Ixazomib Research Articles

Post-Marketing Analysis of Peripheral Neuropathy Burden with New-Generation Proteasome Inhibitors Using the FDA Adverse Event Reporting System

Proteasome inhibitors (PIs) are an integral component of multiple myeloma therapies. Peripheral neuropathy (PN) is a well-known consequence of PIs, most frequently reported with earlier generations such as bortezomib (BTZ). There is a paucity of data highlighting the risk of developing PN with the new-generation PIs carfilzomib (CFZ) and ixazomib (IZB). This study evaluated reports of PN encountered with all three PIs using the Food and Drug Administration Adverse Event (AE) Reporting System (FAERS). Signal disproportionality analysis was reported using the reporting odds ratio (ROR) with 95% confidence interval (CI). PN was reported in a total of 2.1%, 5.0%, and 10.9% of AEs with CFZ, IZB, and BTZ, respectively. The ROR (95% CI) for PN secondary to BTZ, CFZ, and IZB was 34.10 (32.76-35.49), 6.37 (5.50-7.37), and 14.97 (13.63-16.44), respectively. Compared to BTZ, CFZ and IZB have lower rates of reported PN, with RORs of 0.19 (0.16-0.22) and 0.48 (0.43-0.54), respectively.

Pre-Clinical Evaluation of the Proteasome Inhibitor Ixazomib against Bortezomib-Resistant Leukemia Cells and Primary Acute Leukemia Cells

At present, 20–30% of children with acute leukemia still relapse from current chemotherapy protocols, underscoring the unmet need for new treatment options, such as proteasome inhibition. Ixazomib (IXA) is an orally available proteasome inhibitor, with an improved safety profile compared to Bortezomib (BTZ). The mechanism of action (proteasome subunit inhibition, apoptosis induction) and growth inhibitory potential of IXA vs. BTZ were tested in vitro in human (BTZ-resistant) leukemia cell lines. Ex vivo activity of IXA vs. BTZ was analyzed in 15 acute lymphoblastic leukemia (ALL) and 9 acute myeloid leukemia (AML) primary pediatric patient samples. BTZ demonstrated more potent inhibitory effects on constitutive β5 and immunoproteasome β5i proteasome subunit activity; however, IXA more potently inhibited β1i subunit than BTZ (70% vs. 29% at 2.5 nM). In ALL/AML cell lines, IXA conveyed 50% growth inhibition at low nanomolar concentrations, but was ~10-fold less potent than BTZ. BTZ-resistant cells (150–160 fold) displayed similar (100-fold) cross-resistance to IXA. Finally, IXA and BTZ exhibited anti-leukemic effects for primary ex vivo ALL and AML cells; mean LC50 (nM) for IXA: 24 ± 11 and 30 ± 8, respectively, and mean LC50 for BTZ: 4.5 ± 1 and 11 ± 4, respectively. IXA has overlapping mechanisms of action with BTZ and showed anti-leukemic activity in primary leukemic cells, encouraging further pre-clinical in vivo evaluation.

All-Trans-Retinoic Acid Prevents Carfilzomib-Induced Cardiotoxicity By Decreasing Activation of the Renin-Angiotensin System

