Venetoclax for the Treatment of Relapsed or Refractory Acute Myeloid Leukemia or Myelodysplastic Syndrome Harboring Mutant RUNX1.? ClinicalTrials.gov Identifier: NCT04874194 (not yet recruiting); - ?Omacetaxine and Venetoclax for the Treatment of Relapsed or Refractory Acute Myeloid Leukemia or Myelodysplastic Syndrome Harboring Mutant RUNX1.? ClinicalTrials.gov Identifier: NCT04874194 (not yet recruiting);
- ?Safety and Efficacy of Venetoclax With Escalating Doses of Omacetaxine in Patients With Acute Myeloid Leukemia (VEN-OM).? ClinicalTrials.gov Identifier: NCT04926285 (not yet recruiting). - ?Safety and Efficacy of Venetoclax With Escalating Doses of Omacetaxine in Patients With Acute Myeloid Leukemia (VEN-OM).? ClinicalTrials.gov Identifier: NCT04926285 (not yet recruiting).
Ad oggi, quindi, la LAM rimane una necessit? medica non soddisfatta (unmet need), in particolare nei pazienti non idonei a regimi di chemioterapia intensiva. To date, therefore, LAM remains a necessity? unmet medical need, particularly in patients unsuitable for intensive chemotherapy regimens.
Inoltre, nonostante negli ultimi 5 anni siano stati sviluppati diversi nuovi farmaci mirati contro specifiche lesioni genetiche per il trattamento della LAM, in molti sottotipi di LAM la lesione genetica sottostante non ? aggredibile. Pertanto, la terapia deve basarsi su farmaci che vadano possibilmente a colpire pathway cellulari alternativi essenziali per la sopravvivenza della leucemia. Furthermore, although several new drugs targeting specific genetic lesions have been developed in the last 5 years for the treatment of AML, in many subtypes of AML the underlying genetic lesion is not attackable. Therefore, therapy must be based on drugs that possibly target alternative cellular pathways essential for leukemia survival.
La leucemia acuta mieloide con mutazione della nucleofosmina (NPM1) ? un sottotipo di LAM. In particolare, la mutazione del gene NPM1 ? la lesione genetica pi? frequente nella LAM. Acute myeloid leukemia with nucleophosmin mutation (NPM1)? a subtype of AML. In particular, the mutation of the NPM1 gene? the most genetic lesion? frequent in LAM.
Diverse evidenze biologiche e cliniche indicano che la presenza della mutazione del gene NPM1 rappresenti uno dei principali fattori predittivi di risposta favorevole alla terapia con venetoclax nella LAM. Come detto sopra, venetoclax ? un inibitore della proteina anti-apoptotica Bcl-2, che ? altamente espressa nella maggior parte della LAM, inclusa la LAM NPM1-mutata. Venetoclax ? risultato essere estremamente efficace in combinazione con agenti ipometilanti come prima linea di terapia, in particolare nella LAM con mutazione di NPM1, nei pazienti anziani non eleggibili alla chemioterapia intensiva (4,5). I meccanismi alla base di questa peculiare sensibilit? ad oggi non sono ancora stati chiariti. Tuttavia, la presenza di altre mutazioni concomitanti, ed in particolare la mutazione FLT3-ITD (che ? presente in circa il 35-40% dei casi di LAM NPM1-mutata), si rende responsabile della resistenza al trattamento (5,6). Various biological and clinical evidence indicates that the presence of the NPM1 gene mutation represents one of the main predictive factors of a favorable response to venetoclax therapy in AML. As mentioned above, venetoclax ? an inhibitor of the anti-apoptotic protein Bcl-2, which is highly expressed in most AML, including NPM1-mutated AML. Venetoclax? proved to be extremely effective in combination with hypomethylating agents as first line of therapy, particularly in AML with NPM1 mutation, in elderly patients not eligible for intensive chemotherapy (4,5). The mechanisms underlying this peculiar sensitivity? to date they have not yet been clarified. However, the presence of other concomitant mutations, and in particular the FLT3-ITD mutation (which is present in approximately 35-40% of cases of NPM1-mutated AML), is responsible for resistance to treatment (5,6).
Alla luce di quanto sopra, appare evidente la necessit? di fornire nuovi trattamenti per la leucemia acuta mieloide (LAM) con mutazione della nucleofosmina (NPM1), che superino gli svantaggi ai trattamenti noti. In light of the above, the need is evident to provide new treatments for acute myeloid leukemia (AML) with nucleophosmin (NPM1) mutation, which overcome the disadvantages to known treatments.
Nell'ambito della medicina di precisione nella terapia della LAM, nella LAM con mutazione di NPM1 la proteina leucemica NPM1 mutata rappresenterebbe il bersaglio ideale. Tuttavia, come per la maggior parte delle mutazioni del cancro, NPM1 non ? un bersaglio direttamente aggredibile farmacologicamente. Una strategia per superare questo problema pu? consistere nell'indirizzare l'oncoproteina NPM1 mutata alla degradazione. Il noto modello di una particolare forma di LAM, la leucemia acuta promielocitica (APL) dimostra le grandi potenzialit? di questa strategia. Infatti la terapia combinata con un derivato della vitamina A (acido all-trans-retinoico, ATRA) e triossido di arsenico (ATO) induce degradazione della proteina tumorale specifica della APL (PML-RARa) con conseguente sblocco della differenziazione e induzione di morte cellulare. A questi eventi si associa una elevatissima percentuale (>90%) di guarigione. Inoltre, ? stato scoperto che la combinazione di ATRA e ATO induce in vitro la degradazione mediata dal proteasoma specificamente del mutante NPM1, seguita da arresto della crescita cellulare e apoptosi [US20160317579A1] (7). Tuttavia, l'efficacia clinica di ATRA/ATO nei pazienti con LAM NPM1-mutata appare limitata. In the context of precision medicine in the therapy of AML, in AML with NPM1 mutation the mutated NPM1 leukemic protein would represent the ideal target. However, as with most cancer mutations, NPM1 is not ? a target that can be directly attacked pharmacologically. A strategy to overcome this problem can? consists in directing the mutated NPM1 oncoprotein to degradation. The well-known model of a particular form of AML, acute promyelocytic leukemia (APL), demonstrates the great potential of this strategy. In fact, combined therapy with a vitamin A derivative (all-trans-retinoic acid, ATRA) and arsenic trioxide (ATO) induces degradation of the APL-specific tumor protein (PML-RARa) with consequent unlocking of differentiation and induction of cell death . These events are associated with a very high percentage (>90%) of recovery. Furthermore, ? The combination of ATRA and ATO was found to induce in vitro proteasome-mediated degradation specifically of the NPM1 mutant, followed by cell growth arrest and apoptosis [US20160317579A1] (7). However, the clinical efficacy of ATRA/ATO in patients with NPM1-mutated AML appears limited.
Queste osservazioni forniscono le basi per esplorare altri farmaci/composti pi? efficaci nel prendere di mira i livelli di proteina NPM1 mutata e probabilmente con una maggiore potenza anti-leucemica, legata anche ad altri meccanismi. Oltre alla riduzione dei livelli di proteina NPM1 mutata, infatti, potrebbe essere necessario colpire altri percorsi essenziali che contribuiscono allo sviluppo della leucemia e al mantenimento della crescita per eradicare le cellule leucemiche e ottenere risposte clinicamente rilevanti nei pazienti. These observations provide the basis for exploring other drugs/compounds more? effective in targeting mutated NPM1 protein levels and probably with greater anti-leukemic potency, also linked to other mechanisms. In addition to reducing the levels of mutated NPM1 protein, it may be necessary to target other essential pathways that contribute to leukemia development and growth maintenance to eradicate leukemic cells and obtain clinically relevant responses in patients.
Tra le vie sensibili nella LAM con mutazione di NPM1, vi ? l'esportazione nucleare di proteine Crm-1/Exportin 1-dipendente che media l'accumulo aberrante della proteina mutata NPM1 nel citoplasma delle cellule leucemiche, come evento critico nello sviluppo e mantenimento della leucemia. In effetti, ? stato riportato che il ritorno della proteina NPM1 al nucleo mediante correzione genetica della mutazione o con inibitori di Crm1 (ad esempio selinexor) determina una differenziazione terminale e una crescita delle cellule leucemiche significativamente ridotta in studi preclinici sia in vitro che in vivo sulla LAM con mutazione di NPM1 (8). Among the sensitive pathways in AML with NPM1 mutation, there is? Crm-1/Exportin 1-dependent nuclear export of proteins that mediates the aberrant accumulation of the mutated NPM1 protein in the cytoplasm of leukemia cells, as a critical event in the development and maintenance of leukemia. Effectively, ? Return of the NPM1 protein to the nucleus by genetic correction of the mutation or with Crm1 inhibitors (e.g. selinexor) has been reported to result in significantly reduced terminal differentiation and growth of leukemia cells in both in vitro and in vivo preclinical studies of mutated AML. of NPM1 (8).
In questo contesto viene ad inserirsi la soluzione secondo la presente invenzione, che si propone di fornire una combinazione di farmaci efficace per il trattamento della leucemia acuta mieloide (LAM) con mutazione della nucleofosmina (NPM1). The solution according to the present invention fits into this context, which aims to provide an effective combination of drugs for the treatment of acute myeloid leukemia (AML) with nucleophosmin mutation (NPM1).
In particolare, secondo la presente invenzione ? stato ora sorprendentemente trovato che la combinazione di due farmaci non chemioterapici, ossia omacetaxina mepesuccinato e venetoclax, approvati per l'impiego in altre malattie oncoematologiche, pu? essere vantaggiosamente impiegata nel trattamento della leucemia acuta mieloide con mutazione di NPM1. In particular, according to the present invention ? It has now been surprisingly found that the combination of two non-chemotherapeutic drugs, namely omacetaxine mepesuccinate and venetoclax, approved for use in other oncohematological diseases, can be advantageously used in the treatment of acute myeloid leukemia with NPM1 mutation.
Pi? dettagliatamente, i risultati sperimentali mostrati pi? avanti hanno mostrato che questa nuova combinazione di farmaci ha una potente attivit? sinergica contro questa forma di leucemia, sia in esperimenti eseguiti su modelli cellulari in laboratorio sia in esperimenti eseguiti su modelli di leucemia umana nel topo. In particolare, nel modello animale, la combinazione ha dimostrato un marcato effetto anti-leucemico con arresto della crescita della leucemia, che si ? tradotto in un significativo aumento della sopravvivenza dei topi trattati con la combinazione rispetto quelli trattati con i singoli farmaci. Inoltre, la combinazione si ? mostrata relativamente ben tollerata. Questi dati hanno fornito supporto per la sperimentazione clinica pilota approvata dagli organi italiani competenti, Istituto Superiore di Sanit? (ISS) e Agenzia Italiana del Farmaco (AIFA), quale studio di fase I volto a valutare la sicurezza e l'efficacia preliminare di omacetaxina mepesuccinato in associazione a venetoclax in pazienti con leucemia mieloide acuta con mutazione del gene NPM1 recidiva o refrattaria a precedenti terapie. Lo studio ha arruolato il primo paziente (UPN01), con risultati promettenti (riportati pi? avanti). More? in detail, the experimental results shown more? forward have shown that this new combination of drugs has a powerful activity? synergistic against this form of leukemia, both in experiments performed on cellular models in the laboratory and in experiments performed on human leukemia models in mice. In particular, in the animal model, the combination demonstrated a marked anti-leukemic effect with arrest of the growth of the leukemia, which is translated into a significant increase in the survival of mice treated with the combination compared to those treated with the individual drugs. Furthermore, the combination yes ? shown to be relatively well tolerated. These data provided support for the pilot clinical trial approved by the competent Italian bodies, Istituto Superiore di Sanit? (ISS) and the Italian Medicines Agency (AIFA), as a phase I study aimed at evaluating the safety and preliminary efficacy of omacetaxine mepesuccinate in association with venetoclax in patients with acute myeloid leukemia with relapsed or refractory to previous NPM1 gene mutation therapies. The study enrolled the first patient (UPN01), with promising results (reported later).
Nel dettaglio, secondo la presente invenzione, l?efficacia terapeutica della combinazione omacetaxina mepesuccinato e venetoclax ? stata testata in vivo utilizzando 2 diversi modelli estremamente aggressivi di PDX di LAM con mutazione di NPM1 (entrambi triplomutati per DNMT3A/NPM1/FLT3-ITD) modificati per esprimere la luciferasi. Questa tecnologia consente di tracciare la malattia in bioluminescenza in vivo (BLI) ? mediante l?impiego della strumentazione IVIS Lumina -durante il trattamento e valutare la risposta al farmaco. Sorprendentemente, sulla malattia conclamata la terapia combinatoria con HHT e venetoclax ha determinato un forte effetto anti-leucemico e sinergico, risultando in assenza di malattia rilevabile in una coorte di topi analizzati dopo il primo ciclo di trattamento (Figure 1 e 2), e in un vantaggio significativo in termini di sopravvivenza in una coorte analizzata per sopravvivenza rispetto ad animali trattati con il singolo farmaco o con veicolo (Figura 2). In detail, according to the present invention, the therapeutic efficacy of the combination omacetaxine mepesuccinate and venetoclax is? was tested in vivo using 2 different extremely aggressive PDX models of NPM1-mutated AML (both triple mutated for DNMT3A/NPM1/FLT3-ITD) modified to express luciferase. Does this technology enable in vivo bioluminescence (BLI) disease tracking? through the use of IVIS Lumina instrumentation - during treatment and evaluate the response to the drug. Surprisingly, on established disease, combinatorial therapy with HHT and venetoclax resulted in a strong anti-leukemic and synergistic effect, resulting in the absence of detectable disease in a cohort of mice analyzed after the first treatment cycle (Figures 1 and 2), and in a significant survival advantage in a cohort analyzed for survival compared to animals treated with the single drug or vehicle (Figure 2).
Inoltre, la presente invenzione, oltre a fornire una combinazione mai sperimentata prima per il trattamento LAM con mutazione di NPM1, concerne anche uno nuovo schema di trattamento e un razionale di uso specifico nella leucemia mieloide acuta con mutazione della nucleofosmina, sulla base dei dati sperimentali sia su modelli cellulari in laboratorio sia sui topi e dei dati preliminari di sperimentazione nell?uomo riportati pi? avanti (Figure 3-6). Furthermore, the present invention, in addition to providing a combination never tested before for the treatment of AML with NPM1 mutation, also concerns a new treatment scheme and a rationale for specific use in acute myeloid leukemia with nucleophosmin mutation, based on experimental data both on cellular models in the laboratory and on mice and preliminary data from human trials reported further? forward (Figures 3-6).
In particolare, la scelta della dose dell?omacetaxina ? stata fatta basandosi su quella approvata dalla FDA per la LMC (ciclo di induzione, 1.25 mg/m<2 >iniezione sottocutanea due volte al giorno per 14 giorni consecutivi per un ciclo di 28 giorni). Secondo la presente invenzione, l?omacetaxina pu? essere somministrata inizialmente (livello 1) due volte al giorno ad una concentrazione di 0.625 mg/m<2>, anche in questo caso la via sar? sottocutanea e il trattamento continuer? per 14 giorni di un ciclo di 28. Secondo la presente invenzione, alla omacetaxina viene aggiunto il venetoclax, preferibilmente alla dose di 400 mg al giorno per 21 giorni (per via orale) (Figura 4). In particular, the choice of the dose of omacetaxine? was based on that approved by the FDA for CML (induction cycle, 1.25 mg/m<2 >subcutaneous injection twice daily for 14 consecutive days for a 28-day cycle). According to the present invention, omacetaxine can be administered initially (level 1) twice a day at a concentration of 0.625 mg/m<2>, also in this case the route will be subcutaneously and the treatment will continue? for 14 days of a cycle of 28. According to the present invention, venetoclax is added to omacetaxine, preferably at a dose of 400 mg per day for 21 days (orally) (Figure 4).
Nell?uso proposto secondo la presente invenzione la somministrazione di omacetaxina per un periodo di tempo di 14 giorni ? basata sul fatto che il farmaco ha una azione inibente la sintesi proteica transitoria, non irreversibile, mentre si vuole raggiungere l?obiettivo di una inibizione sostenuta della sintesi proteica di proteine essenziali alla sopravvivenza della cellula leucemica, con particolare riferimento alla NPM1-mutated AML. In the proposed use according to the present invention, the administration of omacetaxine for a period of 14 days is based on the fact that the drug has an inhibitory action on transient, non-irreversible protein synthesis, while the aim is to achieve the objective of a sustained inhibition of the protein synthesis of proteins essential to the survival of the leukemic cell, with particular reference to NPM1-mutated AML.