INTRODUCTION Bortezomib (BTZ), Carfilzomib (CFZ) and Ixazomib (IXA) are proteasome inhibitors (PI) approved for Multiple Myeloma (MM) treatment. By design, they all target the rate-limiting proteasome ß5 subunit. CFZ treatment increases the survival of patients with relapsed/refractory MM compared to BTZ. However, CFZ treatment is associated with heart failure not commonly observed for BTZ. The molecular basis for CFZ-induced cardiotoxicity is poorly understood. We have shown before that CFZ co-inhibits the ß5 and ß2 proteasome subunits compared to co-inhibition of the ß5/ß1 subunits by BTZ. We hypothesized that the unique ß5/ß2 subunit inhibition pattern of CFZ likewise explains the CFZ related acute cardiotoxicity. METHODS Isolated primary murine cardiomyocytes treated with BTZ, CFZ or ß5, ß2 and ß1 subunit-selective PI were used as in vitro model. Selectivity of proteasome subunit inhibition was confirmed with activity based chemical probes. Cardiomyocyte contractility was assessed in vitro using motion vector image analysis. The accumulation of individual proteins in cardiomyocytes undergoing 1h treatment with different PI was analyzed by liquid chromatography/mass spectrometry (LC/MS). In vivo analysis of the effects of differential proteasome subunit inhibition patterns was performed by electrocardiography (ECG) monitoring and untargeted metabolomics analysis from murine heart and plasma using R v.3.5.1 (2018-07-02) and Matlab 2020a. RESULTS Co-inhibition of ß5/ß2 proteasome subunits by CFZ or by respective subunit-specific PI for 1h resulted in increased proteasome inhibition and proteotoxic stress in cardiomyocytes compared to ß5/ß1 inhibition induced by BTZ. Likewise, CFZ or the ß5/ß2 type co-inhibition of the proteasome impaired contractility of cardiomyocytes in vitro, in contrast to BTZ or to ß5/ß1-selective proteasome inhibition. LC/MS-based proteome analysis of murine primary cardiomyocytes treated with either CFZ-type or BTZ-type proteasome inhibition identified accumulation of Retinal Dehydrogenase 1 (ALDH1A1) in PI-treated cardiomyocytes, with CFZ showing a slightly stronger effect. ALDH1A1 is involved in retinoic acid metabolism. Co-treatment of cardiomyocytes with its enzymatic end-product, all-trans-Retinoic-Acid (atRA), restored contractility of cardiomyocytes undergoing CFZ treatment in vitro. Leucyl-Cistinyl Aminopeptidase (LCAP) was identified as the protein that accumulated in CFZ-versus BTZ-treated cardiomyocytes (Log2 CFZ = 1.1, Log2 BTZ = -0.49, p=0.002). LCAP is involved in the Renin-Angiotensin System (RAS) and its increase suggests selective activation of RAS with increased degradation of RAS metabolites via LCAP in cardiomyocytes undergoing CFZ treatment. Indeed, mRNA expression for angiotensin converting enzyme 2 (Ace2) and angiotensin receptor 1 (Agtr1) was increased in beating cardiomyocytes treated with CFZ. Conversely, treatment with aminopeptidase inhibitor Bestatin prevented CFZ-induced cardiomyocyte toxicity in vitro. Consistent with our model, co-treatment of cardiomyocytes with CFZ and Valsartan, an Agtr1 inhibitor, likewise prevented CFZ- induced toxicity. In vivo, 1h CFZ treatment selectively led to acute bradycardia with local activation of the RAS and accumulation of angiotensin in the heart. CFZ/atRA co-treatment decreased local angiotensin levels and prevented CFZ-induced bradycardia. There were no differences in angiotensin plasma levels in between the treated mice, suggesting that the local RAS in the heart rather than systemic effects of the RAS mediates the cardiotoxicity of CFZ. CONCLUSION Our data suggest that CFZ specifically impairs cardiac contractility in a dose-dependent manner that is caused by its unique ß5/ß2 proteasome subunit co-inhibition pattern. CFZ leads to more efficient functional proteasome inhibition in cardiomyocytes, resulting in accumulation of LCAP that subsequently activates the local RAS and induce acute cardiotoxicity. Based on our findings, CFZ-induced cardiotoxicity may be prevented or mitigated by either blocking RAS activation at the angiotensin II receptor level using selective drugs like Valsartan, or by reducing angiotensin levels with atRA. This provides a rational strategy to prevent CFZ-induced acute cardiac toxicity by modulating the activity of the local RAS with approved drugs. Disclosures No relevant conflicts of interest to declare.

A phase 0 analysis of ixazomib in patients with glioblastoma.

Improving overall survival in recurrent glioblastoma remains a challenge, and drugs acting by unique mechanisms are urgently required. Ixazomib is an orally-administered proteasome inhibitor used in combination with lenalidomide and dexamethasone to treat patients with multiple myeloma who have received at least one prior therapy. However, ixazomib's ability to reach brain tumors has not been studied during its development. The aim of the present study (ClinicalTrials.gov, NCT02630030) was to establish and quantify ixazomib's presence in glioblastoma. The present study investigated 3 patients with recurrent glioblastoma after administration of oral ixazomib citrate (MLN 9708) at a fixed 4.0 mg dose within a 3-hpreoperative window. A total of 2 blood samples were taken from each patient at the time of incision, tumor sampling and closure. Brain tumor samples were collected during tumor resection. These samples were then used to measure the plasma and brain tumor tissue concentration of the biologically-active form of ixazomib (MLN 2238). Patient 1 had plasma concentrations of ixazomib averaging 26.2, 21.8 and 15.3 ng/ml at incision, tumor sampling and closure, respectively. The brain tumor tissue concentration was 7.88 ng/g. Patient 2 had the same interval and brain tumor tissue measurements of 19.0, 18.0 and 8.93 ng/ml, and 2.03 ng/g. Patient 3 had plasma concentration interval measurements of 25.6, 36.2 and 28.7 ng/ml. Multiple brain tumor tissue samples were taken in patient 3, with an average tissue ixazomib concentration of 3.37 ng/g. Ixazomib was found at plasma concentrations commensurate with its previously established pharmacokinetic profile without clinically relevant drug-related adverse events. Ixazomib reaches glioblastoma tissues at measurable concentrations at the time of tumor resection, confirming target tissue delivery. This justifies the phase I study of ixazomib in recurrent glioblastoma currently in development.

Inhibition of chymotrypsin-like activity of the proteasome by ixazomib prevents mitochondrial dysfunction during myocardial ischemia.