Secondo la presente invenzione, la somministrazione di venetoclax per un periodo di 21 giorni in cicli di 28 giorni in associazione alla modalit? di somministrazione di omacetaxina mepesuccinato descritta sopra ? unica e ha il vantaggio di potenziare l?effetto antileucemico e al contempo, con una settimana di wash-out dai farmaci, di ridurre la tossicit? prevista dalla associazione di due farmaci con tossicit? ematologica in parte sovrapponibile. According to the present invention, the administration of venetoclax for a period of 21 days in cycles of 28 days in association with the modality of administration of omacetaxine mepesuccinate described above? unique and has the advantage of enhancing the antileukemic effect and at the same time, with a week of drug wash-out, reducing toxicity. predicted by the association of two drugs with toxicity? partially overlapping hematology.
Infine, l?impiego di HHT ? ritenuto vantaggioso, oltre che per la sua azione come "inibitore di Mcl-1" (mirando ai suoi livelli proteici), per i seguenti motivi: Finally, the use of HHT is considered advantageous, as well as for its action as a "Mcl-1 inhibitor" (targeting its protein levels), for the following reasons:
i) ? un farmaco approvato dalla FDA, nel contesto della leucemia mieloide cronica (LMC); i) ? an FDA-approved drug, in the context of chronic myelogenous leukemia (CML);
ii) ha mostrato una certa attivit? negli studi clinici sulla LAM; ii) showed some activity? in clinical trials on LAM;
iii) ha dimostrato di essere un trattamento sicuro, quando valutato anche in associazione alla chemioterapia, sia a regime standard intensivo o a basso dosaggio nella LAM; iii) has proven to be a safe treatment, when also evaluated in association with chemotherapy, either as a standard intensive or low-dose regimen in AML;
iv) viene somministrato per via sottocutanea nella sua formulazione approvata dalla FDA; iv) is administered subcutaneously in its FDA-approved formulation;
v) efficacia della combinazione secondo la presente invenzione in modelli di xenotrapianto di cellule primarie di pazienti di LAM con mutazione di NPM1 (modelli patient-derived-xenograft, PDX, Figure 1 e 2); v) efficacy of the combination according to the present invention in xenograft models of primary cells from AML patients with NPM1 mutation (patient-derived-xenograft models, PDX, Figures 1 and 2);
vi) HHT ha meccanismo di azione con potenziali effetti specifici identificati nella LAM con mutazione di NPM1 secondo la presente invenzione (ossia riduzione dei livelli di NPM1 e altri fattori essenziali, induzione della differenziazione cellulare, Figura 3). vi) HHT has mechanism of action with potential specific effects identified in AML with NPM1 mutation according to the present invention (i.e. reduction of levels of NPM1 and other essential factors, induction of cellular differentiation, Figure 3).
Forma pertanto oggetto specifico della presente invenzione una combinazione di omacetaxina mepesuccinato (o omoarringtonina) con venetoclax o con suoi sali o con suoi derivati che siano inibitori delle proteine anti-apoptotiche Bcl-2 e farmaceuticamente accettabili, come ad esempio Venetoclax-M27, per l?uso separato o sequenziale nel trattamento della leucemia acuta mieloide (LAM) con mutazione della nucleofosmina (mutazione NPM1). Therefore, the specific object of the present invention is a combination of omacetaxine mepesuccinate (or homoharringtonine) with venetoclax or with its salts or with its derivatives which are inhibitors of the anti-apoptotic proteins Bcl-2 and pharmaceutically acceptable, such as for example Venetoclax-M27, for the Separate or sequential use in the treatment of acute myeloid leukemia (AML) with nucleophosmin mutation (NPM1 mutation).
Secondo la presente invenzione, per ?uso separato?, si intende la somministrazione, nello stesso momento, dei due composti della combinazione secondo l?invenzione in forme farmaceutiche distinte. According to the present invention, "separate use" means the administration, at the same time, of the two compounds of the combination according to the invention in distinct pharmaceutical forms.
Secondo la presente invenzione, per ?uso sequenziale? si intende la somministrazione successiva dei due composti della combinazione secondo l?invenzione, ciascuno in una forma farmaceutica distinta. According to the present invention, for ?sequential use? means the subsequent administration of the two compounds of the combination according to the invention, each in a distinct pharmaceutical form.
Secondo la presente invenzione, detta leucemia acuta mieloide con mutazione della nucleofosmina pu? comprendere ulteriormente la mutazione FLT3-ITD. According to the present invention, said acute myeloid leukemia with nucleophosmin mutation can further understand the FLT3-ITD mutation.
Inoltre la combinazione secondo la presente invenzione, per l?uso come definito sopra, pu? essere impiegata in pazienti di qualunque et? non candidabili a chemioterapie intensive, sia pretrattati con altre terapie che non, in pazienti di et? ? 75 anni, sia pretrattati con altre terapie che non, in pazienti di qualunque et? con persistenza di malattia, anche minima, candidabili a trapianto di cellule staminali emopoietiche. Furthermore, the combination according to the present invention, for use as defined above, can be used in patients of any age? not candidates for intensive chemotherapy, whether pre-treated with other therapies or not, in patients aged ? 75 years, whether pre-treated with other therapies or not, in patients of any age? with persistence of disease, even minimal, eligible for hematopoietic stem cell transplant.
Secondo la presente invenzione l?omacetaxina mepesuccinato pu? essere somministrata per via sottocutanea o per via orale, e venetoclax pu? essere somministrato per via orale. According to the present invention, omacetaxine mepesuccinate can be administered subcutaneously or orally, and venetoclax can? be administered orally.
Inoltre, secondo la presente invenzione, detta combinazione pu? essere somministrata in un ciclo di trattamento della durata complessiva da 21 a 45 giorni, preferibilmente 28 giorni, in cui Furthermore, according to the present invention, said combination can? be administered in a treatment cycle lasting a total of 21 to 45 days, preferably 28 days, in which
- omacetaxina mepesuccinato pu? essere somministrata, in un primo ciclo di trattamento e nei cicli di trattamento successivi, a un dosaggio da 0,5 a 2 mg/m<2 >due volte al giorno dal giorno 1 al giorno 14 oppure dal giorno 1 al giorno 7, preferibilmente al dosaggio di 0,625 mg/m<2 >oppure al dosaggio di 1,25 mg/m<2 >dal giorno 1 al giorno 14 oppure dal giorno 1 al giorno 7; - omacetaxine mepesuccinate can? be administered, in a first treatment cycle and in subsequent treatment cycles, at a dosage of 0.5 to 2 mg/m<2 > twice daily from day 1 to day 14 or from day 1 to day 7, preferably at a dosage of 0.625 mg/m<2 >or at a dosage of 1.25 mg/m<2 >from day 1 to day 14 or from day 1 to day 7;
- venetoclax, o suoi sali o suoi derivati, pu? essere somministrato in un primo ciclo di trattamento a un dosaggio da 50 a 800 mg dal giorno 2 ad almeno il giorno 21, in cui il dosaggio viene aumentato da un dosaggio iniziale a un dosaggio finale, preferibilmente essendo somministrato a un dosaggio di 50 mg al giorno 2, 100 mg al giorno 3, 200 mg al giorno 4, 400 mg dal giorno 5 al giorno 21; - venetoclax, or its salts or derivatives, can? be administered in a first treatment cycle at a dosage of 50 to 800 mg from day 2 to at least day 21, where the dosage is increased from an initial dosage to a final dosage, preferably being administered at a dosage of 50 mg per day 2, 100 mg on day 3, 200 mg on day 4, 400 mg from day 5 to day 21;
- venetoclax, o suoi sali o suoi derivati, pu? essere somministrato nei cicli di trattamento successivi al primo a un dosaggio da 50 a 800 mg dal giorno 1 al giorno 14, oppure dal giorno 1 al giorno 35, oppure preferibilmente dal giorno 1 al giorno 21, preferibilmente ? somministrato al dosaggio di 400 mg dal giorno 1 al giorno 21; e in cui - venetoclax, or its salts or derivatives, can? be administered in the treatment cycles following the first at a dosage of 50 to 800 mg from day 1 to day 14, or from day 1 to day 35, or preferably from day 1 to day 21, preferably ? administered at a dosage of 400 mg from day 1 to day 21; and in which
- omacetaxina mepesuccinato, venetoclax o suoi sali o suoi derivati, possono non essere somministrati per un periodo di tempo da 7 a 10 giorni (?offtherapy?) prima di iniziare un eventuale ciclo successivo di trattamento, preferibilmente n? omacetaxina mepesuccinato n? venetoclax sono somministrati dal giorno 22 al giorno 28 oppure dal giorno 36 al giorno 45. - omacetaxine mepesuccinate, venetoclax or its salts or derivatives, may not be administered for a period of time from 7 to 10 days (?offtherapy?) before starting a possible subsequent cycle of treatment, preferably n? omacetaxine mepesuccinate n? venetoclax are administered from day 22 to day 28 or from day 36 to day 45.