The heart is critically dependent on mitochondrial respiration for energy supply. Ischemia decreases oxygen availability, with catastrophic consequences for cellular energy systems. After a few minutes of ischemia, the mitochondrial respiratory chain halts, ATP levels drop and ion gradients across cell membranes collapse. Activation of cellular proteases and generation of reactive oxygen species by mitochondria during ischemia alter mitochondrial membrane permeability, causing mitochondrial swelling and fragmentation and eventually cell death. The mitochondria, therefore, are important targets of cardioprotection against ischemic injury. We have previously shown that ixazomib (IXA), a proteasome inhibitor used for treating multiple myeloma, effectively reduced the size of the infarct produced by global ischemia in isolated rat hearts and prevented degradation of the sarcoplasmic reticulum calcium release channel RyR2. The aim of this work was to further characterize the protective effect of IXA by determining its effect on mitochondrial morphology and function after ischemia. We also quantified the effect of IXA on levels of mitofusin-2, a protein involved in maintaining mitochondrial morphology and mitochondria-SR communication. We found that mitochondria were significantly preserved and functional parameters such as oxygen consumption, the ability to generate a membrane potential, and glutathione content were improved in mitochondria isolated from hearts perfused with IXA prior to ischemia. IXA also blocked the release of cytochrome c observed in ischemia and significantly preserved mitofusin-2 integrity. These beneficial effects resulted in a significant decrease in the left ventricular end diastolic pressure upon reperfusion and a smaller infarct in isolated hearts.

Ixazomib Modulates Bone Remodeling and Actives Sonic Hedgehog Pathways

Multiple myeloma (MM) is a clonal B-cell malignancy characterized by an accumulation of clonal plasma cells (PC) in the bone marrow (BM) leading to bone destruction and BM failure. Osteolytic bone disease is a common manifestation of MM that leads to progressive skeleton destruction and is the most severe cause of morbidity in MM patients. Pathogenetic mechanisms of MM bone destruction are closely linked to MM PC and osteoclasts (OCs) hyperactivity coupled with defective osteoblast (OB) function unable to counteract bone resorption. We recently demonstrated that the proteasome inhibitor (PI) Bortezomib, commonly used to treat MM, was capable to inhibit osteoclastic differentiation modulating chitinase family genes. In this work we investigated the effect of Ixazomib (IXA), a third generation PI, on osteoclastogenesis and osteogenic differentiation. Human monocytes were differentiated in OCs in presence of OC medium (supplemented with RANKL and M-CSF), and/or 10nM IXA. We observed that IXA was able to inhibit the expression of different OCs markers such as RANK, CTSK, TRAP, and MMP9 when added in OC medium in respect to OC medium alone (p<0.001). In addition, IXA treatment reduced CHIT1 enzymatic activity and downregulated CHIT1 and YKL40 (both mRNA and proteins). Immunofluorescence evaluation confirmed that IXA inhibited the mature OCs formation with five or more nuclei. Moreover, IXA was able to stimulate osteogenic differentiation of human mesenchymal stromal cells (MSCs). After 21 days of treatment, IXA alone or added to osteogenic medium increased the osteogenic markers genes (BMP2, RUNX2, Osteocalcin and Osteonectin). Immunofluorescence assay confirmed the increase of BMP2 after IXA treatment alone or in combination with osteogenic medium. Sonic Hedgehog (Shh) is one of the intricate signal transduction mechanisms that govern the precisely regulated developmental processes of multicellular organisms. Its signaling cascade plays an important role in bone homeostasis, and reducing Shh pathway protects against age-related bone loss. In this work we observed that IXA, but not Bortezomib, was able to bind the Smoothened (Smo) receptor which is the transducing molecule of the extracellular signal of Shh. Interestingly, using cellular thermal shift assay (CETSA), we demonstrated that IXA was able to bind directly Smo receptor activating the Shh cascade that leads to the nuclear translocation of Gli1 an effector of this pathway. Inhibiting Shh signaling by using an inhibitor of Smo activation (Cyclopamine), IXA-activated ostegenic differentiation-related genes were down-regulated. In conclusion, our data demonstrated that IXA regulates bone remodeling decreasing osteoclastogenesis and promoting osteogenic differentiation and therefore represents a good therapeutic option to improve the complex pathological condition of patients with MM. Disclosures Conticello: Celgene: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding. Palumbo:Amgen: Honoraria; Celgene: Honoraria; Hospira: Honoraria; Janssen: Honoraria; Novartis: Honoraria; Teva: Honoraria. Di Raimondo:Amgen: Consultancy, Honoraria, Research Funding; Takeda: Consultancy; Celgene: Consultancy, Honoraria, Research Funding.

Ixazomib, Rituximab and Dexamethasone (IRD) in Patients with Relapsed or Progressive Waldenstrom's Macroblobulinemia: Results of the Prospective Phase I/II HOVON 124/Ecwm-R2 Trial

Ixazomib, Rituximab and Dexamethasone (IRD) in Patients with Relapsed or Progressive Waldenstrom's Macroblobulinemia: Results of the Prospective Phase I/II HOVON 124/Ecwm-R2 Trial

Carfilzomib Induces Cardiotoxicity Via ß5/ß2-Specific Proteasome Subunit Inhibition Pattern

BACKGROUND All proteasome inhibitors (PI) approved for Multiple Myeloma (MM) treatment, Bortezomib (BTZ), Ixazomib (IXA) and Carfilzomib (CFZ), by design target the rate-limiting ß5 proteolytic proteasome subunit, but differ in their co-inhibition of the ß1 or ß2 proteolytic subunits at higher doses. CFZ is unique in inducing ß5/ß2 co-inhibition in a dose-dependent manner, which results in increased proteotoxic stress and higher cytotoxicity compared to the ß5/ß1 co-inhibition pattern mediated by BTZ or IXA. CFZ treatment, in particular at higher doses, is not only associated with increased clinical activity, but also with significantly higher cardiac toxicity compared to BTZ. The molecular basis for this difference is poorly understood, and mechanism-based pharmacological alleviation strategies are lacking. We hypothesized that the acute cardiotoxicity of CFZ is related to its unique ß5/ß2 inhibition pattern. METHODS Isolated primary murine cardiomyocytes showing spontaneous rhythmical contractions were used to assess the functional cardiotoxicity of PI in vitro. Cardiomyocytes were treated with the PI BTZ, CFZ or with highly-selective, subunit (ß1, ß2, ß5)-specific PI for 1h. Proteasome inhibition was directly verified using subunit-selective activity-based chemical probes. Contractility was assessed with motion vector image analysis, and calcium transients were examined by confocal microscopy after transduction of cardiomyocytes with a GCaMP vector. Differentially accumulated proteins after 1h PI treatment were quantitatively compared by liquid chromatography/mass spectrometry (LC/MS). In vivo, electrical changes and alterations in heart rate were monitored by electrocardiography (ECG). Calcium transients, ECG signals and LC/MS data were analyzed with R software version 3.5.1 (2018-07-02). RESULTS Treatment of cardiomyocytes with CFZ or with the combination of ß5/ß2-targeting subunit-specific PI impaired contractility in vitro in contrast to BTZ, or to co-inhibition of ß5/ß1 proteasome subunits. In vivo, the CFZ-type proteasome inhibition triggered acute bradycardia. Treatment of cardiomyocytes with CFZ induced a shift of intracellular calcium pools from the endoplasmic reticulum (ER) to the cytosol, consistent with ER-to-cytosol translocation of calcium described upon ER stress induction. Co-treatment with CFZ and cycloheximide, an inhibitor of protein synthesis, rescued cardiomyocytes from CFZ-induced functional impairment in vitro, suggesting that the accumulation of specific proteins is involved in CFZ-induced cardiomyocyte dysfunction. Quantitative proteomic comparison of primary cardiomyocytes treated by either CFZ-type or BTZ-type proteasome inhibition for 1h identified a selective accumulation of proteins of the retinoic acid pathway in CFZ-treated cardiomyocytes. Interestingly, co-treatment of cardiomyocytes with all-trans-retinoic acid (ATRA) prevented CFZ-induced acute cardiotoxicity in vitro. CONCLUSION Our data suggests that CFZ specifically impairs cardiac contractility through its unique ß5/ß2 proteasome subunit inhibition pattern, which results in more effective functional proteasome inhibition, protein accumulation and proteotoxic stress. The shift of intracellular calcium pools in cardiomyocytes upon CFZ treatment mirrors contractility impairment, and the specific changes identified in the retinoic acid pathway suggest ATRA as a potential drug candidate to alleviate CFZ-induced cardiac toxicity. Disclosures No relevant conflicts of interest to declare.

Treatment of Myeloma Patients Progressing on Lenalidomide-Based Maintenance after ASCT Performed As Part of 1st, 2nd or 3rd Line Therapy: Real-World Results from the National Myeloma Canada Research Network (MCRN) Database

Treatment of Myeloma Patients Progressing on Lenalidomide-Based Maintenance after ASCT Performed As Part of 1st, 2nd or 3rd Line Therapy: Real-World Results from the National Myeloma Canada Research Network (MCRN) Database

Treatment of Multiple Myeloma Patients Progressing on Lenalidomide-Based Regimens

Treatment of Multiple Myeloma Patients Progressing on Lenalidomide-Based Regimens

Proteasome inhibition by Carfilzomib, but not by Bortezomib, induces a specific, proteasome subunit-dependent impairment in cardiac contractility.

All approved proteasome inhibitors (PI), Bortezomib (BTZ), Ixazomib (IXA) and Carfilzomib (CFZ), by design target the rate-limiting B5 proteasome subunit. Only CFZ at higher doses shows co-inhibition of B5/B2. The CFZ-type B5/B2 co-inhibition induces increased proteotoxic stress and higher cytotoxicity in MM than the BTZ-type, B5/B1 inhibition, consistent with increased survival of relapsed/refractory MM patients. However, higher doses of CFZ produce acute heart failure not commonly seen with the first-generation PI, which is mechanistically poorly understood.