Secondo una forma di realizzazione preferita della presente invenzione, in un ciclo di trattamento con una durata complessiva di 28 giorni According to a preferred embodiment of the present invention, in a treatment cycle with a total duration of 28 days
- omacetaxina mepesuccinato ? somministrata al dosaggio di 0,625 mg/m<2 >oppure al dosaggio di 1,25 mg/m<2 >dal giorno 1 al giorno 14 oppure dal giorno 1 al giorno 7; - omacetaxine mepesuccinate? administered at a dosage of 0.625 mg/m<2 >or at a dosage of 1.25 mg/m<2 >from day 1 to day 14 or from day 1 to day 7;
- venetoclax ? somministrato al dosaggio di 50 mg al giorno 2, 100 mg al giorno 3, 200 mg al giorno 4, 400 mg dal giorno 5 al giorno 21 quando detto ciclo di trattamento ? un primo ciclo di trattamento oppure al dosaggio di 400 mg dal giorno 1 al giorno 21 quando detto ciclo di trattamento ? un ciclo successivo al primo ciclo di trattamento; - venetoclax ? administered at a dosage of 50 mg on day 2, 100 mg on day 3, 200 mg on day 4, 400 mg from day 5 to day 21 when said treatment cycle is? a first treatment cycle or at a dosage of 400 mg from day 1 to day 21 when said treatment cycle is? a cycle following the first treatment cycle;
- dal giorno 22 al giorno 28 non viene somministrato n? omacetaxina mepesuccinato n? venetoclax o suoi sali o suoi derivati. - from day 22 to day 28 no? omacetaxine mepesuccinate n? venetoclax or its salts or derivatives.
La presente invenzione concerne inoltre una composizione farmaceutica comprendente omacetaxina mepesuccinato e venetoclax o suoi sali o suoi derivati che siano inibitori delle proteine anti-apoptotiche Bcl-2 e farmaceuticamente accettabili, come ad esempio Venetoclax-M27, assieme a eccipienti e/o adiuvanti farmaceuticamente accettabili, per l?uso nel trattamento della leucemia acuta mieloide (LAM) con mutazione della nucleofosmina (mutazione NPM1). The present invention also concerns a pharmaceutical composition comprising omacetaxine mepesuccinate and venetoclax or its salts or its derivatives which are inhibitors of the anti-apoptotic proteins Bcl-2 and pharmaceutically acceptable, such as for example Venetoclax-M27, together with pharmaceutically acceptable excipients and/or adjuvants , for use in the treatment of acute myeloid leukemia (AML) with nucleophosmin mutation (NPM1 mutation).
Secondo la presente invenzione, detta leucemia acuta mieloide con mutazione della nucleofosmina pu? comprendere ulteriormente la mutazione FLT3-ITD. According to the present invention, said acute myeloid leukemia with nucleophosmin mutation can further understand the FLT3-ITD mutation.
Inoltre, la composizione farmaceutica secondo la presente invenzione, per l?uso come definito sopra, pu? essere impiegata in pazienti di qualunque et? non candidabili a chemioterapie intensive, sia pretrattati con altre terapie che non, in pazienti di et? ? 75 anni, sia pretrattati con altre terapie che non, in pazienti di qualunque et? con persistenza di malattia, anche minima, candidabili a trapianto di cellule staminali emopoietiche. Furthermore, the pharmaceutical composition according to the present invention, for use as defined above, can be used in patients of any age? not candidates for intensive chemotherapy, whether pre-treated with other therapies or not, in patients aged ? 75 years, whether pre-treated with other therapies or not, in patients of any age? with persistence of disease, even minimal, eligible for hematopoietic stem cell transplant.
Secondo la presente invenzione, detta composizione farmaceutica pu? essere in una forma per la somministrazione orale. According to the present invention, said pharmaceutical composition can be in a form for oral administration.
La presente invenzione verr? ora descritta, a titolo illustrativo, ma non limitativo, secondo una sua forma preferita di realizzazione, con particolare riferimento agli esempi e alle figure dei disegni allegati, in cui: Will this invention come? now described, by way of illustration, but not by way of limitation, according to one of its preferred embodiments, with particular reference to the examples and figures of the attached drawings, in which:
- la Figura 1 mostra risultati esemplificativi di un esperimento in vivo su modello PDX valutato mediate BLI. Cellule primarie di paziente di LAM NPM1-mutata ripopolate nel topo immunocompromesso (PDX2) esprimenti il gene della luciferasi sono state inoculate in un gruppo di topi e dopo 6 giorni ? stato valutato mediante bioluminescenza in vivo (BLI) l?attecchimento di malattia. I topi sono stati divisi in 4 coorti di trattamento (veicolo, ABT, HHT e HHT/ABT) e monitorati nel tempo. Nella immagine in bianco e nero, in corrispondenza della immagine del topo, il segnale nero indica presenza di elevata quantit? di malattia e quello bianco indica assenza di malattia. Come si pu? osservare, il gruppo HHT/ABT non ha malattia rilevabile a 17 giorni (D17) dal trapianto, dopo 2 settimane di trattamento, e ancora minima risulta al D31 (alla fine del I ciclo di 4 settimane di trattamento). Un marcato incremento del segnale (e quindi di malattia) si osserva invece negli altri gruppi. In particolare, al D31, 3 dei 5 topi del gruppo non trattato (veicolo) erano gi? deceduti per malattia. Veicolo: veicolo in assenza di farmaco; ABT: ABT-199 (venetoclax); HHT: omoarringtonina. - Figure 1 shows exemplary results of an in vivo experiment on a PDX model evaluated using BLI. Primary NPM1-mutated AML patient cells repopulated in the immunocompromised mouse (PDX2) expressing the luciferase gene were inoculated into a group of mice and after 6 days ? The establishment of the disease was evaluated using in vivo bioluminescence (BLI). Mice were divided into 4 treatment cohorts (vehicle, ABT, HHT, and HHT/ABT) and monitored over time. In the black and white image, corresponding to the image of the mouse, the black signal indicates the presence of a high quantity? of disease and the white one indicates absence of disease. How can you? observe, the HHT/ABT group has no detectable disease at 17 days (D17) after transplantation, after 2 weeks of treatment, and is still minimal at D31 (at the end of the first cycle of 4 weeks of treatment). A marked increase in the signal (and therefore in disease) is instead observed in the other groups. In particular, at D31, 3 of the 5 mice in the untreated group (vehicle) were already died due to illness. Vehicle: vehicle in the absence of drug; ABT: ABT-199 (venetoclax); HHT: homoharringtonine.
- la Figura 2 mostra risultati di esperimenti in vivo su modelli PDX in termini di valutazione di infiltrazione di malattia e sopravvivenza dei topi. A) Esperimento su modello PDX2 (NPM1/DNMT3A/FLT3-ITD triplo mutato). B) Esperimento su modello PDX3 (NPM1/DNMT3A/FLT3-ITD triplo mutato). A-B) Infiltrazione di malattia dopo il I ciclo (sinistra): una coorte di topi ? stata analizzata al termine del I ciclo di trattamento (d28) mediante valutazione in citofluorimetria della percentuale di infiltrazione midollare da parte di cellule leucemiche umane CD45-positive (grafico a sinistra). Curve di sopravvivenza (destra): un?altra coorte di topi ? stata assegnata al trattamento completo previsto di 2 2 cicli, e analizzata per sopravvivenza. - Figure 2 shows results of in vivo experiments on PDX models in terms of evaluating disease infiltration and survival of mice. A) Experiment on PDX2 model (NPM1/DNMT3A/FLT3-ITD triple mutated). B) Experiment on PDX3 model (NPM1/DNMT3A/FLT3-ITD triple mutated). A-B) Disease infiltration after the first cycle (left): a cohort of mice? was analyzed at the end of the first treatment cycle (d28) by flow cytometric evaluation of the percentage of bone marrow infiltration by CD45-positive human leukemic cells (graph on the left). Survival curves (right): another cohort of mice? was assigned to the planned full treatment of 2 2 cycles, and analyzed for survival.
- la Figura 3 mostra la teoria alla base della azione specifica di omacetaxina (HHT) venetoclax (Ven) nella LAM con mutazione di NPM1 (NPM1m) e dati sperimentali in vitro. A) Meccanismo di azione congiunta di HHT e Ven nella LAM con mutazione di NPM1. La combinazione colpisce elementi chiave per la sopravvivenza e proliferazione della cellula leucemica NPM1-mutata. In particolare, FLT3 ? un gene frequentemente mutato (FLT3-ITD) in associazione a NPM1 aumentando la crescita della leucemia e peggiorandone notevolmente la prognosi. Crm-1 (exportin 1/XPO1) ? una proteina carrier la cui presenza e funzione ? essenziale per mantenere la proteina leucemica NPM1 mutata nel citoplasma. La loro interazione ? dimostrata essere alla base del mantenimento delle caratteristiche della leucemia NPM1-mutata e la loro inibizione comporta differenziazione cellulare e arresto della crescita. B) Analisi in western blot con anticorpi specifici diretti contro la proteina NPM1 mutata (NPM1 mutante), la proteina NPM1 originaria, la proteina carrier esportina 1 (Crm1) e la proteina antiapoptotica Mcl-1. La proteina histone H3, ? usata come parametro di caricamento proteico omogeneo tra i diversi campioni analizzati. I campioni corrispondono a lisati proteici della linea cellulare di leucemia mieloide acuta IMS-M2, recante la mutazione di NPM1, non trattati (-) o trattati con diverse dosi di HHT (3.5 e 7 nM) per 2 (d2) o 6 (d6) giorni. Da questa analisi si osserva che in particolar modo i livelli della proteina leucemica NPM1, cos? come quelli di Crm1 e Mcl-1, appaiono ridotti dalla inibizione sostenuta della sintesi proteica da parte di HHT, con effetto dose- e tempo-dipendente. C) Cellule IMS-M2 sono state esposte a diverse dosi di HHT per 6 giorni con rinnovo del terreno di coltura e del farmaco a 72 ore (72h 72h) e analizzate mediante citometria a flusso per l?espressione dei marker di superficie CD14 e CD11b (anti-CD14-FITC; anti-CD11b-PE). I valori di fluorescenza mediana (MF) sono indicati. Con il trattamento si osserva un progressivo incremento dei livelli di MF per i marker CD14 e CD11b, segno di differenziazione cellulare in senso mielomonocitario. - Figure 3 shows the theory underlying the specific action of omacetaxine (HHT) venetoclax (Ven) in AML with NPM1 mutation (NPM1m) and in vitro experimental data. A) Mechanism of joint action of HHT and Ven in AML with NPM1 mutation. The combination affects key elements for the survival and proliferation of the NPM1-mutated leukemia cell. In particular, FLT3 ? a frequently mutated gene (FLT3-ITD) in association with NPM1 increasing the growth of leukemia and significantly worsening its prognosis. Crm-1 (exportin 1/XPO1) ? a carrier protein whose presence and function? essential for maintaining the mutated leukemic NPM1 protein in the cytoplasm. Their interaction? demonstrated to be at the basis of the maintenance of the characteristics of NPM1-mutated leukemia and their inhibition leads to cellular differentiation and growth arrest. B) Western blot analysis with specific antibodies directed against the mutated NPM1 protein (mutant NPM1), the original NPM1 protein, the carrier protein exportin 1 (Crm1) and the antiapoptotic protein Mcl-1. The histone H3 protein, ? used as a homogeneous protein loading parameter between the different samples analyzed. The samples correspond to protein lysates of the acute myeloid leukemia cell line IMS-M2, bearing the NPM1 mutation, untreated (-) or treated with different doses of HHT (3.5 and 7 nM) for 2 (d2) or 6 (d6 ) days. From this analysis it is observed that in particular the levels of the leukemic protein NPM1, so like those of Crm1 and Mcl-1, appear reduced by the sustained inhibition of protein synthesis by HHT, with a dose- and time-dependent effect. C) IMS-M2 cells were exposed to different doses of HHT for 6 days with renewal of the culture medium and drug at 72 hours (72h 72h) and analyzed by flow cytometry for the expression of the surface markers CD14 and CD11b (anti-CD14-FITC; anti-CD11b-PE). Median fluorescence (MF) values are indicated. With treatment, a progressive increase in MF levels for the CD14 and CD11b markers is observed, a sign of cellular differentiation in the myelomonocytic sense.
- la Figura 4 mostra lo schema di trattamento del protocollo di sperimentazione nell?uomo, denominato SynVen-AML. Le dosi e la via di somministrazione di omacetaxina mepesuccinato (Synribo) sono state scelte in base al suo impiego e all'approvazione della FDA nel contesto della LMC, e sulla base del meccanismo previsto sui livelli della proteina NPM1 leucemica. Poich? ? probabile che sia necessaria un'esposizione prolungata al farmaco per ottenere un'azione antileucemica clinicamente rilevante senza l'aiuto di un farmaco citotossico concomitante, abbiamo scelto di esplorare per la sicurezza e l'efficacia preliminare un programma di 14 giorni consecutivi (14 giorni) di trattamento prima con Synribo e poi, in caso di non tollerabilit?, per almeno 7 giorni (7 giorni). - Figure 4 shows the treatment scheme of the human trial protocol, called SynVen-AML. The doses and route of administration of omacetaxine mepesuccinate (Synribo) were chosen based on its use and FDA approval in the setting of CML, and based on the predicted mechanism of leukemic NPM1 protein levels. Since? ? It is likely that prolonged drug exposure is required to achieve clinically relevant antileukemic action without the aid of a concomitant cytotoxic drug, we chose to explore a 14 consecutive day (14-day) schedule for safety and preliminary efficacy. of treatment first with Synribo and then, in case of intolerability, for at least 7 days (7 days).
L'aggiunta di venetoclax, data la sua azione proapoptotica come inibitore di bcl-2, alla dose di 400 mg ha il razionale scientifico oltre che sperimentale, come spiegato sopra. Per il primo ciclo ? prevista una fase di progressivo incremento della dose (ramp-up) con venetoclax a dosi crescenti per prevenire la sindrome da lisi tumorale, che potrebbe verificarsi in presenza di elevata massa di malattia quale espressione della attivit? sinergica dei due farmaci. The addition of venetoclax, given its proapoptotic action as a bcl-2 inhibitor, at a dose of 400 mg has a scientific as well as experimental rationale, as explained above. For the first cycle? a phase of progressive dose increase (ramp-up) is foreseen with venetoclax at increasing doses to prevent tumor lysis syndrome, which could occur in the presence of a high mass of disease as an expression of the activity synergy of the two drugs.
- la Figura 5 mostra le immagini dell?aspirato midollare pre (basale) e post (d28) I ciclo di terapia nel paziente UPN01 (protocollo di fase 1 SynVen-AML, EudraCT n. 2019-001821-29. Mentre nella condizione basale si apprezza una infiltrazione diffusa del midollo osseo da parte di blasti leucemici che appaiono come una popolazione cellulare di media taglia ed omogenea (75-80%), alla valutazione post-I ciclo (d28) si osserva una marcata riduzione dei blasti (6-10%) con cellularit? midollare normale e una ripresa emopoietica trilineare, che appare come una eterogeneit? della popolazione cellulare (con un elemento di grandi dimensioni al centro, corrispondente ad un megacariocita, e cellule pi? piccole con nucleo segmentato o a banda corrispondenti a cellule normali della linea granulocitaria). Il quadro midollare depone per Risposta Parziale (RP) di malattia. La valutazione della frequenza dell?allele mutato (VAF=variant allelic frequency), sempre in eterozigosi (vale a dire solo 1 dei 2 presenti in ciascuna cellula), conferma la percentuale di cellule leucemiche con mutazione di NPM1 infiltranti il midollo e la loro marcata riduzione dopo trattamento. - Figure 5 shows the images of the bone marrow aspirate before (basal) and after (d28) the first cycle of therapy in patient UPN01 (SynVen-AML phase 1 protocol, EudraCT n. 2019-001821-29. While in the basal condition appreciates a widespread infiltration of the bone marrow by leukemic blasts which appear as a medium-sized and homogeneous cell population (75-80%), at the post-I cycle evaluation (d28) a marked reduction of the blasts is observed (6-10 %) with normal medullary cellularity and a trilinear hematopoietic recovery, which appears as a heterogeneity of the cell population (with a large element in the center, corresponding to a megakaryocyte, and smaller cells with a segmented or banded nucleus corresponding to normal granulocytic lineage). ), confirms the percentage of leukemic cells with NPM1 mutation infiltrating the marrow and their marked reduction after treatment.