Abstract 285: Ixazomib combined with the nuclear export inhibitors selinexor or eltanexor for the treatment of multiple myeloma

Abstract Introduction In the past decade, response and survival rates in multiple myeloma (MM) patients have been greatly increased by newer therapies. Nevertheless, most patients with MM eventually die from relapsed/refractory disease. Previous studies in our lab and in others have shown that exportin 1 inhibitors (XPO1i) are potent anti-MM drugs, whether used as single agents or in combination with proteasome inhibitors (PIs), immunomodulator drugs (IMiDs), anthracyclines, or alkylating agents. Ixazomib (IXA), an orally available PI, has been shown to be effective when combined with IMiDs, dexamethasone, alkylating agents, and/or prednisone for the treatment of patients with MM. We investigated whether the clinical XPO1i selinexor (SEL) or eltanexor (ELT) combined with IXA could overcome PI resistance in MM cell lines and in ex vivo bone marrow aspirates from PI-refractory patients. Materials and Methods PI-resistant MM cell lines 8226B25 and U226PSR and their respective parental cell lines, 8226 and U266, were treated in vitro and in vivo (mice) with SEL/IXA or ELT/IXA combinations or with each drug as a single agent. Bone marrow aspirates from newly diagnosed and PI-refractory MM patients were treated ex vivo with SEL/IXA or ELT/IXA and assayed for apoptosis. Mechanistic studies included NFkB and IkBα protein assays, immunofluorescence microscopy, ImageStream flow-cytometry, and proximity-ligation assay. IkBα knockdown and NFkB transcriptional activity were measured in SEL/IXA- or ELT/IXA-treated MM cells. Results SEL and ELT each sensitized parental and, to a lesser extent, PI-resistant MM cells to IXA, as measured by apoptosis. XPO1i/IXA treatments inhibited U266 and 8226 MM tumor growth and increased survival in mice; however, PI-resistant tumors were not sensitized by XPO1i/IXA treatment. Myeloma cells isolated from both newly diagnosed and PI-refractory MM patients were sensitized by XPO1i to IXA, as compared to untreated and single agent-treated MM cells, without affecting non-MM cells. Immunofluorescence microscopy, Western blot, and ImageStream analyses of MM cells showed increased nuclear IkBα with the XPO1i/PI treatments. Proximity ligation found increased IkBα-NFkB complexes in treated MM cells. IkBα knockdown abrogated XPO1i/PI-induced cytotoxicity in MM cells. XPO1i/PI treatment decreased NFkB transcriptional activity. Conclusions In previous studies, we found that SEL sensitized PI-resistant cell lines to carfilzomib and bortezomib; however, IXA was less effective in these same MM cells. SEL or ELT used in combination with IXA have the potential to overcome PI drug resistance in MM patients. Sensitization may be due to inactivation of the NFkB pathway by IkBα. Further investigation is underway to examine these mechanisms. The results presented in this study support combinatorial clinical trials in relapsed and refractory MM patients who use PI therapies. Citation Format: Joel G. Turner, Jana Dawson, Alexis Bauer, Juan Gomez, Erkan Baloglu, Yosef Landesman, Daniel M. Sullivan. Ixazomib combined with the nuclear export inhibitors selinexor or eltanexor for the treatment of multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 285.

Novel cell line models to study mechanisms and overcoming strategies of proteasome inhibitor resistance in multiple myeloma

Experimental data on resistance mechanisms of multiple myeloma (MM) to ixazomib (IXA), a second-generation proteasome inhibitor (PI), are currently lacking. We generated MM cell lines with a 10-fold higher resistance to IXA as their sensitive counterparts, and observed cross-resistance towards the PIs carfilzomib (CFZ) and bortezomib (BTZ). Analyses of the IXA-binding proteasome subunits PSMB5 and PSMB1 show increased PSMB5 expression and activity in all IXA-resistant MM cells, and upregulated PSMB1 expression in IXA-resistant AMO1 cells. In addition, sequence analysis of PSMB5 revealed a p.Thr21Ala mutation in IXA-resistant MM1.S cells, and a p.Ala50Val mutation in IXA-resistant L363 cells, whereas IXA-resistant AMO1 cells lack PSMB5 mutations. IXA-resistant cells retain their sensitivity to therapeutic agents that mediate cytotoxic effects via induction of proteotoxic stress. Induction of ER stress and apoptosis by the p97 inhibitor CB-5083 was strongly enhanced in combination with the PI3Kα inhibitor BYL-719 or the HDAC inhibitor panobinostat suggesting potential therapeutic strategies to circumvent IXA resistance in MM. Taken together, our newly established IXA-resistant cell lines provide first insights into resistance mechanisms and overcoming treatment strategies, and represent suitable models to further study IXA resistance in MM.

Effects of Proteasome Inhibition in the Anti-Tumor Activity of Venetoclax in Mantle Cell Lymphoma Pre-Clinical Models