- la Figura 6 mostra evidenza morfologica di differenziazione cellulare delle cellule leucemiche nel Paziente UPN01 durante il trattamento. Durante il trattamento con omacetaxina mepesuccinato e venetoclax (d14) all?aspirato midollare si ? osservata una marcata differenziazione dei blasti in senso monocitario, con cellule dai nuclei ripiegati e bizzarri, a forma di quadrifoglio a tratti. Contemporaneamente, compariva una marcata ipertrofia gengivale, con tutta probabilit? attribuibile alla infiltrazione da parte dei blasti monocitoidi, come ? noto accadere in tali casi. Con il proseguire del trattamento (d28, fine I ciclo), l?ipertrofia gengivale si ? progressivamente attenuata. - Figure 6 shows morphological evidence of cellular differentiation of leukemic cells in Patient UPN01 during treatment. During treatment with omacetaxine mepesuccinate and venetoclax (d14) the bone marrow aspirate was defective. a marked differentiation of the blasts in a monocytic direction was observed, with cells with folded and bizarre nuclei, sometimes in the shape of a four-leaf clover. At the same time, a marked gingival hypertrophy appeared, most likely? attributable to infiltration by monocytoid blasts, how? known to happen in such cases. As the treatment continues (d28, end of the first cycle), the gingival hypertrophy decreases. progressively attenuated.
ESEMPIO 1. Studio in vitro degli effetti di omacetaxina mepesuccinato sui livelli di proteine essenziali nella LAM con mutazione di NPM1 e sulla differenziazione cellulare. EXAMPLE 1. In vitro study of the effects of omacetaxine mepesuccinate on the levels of essential proteins in AML with NPM1 mutation and on cell differentiation.
Materiali e Metodi Materials and methods
Farmaci: Per gli esperimenti preclinici in vitro abbiamo impiegato omoarringtonina (HHT), acquistato da Selleckchem (Houston, TX USA, Cat #S9015). ABT-199 (venetoclax) ? stato acquistato da Selleckchem (Houston, TX USA, Cat # S8048). Drugs: For preclinical in vitro experiments we used homoharringtonine (HHT), purchased from Selleckchem (Houston, TX USA, Cat #S9015). ABT-199 (venetoclax) ? was purchased from Selleckchem (Houston, TX USA, Cat # S8048).
Cellule: La linea cellulare IMS-M2 esprimente la mutazione di NPM1 ? stata gentilmente fornita dal prof. Daniel G. Tenen (from: Cancer Science Institute of Singapore, National University of Singapore, Singapore) e precedentemente descritta (7-9). Cells: The IMS-M2 cell line expressing the NPM1 mutation? was kindly provided by the prof. Daniel G. Tenen (from: Cancer Science Institute of Singapore, National University of Singapore, Singapore) and previously described (7-9).
Analisi in Western blot: In breve, le cellule sono state lisate in tampone Laemmli (1,5M Tris-HCL Ph 6.8, glicerolo, ?-mercaptoetanolo, SDS, blu di bromofenolo) e le proteine sono state separate su gel SDS/PAGE Precast 4-15% gradiente (Biorad, Hercules, CA, USA) e trasferite su membrane di nitrocellulosa (GE Healthcare, Piscataway, NJ, USA). Dopo l'incubazione con gli anticorpi primari indicati, le membrane sono state sviluppate mediante chemiluminescenza potenziata (ECL, GE Healthcare, Piscataway, NJ, USA) secondo le istruzioni del produttore e visualizzate utilizzando il sistema Hyperprocessor (GE Healthcare, Piscataway, NJ, USA). L'istone H3 ? stato utilizzato come controllo di caricamento. Western blot analysis: Briefly, cells were lysed in Laemmli buffer (1.5M Tris-HCL Ph 6.8, glycerol, ?-mercaptoethanol, SDS, bromophenol blue) and proteins were separated on SDS/PAGE Precast gels 4-15% gradient (Biorad, Hercules, CA, USA) and transferred onto nitrocellulose membranes (GE Healthcare, Piscataway, NJ, USA). After incubation with the indicated primary antibodies, membranes were developed by enhanced chemiluminescence (ECL, GE Healthcare, Piscataway, NJ, USA) according to the manufacturer's instructions and visualized using the Hyperprocessor system (GE Healthcare, Piscataway, NJ, USA ). Histone H3? was used as a loading control.
I seguenti anticorpi sono stati usati come indicato: anticorpo policlonale di coniglio mutante anti-NPM1 (prodotto da B.F.); anticorpo monoclonale di topo anti-NPM1 wild-type (diretto contro il C-terminale di NPM1 wild-type, #32-5200, Invitrogen); anticorpo anti-Crm-1 (Exportin-1/CRM1 (D6V7N) Rabbit mAb #46249, Cell Signaling Danvers, Massachusetts, USA); anticorpo anti-Mcl-1 (Mcl-1 (D5V5L) Rabbit mAb #39224, Cell Signaling); anticorpo anti-H3 (Histone H3 (3H1) Rabbit mAb #9717, Cell Signaling). Dopo l'incubazione con l'anticorpo secondario appropriato, il segnale ? stato rivelato da una chemioluminescenza potenziata (substrato Immobilon Crescendo Western HRP, Millipore). The following antibodies were used as indicated: anti-NPM1 mutant rabbit polyclonal antibody (produced by B.F.); mouse monoclonal anti-wild-type NPM1 antibody (directed against the C-terminus of wild-type NPM1, #32-5200, Invitrogen); anti-Crm-1 antibody (Exportin-1/CRM1 (D6V7N) Rabbit mAb #46249, Cell Signaling Danvers, Massachusetts, USA); anti-Mcl-1 antibody (Mcl-1 (D5V5L) Rabbit mAb #39224, Cell Signaling); anti-H3 antibody (Histone H3 (3H1) Rabbit mAb #9717, Cell Signaling). After incubation with the appropriate secondary antibody, the signal is was revealed by enhanced chemiluminescence (Immobilon Crescendo Western HRP substrate, Millipore).
Risultati Results
Risultati esemplificativi sono illustrati nella Figura 3, B e C. L?analisi in western blot su estratti proteici dalla linea cellulare di LAM umana IMS-M2, recante la mutazione di NPM1, con anticorpi specifici diretti contro la proteina NPM1 mutata, la proteina Crm1 e la proteina anti-apoptotica Mcl-1, mostra che i livelli della proteina leucemica NPM1, cos? come quelli di Crm1 e Mcl-1, appaiono ridotti dalla inibizione sostenuta della sintesi proteica da parte di HHT, con effetto dose- e tempo-dipendente (Figura 3, B). La proteina histone H3, come controllo, indica un caricamento proteico omogeneo tra i diversi campioni analizzati. Inoltre, le cellule IMS-M2 quando esposte a diverse dosi di HHT per 6 giorni mostrano un progressivo incremento dell?espressione degli antigeni di superficie CD14 e CD11b, segno di differenziazione cellulare in senso mielomonocitario (Figura 3, C). Exemplary results are shown in Figure 3, B and C. Western blot analysis of protein extracts from the human AML cell line IMS-M2, carrying the NPM1 mutation, with specific antibodies directed against the mutated NPM1 protein, the Crm1 protein and the anti-apoptotic protein Mcl-1, shows that the levels of the leukemic protein NPM1, so? like those of Crm1 and Mcl-1, appear reduced by the sustained inhibition of protein synthesis by HHT, with a dose- and time-dependent effect (Figure 3, B). The histone H3 protein, as a control, indicates a homogeneous protein loading between the different samples analyzed. Furthermore, IMS-M2 cells when exposed to different doses of HHT for 6 days show a progressive increase in the expression of the surface antigens CD14 and CD11b, a sign of cellular differentiation in the myelomonocytic sense (Figure 3, C).
ESEMPIO 2. Studio in vivo dell?efficacia della combinazione di omacetaxina mepesuccinato e venetoclax su topi. EXAMPLE 2. In vivo study of the efficacy of the combination of omacetaxine mepesuccinate and venetoclax on mice.
Materiali e Metodi Materials and methods
In relazione al prelievo e all?utilizzazione di materiale biologico di origine umana, si dichiara che ? stato ottenuto l?espresso consenso, libero e informato, a tale prelievo e utilizzazione, del soggetto a cui ? stato prelevato tale materiale, in base alla normativa vigente. In relation to the collection and use of biological material of human origin, it is declared that? The express, free and informed consent to such sampling and use has been obtained from the subject to whom it is provided. such material was collected, in accordance with current legislation.
Farmaci: Per gli esperimenti preclinici in vivo nel modello murino abbiamo impiegato omoarringtonina (HHT), acquistato da Selleckchem (Houston, TX USA, Cat #S9015). ABT-199 (venetoclax) ? stato acquistato da Selleckchem (Houston, TX USA, Cat # S8048). Per l?uso in vivo nel topo, l'ABT-199 ? stato sciolto settimanalmente in 100% DMSO a 10 mg/ml ed ? stato preparato giornalmente con 15% etanolo, 60% kolliphor, e l'HHT ? stato sciolto settimanalmente in 100% DMSO a 50 mg/ml ed ? stato preparato giornalmente in NaCl0,9%. Drugs: For preclinical in vivo experiments in the mouse model we used homoharringtonine (HHT), purchased from Selleckchem (Houston, TX USA, Cat #S9015). ABT-199 (venetoclax) ? was purchased from Selleckchem (Houston, TX USA, Cat # S8048). For in vivo use in mice, ABT-199 is been dissolved weekly in 100% DMSO at 10 mg/ml and ? was prepared daily with 15% ethanol, 60% kolliphor, and the HHT ? been dissolved weekly in 100% DMSO at 50 mg/ml and ? was prepared daily in 0.9% NaCl.
Cellule: Le cellule di leucemia umana impiegate per i modelli di xenotrapianto murini (patient-derived xenograft, PDX model) sono state ottenute da pazienti con diagnosi di LAM con mutazione di NPM1. Cells: The human leukemia cells used for the mouse xenograft models (PDX model) were obtained from patients diagnosed with NPM1 mutated AML.
Sperimentazione animale (su topi): Tutti gli studi sugli animali sono stati eseguiti in conformit? con i protocolli approvati dal Ministero della salute e dall'OPBA (Comitato locale per la cura e l'uso degli animali). I topi NOD.Cg-PrkdcscidIl2rgtm1Wj1/SzJ, NOD scid gamma (NSG) sono stati originariamente acquistati da Charles River Laboratories (Europe) e poi riprodotti e mantenuti presso il Centro di Servizi e Ricerca Preclinica dell?Universit? di Perugia, Perugia, Italia, in conformit? con le linee guida nazionali/europee. Animal testing (on mice): All animal studies were performed in accordance with with the protocols approved by the Ministry of Health and the OPBA (Local Committee for the Care and Use of Animals). NOD.Cg-PrkdcscidIl2rgtm1Wj1/SzJ, NOD scid gamma (NSG) mice were originally purchased from Charles River Laboratories (Europe) and then bred and maintained at the University's Preclinical Research and Services Center. of Perugia, Perugia, Italy, in accordance? with national/European guidelines.
Topi di otto/dieci settimane sono stati iniettati per via endovenosa (vena della coda) con 1 x 10<6 >di cellule primarie di pazienti (cellule di leucemia mieloide acuta NPM1, DNMT3A e FLT3-ITD mutate) stabilmente trasdotte con il vettore pHIV-Luc-ZsGreen (Addgene, plasmide #39196) che co-esprime la luciferasi di lucciola (Luc2P) e la proteina ZsGreen sotto il promotore EF-1alfa umano (PDX2 e PDX3). Dopo il trapianto, i topi sono stati mantenuti in gabbie dedicate e alimentati con cibo sterile e acqua acidificata, contenente 100 ?g/mL di ciprofloxacina, come antibiotico. L'impiego di cellule che esprimono luciferasi consente di monitorare la malattia mediante imaging di bioluminescenza in vivo (BLI) durante il trattamento farmacologico e valutare la risposta al farmaco. Eight to ten week old mice were injected intravenously (tail vein) with 1 x 10<6> of primary patient cells (NPM1, DNMT3A and FLT3-ITD mutated acute myeloid leukemia cells) stably transduced with the pHIV vector -Luc-ZsGreen (Addgene, plasmid #39196) co-expressing firefly luciferase (Luc2P) and ZsGreen protein under the human EF-1alpha promoter (PDX2 and PDX3). After transplantation, mice were maintained in dedicated cages and fed sterile food and acidified water, containing 100 ?g/mL ciprofloxacin, as an antibiotic. The use of luciferase-expressing cells allows disease monitoring by in vivo bioluminescence imaging (BLI) during drug treatment and to evaluate drug response.
Dopo 6-10 giorni dall?inoculo (stato di malattia conclamata), l?attecchimento veniva confermato e valutato mediante BLI e i topi divisi omogeneamente in quattro gruppi (almeno n = 5 per gruppo) e trattati con veicolo, ABT-199 (venetoclax) (100 mg/kg) per os (5 giorni/settimana per 4 settimane), HHT (1 mg/kg) per via sottocutanea (s.c.) (5 giorni/settimana per 2 settimane, induzione, o 1 settimana, consolidamento) o con la combinazione. I segni clinici, le variazioni del peso corporeo e la crescita del tumore sono stati misurati 2 volte a settimana fino alla conclusione dello studio. Il carico leucemico delle cellule PDX ? stato monitorato mediante imaging di bioluminescenza in pi? punti temporali. In breve, i topi sono stati iniettati per via sottocutanea con luciferina (XenoLight D-Luciferin Potassium Salt, PerkinElmer) e tenuti anestetizzati con isoflurano prima dell'imaging in vivo con IVIS Lumina III In Vivo Imaging System. La quantificazione e l'elaborazione delle immagini sono state eseguite utilizzando il Living Imaging Software (PerkinElmer). After 6-10 days from inoculation (full-blown disease state), engraftment was confirmed and evaluated by BLI and the mice divided evenly into four groups (at least n = 5 per group) and treated with vehicle, ABT-199 (venetoclax) (100 mg/kg) orally (5 days/week for 4 weeks), HHT (1 mg/kg) subcutaneously (s.c.) (5 days/week for 2 weeks, induction, or 1 week, consolidation) or with the combination. Clinical signs, changes in body weight, and tumor growth were measured 2 times per week until the conclusion of the study. The leukemic burden of PDX cells? was monitored using bioluminescence imaging in addition? time points. Briefly, mice were injected subcutaneously with luciferin (XenoLight D-Luciferin Potassium Salt, PerkinElmer) and kept anesthetized with isoflurane before in vivo imaging with the IVIS Lumina III In Vivo Imaging System. Quantification and image processing were performed using Living Imaging Software (PerkinElmer).
Dopo il trattamento per il tempo indicato, i topi sono stati soppressi per dislocazione cervicale, per analisi della infiltrazione di leucemia del midollo osseo (bone marrow, BM), mediante impiego di anti-human CD45 con marcatore di fluorescenza e lettura in citometria a flusso (BD Biosciences), oppure sono stati monitorati per la sopravvivenza. After treatment for the indicated time, the mice were killed by cervical dislocation, for analysis of leukemia infiltration of the bone marrow (BM), using anti-human CD45 with fluorescence marker and flow cytometry reading (BD Biosciences), or were monitored for survival.