Background: At the molecular level, mantle cell lymphoma (MCL) is characterized by the deregulation of Bcl-2 family members (Mcl-1, BIM) and cell cycle (cyclin D1) regulatory proteins. Perhaps related to this, the clinical outcome of MCL continues to be poor specially for those patients with disease progression after high dose chemotherapy and autologous stem cell rescue and/or BTK inhibitors, stressing the need to develop novel therapeutic strategies or optimize current available options. Venetoclax (V), a highly selective Bcl-2 inhibitor, has shown modest activity against relapsed/refractory MCL. Over-expression of Mcl-1 has been postulated to be a mechanism of resistance to V limiting its anti-tumor activity in subtypes of lymphoma including MCL. The lethality by proteasome inhibitors (PIs) has been associated with changes in the Bcl-2 family members (Bax, Noxa, Mcl-1 and Bcl-XL) in lymphoma pre-clinical models, making them ideal agents to combine with V. To this end, we studied the anti-tumor activity of combining PIs with V in MCL pre-clinical models. Materials and Methods: A panel cytarabine sensitive (Rec-1, Jeko, Granta, HBL-2, Z-138 and Mino) and resistant (araC) cell lines (Jeko araC, HBL-2 araC, and Mino araC) were exposed to V, Bortezomib (BTZ), carfilzomib (CFZ), or ixazomib (IXZ) for 24, 48 and 72 hours. Cell viability was calculated measuring the ATP content. IC50 drug concentrations were calculated for each agent. Subsequently, MCL cell lines were exposed to escalating doses of V (0.001uM-5uM) and CFZ (1.5625nM-50nM), BTZ (3.125nM-100nM) or IXZ (3.125nM-100nM). In addition, primary tumor cells isolated from B-cell lymphoma patients (N=21) including MCL patients were exposed to V +/- BTZ or CFZ for 48 hrs. Cell viability was determined by Cell Titerglo. Coefficient of synergy were calculated using CalcuSyn software program. Induction of apoptosis was detected by Annexin V/Propidium iodine staining and PARP cleavage. Changes in Bcl-2 and cell cycle regulatory proteins were evaluated by Western blotting in HBL-2 cells. For in vivo experiments, 6-8 weeks old severe combined immunodeficiency (SCID) mice were inoculated with 10x106 HBL-2 cells via tail vein injection (IV). Subsequently, SCID mice were treated with V (100mg/kg/dose via gastric lavage on days 3-7, 10-14 and 17-21) or IXZ (6mg/kg/dose IV days 3, 6, 10, 13, 17 and 20) or combination of both agents. A group of untreated animals was used as a control. Differences in survival were evaluated between treatment groups. Results: In vitro exposure of MCL cell lines to either V, BTZ, CFZ, and IXZ induced cell death in a dose- and time-dependent manner. Significant synergistic activity was observed by combining both V with CFZ or IXZ at known sub-therapeutic and therapeutic doses of individual agents measured by ATP content and apoptosis potential. Anti-tumor activity was observed in cytarabine sensitive and resistant cell lines. Similar findings were observed in primary tumor cells isolated from B-cell lymphoma patients. In vitro exposure of MCL cell lines with the lowest IC50 (HBL-2) to V and PIs (BTZ, CFZ, or IXZ) resulted in the upregulation of Noxa, BIM, Mcl-1 cleavage form (pro-apoptotic) and downregulation of Bcl-XL leading to PARP cleavage and apoptosis. In vivo treatment of MCL bearing SCID mice with V resulted in significant anti-tumor activity when compared to single agent IXZ treated or control animals. Of interest, MCL bearing SCID animals treated with V and IXZ exhibited a better disease control and the survival was longer than SCID animals treated with V or IXZ single agent (P<0.05). Conclusion: Our data suggests that V exhibits strong synergistic activity with PIs, especially with CFZ (in vitro) or IXZ (in vitro and in vivo). Together, our data supports the evaluation of V in combination with readily available novel PIs (IXZ or CFZ) in relapsed/refractory MCL. (Supported by LRF grant 555463, an NIH grant R01 CA136907-01A1 and a grant from The Roswell Park Cancer Institute Alliance Foundation) DisclosuresNo relevant conflicts of interest to declare.

Tumor-Lysis Syndrome in Relapsed or Refractory Multiple Myeloma Patients Treated with Proteasome Inhibitors

<Introduction>Tumor-lysis syndrome (TLS) describes a group of metabolic disturbances caused by the massive and abrupt release of cellular components into the blood after the rapid lysis of malignant cells. TLS is a potentially life-threatening complication in rapidly proliferating, bulky tumor, or highly chemo-radiosensitive cancers. In contrast, TLS is the rare complication among patients with multiple myeloma (MM). TLS seems to occur more frequently in MM patients receiving bortezomib (BOR) and carfilzomib (CFZ) than in those receiving other cytotoxic agents. We retrospectively investigated the frequency, risk factors, and clinical outcome among the relapsed or refractory MM (RRMM) patients treated with proteasome inhibitors (PI), such as BOR, CFZ, and ixazomib (IXA), in our institute.<Methods>Between November 2005 and December 2017, seventy-two RRMM myeloma patients treated with BOR, CFZ, or IXA, in our institute. The number of patients treated PI was defined as below. Relapse or refractory disease and response criteria were defined according to the International Myeloma Working Group criteria. Patients with monoclonal gammopathy of undetermined significance, smoldering multiple myeloma, and primary plasma cell leukemia were excluded. We evaluated TLS by the Cario-Bishop grading classification. After starting of PI, the following parameters were recorded and evaluated for prognosis: serum hemoglobin concentration (Hb), serum level of lactate dehydrogenase (LDH), creatinine (Cr), uric acid (UA). The cutoff levels of LDH, Cr, and UA were the upper normal limit. Anemia was defined by the CRAB criteria. Hepatosplenomegaly and plasmacytoma were evaluated using computed tomography. We analyze these two groups in terms of patient characteristics, laboratory findings, response, and survival. Fisher's exact tests were used to compare differences between the two groups. We analyzed prognostic factors for survival in significant poor predictors of laboratory parameters and well-established prognostic factors by Cox regression analysis. Overall survival (OS) and time to next treatment (TTNT) were analyzed by the Kaplan-Meier method.<Results>Median age of the patients was 71 years (range, 41-86) at the time of PI treatment. Median number of prior treatments was 2 (range, 1-8). The number of patients treated 53 of BOR, 10 of IXA, 4 of BOR + CFZ, 3 of CFZ + IXA, 1 of BOR + IXA, and 1of BOR + CFZ + IXA, respectively.TLS occurred in 13 patients (18.1%); such as 8 of BOR (13.3%), 4 of CFZ (40.0%), and 1 of IXA (6.7%). TLS occurred more frequently in the patients treated with twice weekly CFZ plus dexamethasone (CFZ 56mg/m2) than twice weekly CFZ, lenalidomide, plus dexamethasone (CFZ 27mg/m2); 50% and 25%, respectively. Median day of TLS after PI was 8 (range, 4-21). Median day of TLS in BOR, CFZ, and IXA were 7.5, 5.5, and 13 days, respectively. Two patients received rasbricase after TLS occurred. There was no patient who underwent hemodialysis for TLS. The mortality related with TLS was none.The significant factors associated TLS were anemia, high LDH level, prior treatments of 2 or more and hepatosplenomegaly according to univariate analysis. In multivariate analysis, high LDH level (Odds ratio; 12.9, P=0.027) and hepatosplenomegaly (Odds ratio; 13.4, P=0.035) was related with TLS significantly.There was no significant difference between overall response rate in the TLS group and non-TLS group (46.4% vs 64.8%, P=0.117). In median follow-up was 12.9 months, the median TTNT of the TLS group was significantly shorter than that of the non-TLS group (1.9 vs 7.0 months, P = 0.009, Figure 1a). The median OS of the TLS group was significantly shorter than that of the non-TLS group as well (5.8 vs 46.4 months, P = 0.017, Figure 1b).<Conclusion>Our analysis revealed that 18.1% of RRMM patients treated with PI developed TLS although TLS was manageable by prophylaxis.gh LDH level and hepatosplenomegaly were identified with significant risk factors for TLS. TLS was not associated with good response of treatment. The TTNT and OS in the TLS group was significantly shorter than those in the non-TLS group. We considered that TLS might be related with progressive disease without activity of treatment in RRMM patients treated with PI. Among the RRMM patients with high LDH level, TLS is one of common adverse events in first cycle of PI but tolerable. DisclosuresNo relevant conflicts of interest to declare.

Determining the Crystal Structures of Peptide Analogs of Boronic Acid in the Absence of Single Crystals: Intricate Motifs of Ixazomib Citrate Revealed by XRPD Guided by ss-NMR

Uncertainties in the structure determination of peptide analogs of boronic acid, exacerbated by the many coordination modes of boron, represent an obstacle in understanding their role in living organisms and thus also in developing the next generation of anticancer drugs. For that reason, we present here a general experimental-computational strategy allowing structure determination of complex boronic acid derivatives with extensive conformational variability. We demonstrate successful solution of the crystal structures of two nonsolvated polymorphs of ixazomib citrate directly from synchrotron powder diffraction data, which is challenging because the two molecules in the asymmetric unit cell that exhibit 32 degrees of conformational freedom push the limits of current solution procedures. We used a novel two-step Rietveld refinement based on DFT-D restraints to improve information quality derived from powder diffraction data to be comparable with that of single-crystal solutions. NMR crystallography was ap...

Cost-effectiveness of Drugs to Treat Relapsed/Refractory Multiple Myeloma in the United States.