Risultati Results
Risultati esemplificativi sono illustrati nelle Figure 1 e 2. Nel modello animale, la combinazione ha dimostrato un marcato effetto anti-leucemico con arresto della crescita della leucemia alla fine del primo ciclo di trattamento (d28), valutata sia mediante BLI (Figura 1) che mediante citofluorimetria a flusso nel midollo estratto dalle ossa di un gruppo di topi sacrificati (Figura 2). In particolare, la malattia risultava assente, o minimamente rilevabile, nei topi trattati con la combinazione rispetto a quelli trattati con i singoli farmaci. Questo effetto antileucemico si ? tradotto in un significativo aumento della sopravvivenza dei topi trattati con la combinazione rispetto quelli trattati con i singoli farmaci (Figura 2). Inoltre, la combinazione si ? mostrata relativamente ben tollerata dagli animali. Exemplary results are illustrated in Figures 1 and 2. In the animal model, the combination demonstrated a marked anti-leukemic effect with arrest of leukemia growth at the end of the first treatment cycle (d28), assessed both by BLI (Figure 1) and by flow cytometry in the marrow extracted from the bones of a group of sacrificed mice (Figure 2). In particular, the disease was absent, or minimally detectable, in mice treated with the combination compared to those treated with the individual drugs. Is this antileukemic effect? translated into a significant increase in survival of mice treated with the combination compared to those treated with the individual drugs (Figure 2). Furthermore, the combination yes ? shown to be relatively well tolerated by animals.
ESEMPIO 3. Case Report (paziente UPN01) da studio clinico di fase I volto a valutare la sicurezza e l'efficacia preliminare della combinazione di omacetaxina mepesuccinato e venetoclax in pazienti con LAM con mutazione di NPM1 recidivante/refrattaria. EXAMPLE 3. Case Report (patient UPN01) from a phase I clinical trial aimed at evaluating the safety and preliminary efficacy of the combination of omacetaxine mepesuccinate and venetoclax in patients with AML with relapsed/refractory NPM1 mutation.
In relazione al prelievo e all?utilizzazione di materiale biologico di origine umana, si dichiara che ? stato ottenuto l?espresso consenso, libero e informato, a tale prelievo e utilizzazione, del soggetto a cui ? stato prelevato tale materiale, in base alla normativa vigente. In relation to the collection and use of biological material of human origin, it is declared that? The express, free and informed consent to such sampling and use has been obtained from the subject to whom it is provided. such material was collected, in accordance with current legislation.
Un uomo di 69 anni, affetto da LAM NPM1-mutata in sesta recidiva di malattia dopo cinque cicli di trattamento, ? stato ricoverato per essere sottoposto al primo ciclo di chemioterapia di induzione secondo il protocollo sperimentale di fase 1 ?SynVen-AML (Figura 4). L?emocromo all?ingresso mostrava: GB 10,070/mmc, Hb 7.8 g/dL, PLT 39,000/mmc. Alla valutazione molecolare: infiltrazione marcata (75-80%) da parte di cellule leucemiche NPM1 mutate (Figura 5). A 69-year-old man, suffering from NPM1-mutated AML in the sixth disease relapse after five cycles of treatment, is was hospitalized to undergo the first cycle of induction chemotherapy according to the experimental phase 1 protocol ?SynVen-AML (Figure 4). The blood count on admission showed: WBC 10,070/mmc, Hb 7.8 g/dL, PLT 39,000/mmc. At molecular evaluation: marked infiltration (75-80%) by mutated NPM1 leukemic cells (Figure 5).
Il paziente ? stato ricoverato per 29 giorni e ha ricevuto I ciclo di terapia con omacetaxina mepesuccinato (Synribo) al dosaggio di 0.625 mg/m<2 >(livello 1) sottocute due volte al giorno dal giorno 1 al giorno 14, e venetoclax per via orale al dosaggio di 50 mg al giorno 2, 100 mg giorno 3, 200 mg al giorno 4, 400 mg dal giorno 5 fino al termine della terapia (g 21), come previsto da protocollo. Il ciclo prevedeva, infine, un periodo di 7 giorni in assenza di somministrazione dei farmaci sperimentali (?offtherapy?, (g 21-28) (Figura 4). The patient ? was hospitalized for 29 days and received the first cycle of therapy with omacetaxine mepesuccinate (Synribo) at a dosage of 0.625 mg/m<2 >(level 1) subcutaneously twice a day from day 1 to day 14, and venetoclax orally on dosage of 50 mg on day 2, 100 mg on day 3, 200 mg on day 4, 400 mg from day 5 until the end of therapy (d 21), as required by the protocol. Finally, the cycle included a period of 7 days in the absence of administration of the experimental drugs (?offtherapy?, (d 21-28) (Figure 4).
Al termine dei 14 giorni di terapia combinata ? stato eseguito esame del midollo osseo, che mostrava riduzione della cellularit? e marcati aspetti di differenziazione monocitaria, in linea con quanto descritto nel modello cellulare in laboratorio. In corrispondenza di e nei giorni successivi, e compatibilmente a tale reperto si documentava la comparsa di spiccata ipertrofia gengivale, che poi si ? attenuata con il passare del tempo e il prosieguo del trattamento (Figura 6). At the end of the 14 days of combined therapy? Bone marrow examination was performed, which showed reduction in cellularity? and marked aspects of monocyte differentiation, in line with what is described in the laboratory cellular model. Corresponding to and in the following days, and compatible with this finding, the appearance of marked gingival hypertrophy was documented, which then became evident. attenuated with the passage of time and the continuation of treatment (Figure 6).
L'obiettivo primario di questo studio di fase 1 ? determinare la tollerabilit? della nuova associazione farmacologica e avere dati preliminari di efficacia. The primary objective of this phase 1 study? determine tolerability? of the new pharmacological combination and have preliminary efficacy data.
Durante il periodo di terapia, come atteso in paziente con LAM in terapia, si sono verificate leucopenia di grado 4 (globuli bianchi, neutrofili <500/mmc), anemia di grado 3 (emoglobina <8 g/dl) e trombocitopenia di grado 4 (piastrine <20000/mmc). L?esigenza trasfusionale tuttavia si ? limitata a 6 unit? di globuli rossi e 5 unit? di piastrine, in assenza di sintomatologia da anemia e/o manifestazioni emorragiche. Considerando la leucopenia di grado 4, ? stata iniziata una profilassi della neutropenia febbrile mediante somministrazione dell?antibiotico levofloxacina per via orale. During the therapy period, as expected in patients with AML on therapy, grade 4 leukopenia (white blood cells, neutrophils <500/mmc), grade 3 anemia (hemoglobin <8 g/dl) and grade 4 thrombocytopenia occurred (platelets <20000/mmc). However, the need for transfusion is limited to 6 units? of red blood cells and 5 units? of platelets, in the absence of symptoms of anemia and/or haemorrhagic manifestations. Considering grade 4 leukopenia, ? prophylaxis of febrile neutropenia was started by administering the antibiotic levofloxacin orally.
Non si sono verificate altre tossicit? significative: in particolare non si sono registrati rialzi termici n? episodi infettivi durante questo ciclo di terapia. Anche dal punto di vista della tossicit? di altri organi e tessuti (fegato, cuore, reni, cute, polmone, etc), nulla da segnalare, neanche a livello di alterazione di esami di laboratorio. No other toxicities occurred? significant: in particular there were no thermal increases n? infectious episodes during this cycle of therapy. Also from the point of view of toxicity? of other organs and tissues (liver, heart, kidneys, skin, lung, etc), nothing to report, not even in terms of alterations in laboratory tests.
Alla valutazione midollare del d28, si documentava una risposta parziale di malattia con una buona ripresa trilineare a livello midollare, nonostante la pancitopenia periferica (vale a dire, bassi valori delle cellule del sangue: globuli rossi, globuli bianchi e piastrine, rispetto alla norma) (attribuita al residuo di malattia leucemica) (Figura 5). At the spinal cord evaluation on d28, a partial disease response was documented with a good trilinear recovery at the spinal cord level, despite peripheral pancytopenia (i.e., low blood cell values: red blood cells, white blood cells and platelets, compared to normal) (attributed to residual leukemic disease) (Figure 5).
Il paziente, pertanto, ? stato sottoposto a II ciclo di terapia secondo lo stesso schema, come previsto da protocollo. Anche questo ciclo ? stato perfettamente tollerato con un periodo di ricovero di soli 17 giorni e un fabbisogno trasfusionale di appena 3 unit? di globuli rossi e 1 unit? di piastrine. Di nuovo, non si sono verificate altre tossicit? significative. The patient, therefore, is underwent a second cycle of therapy according to the same scheme, as required by the protocol. This cycle too? was perfectly tolerated with a hospitalization period of only 17 days and a transfusion requirement of just 3 units? of red blood cells and 1 unit? of platelets. Again, no other toxicities occurred? significant.
Altri pazienti attendono di essere arruolati nel protocollo clinico attivo. Other patients are waiting to be enrolled in the active clinical protocol.
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