New 3-drug regimens have been developed and approved to treat multiple myeloma (MM). The absence of direct comparative data and the high cost of treatment support the need to assess the relative clinical and economic outcomes across all approved regimens. To evaluate the cost-effectiveness of treatments for relapsed and/or refractory MM from a U.S. health system perspective. We developed a partition survival model with 3 health states (progression-free, progression, and death) to evaluate the following regimens: carfilzomib (CFZ), elotuzumab (ELO), ixazomib (IX), daratumumab (DAR), and panobinostat (PAN) in combination with lenalidomide (LEN) or bortezomib (BOR) plus dexamethasone (DEX) in the second and/or third line of therapy. To estimate relative treatment effects, we developed a network meta-analysis and applied progression-free survival hazard ratios to baseline parametric progression-free survival functions derived from pooled data on LEN+DEX. We estimated overall survival using data on the relationship between progression-free survival and overall survival from a large meta-analysis of MM patients. Modeled costs included those related to drug treatment, administration, monitoring, adverse events, and progression. Utilities were from publicly available data and manufacturer data, if published sources were unavailable. Model results showed that regimens containing DAR yielded the highest expected life years (DAR range: 6.71-7.38 vs. non-DAR range: 3.25-5.27) and quality-adjusted life-years (QALY; DAR range: 4.38-5.44 vs. non-DAR range: 2.04-3.46), with DAR+BOR+DEX (second line) and PAN+BOR+DEX (third line) as the most cost-effective options (incremental cost-effectiveness ratio: $50,700 and cost saving, respectively). The applicability of the PAN+BOR+DEX result may be challenging, however, because of ongoing toxicity concerns. In the probabilistic sensitivity analysis, second-line DAR+BOR+DEX and third-line PAN+BOR+DEX had an 89% and 87% probability of being cost-effective at the $150,000 per QALY threshold, respectively. The introduction of newer drugs and regimens to treat second- and third-line relapsed/refractory MM appears to provide clinical benefits by lengthening progression-free and overall survival and improving quality of life. However, only the addition of DAR or PAN may be considered cost-effective options according to commonly cited thresholds, and PAN+BOR+DEX results require cautious interpretation. Achieving levels of value more closely aligned with patient benefit would require substantial discounts from the remaining agents evaluated. Funding for this work was provided in part by the Institute for Clinical and Economic Review, which collaborated on the design, conduct, and reporting of this evaluation. During the conduct of this study, Ollendorf, Synnott, Chapman, and Pearson report grants from Blue Shield of California Foundation, California Health Care Foundation, and Laura and John Arnold Foundation and also report other grants from Aetna, AHIP, Anthem, Blue Shield of California, CVS Caremark, Express Scripts, Harvard Pilgrim Health Care, OmedaRx, United Healthcare, Kaiser Permanente, Premera, AstraZeneca, Genentech, GlaxoSmithKline, Johnson & Johnson, Merck, National Pharmaceutical Council, Takeda, Pfizer, Novartis, Lilly, Spark Therapeutics, Sanofi, Prime Therapeutics, and Health Care Service Corporation outside the submitted work. Carlson reports grants from the Institute for Clinical and Economic Review during the conduct of the study and personal fees from Seattle Genetics, Genentech, and Pfizer outside the submitted work. Russo, Guzauskas, Liu, and Brouwer have nothing to disclose. Study concept and design were contributed by Carlson, Guzauskas, and Ollendorf. Guzauskas, Chapman, Synnott, and Liu collected the data, and Carlson, Guzauskas, Chapman, and Ollendorf contributed to data analysis, along with Synnott and Liu. The manuscript was written by Carlson, Guzauskas, and Brouwer, along with Chapman, Synnott, and Ollendorf, and revised by Carlson, Brouwer, and Guzauskas, along with Chapman, Synnott, and Ollendorf.

Pharmacokinetic drug evaluation of ixazomib citrate for the treatment of multiple myeloma

ABSTRACTIntroduction: multiple myeloma (MM) is a plasma cell disorder that represents the second most frequent hematologic cancer. Although MM is still an incurable disease, prognosis has improved in the last decades thanks to the introduction of novel agents such as proteasome inhibitors (PIs), immunomodulatory drugs, monoclonal antibodies, and histone deacetylase inhibitors.Areas covered: ixazomib is the first oral PI recently approved by Food and Drug Administration (FDA) and European Medicine Agency (EMA) in combination with lenalidomide and dexamethasone as salvage therapy in MM patients. In this paper, we focus on its pharmacokinetics features, as well as its safety and efficacy in clinical studies.Expert opinion: ixazomib can be considered an oral analogue of bortezomib, with 9.5-day half-life, 58% of oral bioavailability, and a large distribution volume of 543L. These features make it a versatile molecule, potentially useful both in combination and as single agent. Oral route of administration and good efficacy/safety profile are its winning characteristics, providing the rationale for a future role also in the maintenance setting.

Research progress of proteasome inhibitors in treatment of multiple myeloma

Multiple myeloma(MM) cases constitute a relatively high proportion of patients suffering from hematological malignant diseases, with its prevalence rate mounting every year. Substantial breakthrough of MM treatment has been achieved due to the development of proteasome inhibitors(PI), which facilitates cellular apoptosis induced by the accumulation of intracellular ubiquitinated and/or misfolded proteins, via the inhibition of the degradation of the apoptosis-associated proteins. The exact pharmaceutical mechanisms of different PI vary, leading to different therapeutic effects and drug adverse reactions. Further understanding of the pharmaceutical mechanisms, therapeutic effects and safety of various PI lays the theoretical foundation for their clinical use. This article focuses the research progress and the clinical application of PIs in terms of MM treatment. Key words: Multiple myeloma; Proteasome inhibitors; Bortezomib; Carfilzomib; Ixazomib

Stem Cell Transplantation in Multiple Myeloma.

High-dose therapy (HDT) followed by autologous stem cell transplantation (ASCT) remains the standard of care for patients younger than 65 years of age with multiple myeloma (MM). However, this therapeutic approach has undergone substantial advances in this last decade, mainly due to the introduction of new drugs such as thalidomide, lenalidomide and bortezomib. These new drugs, in different combinations, have shown to significantly increase response rates after induction therapy and ASCT. Moreover, the positive results obtained with these agents in consolidation and maintenance strategies after ASCT strongly support the concept of continuous therapy, whose ultimate goal is the long-term control of the disease and the improvement of outcome. Preliminary data from studies investigating next generation proteasome inhibitors, such as carfilzomib and ixazomib, used upfront as well as at subsequent therapeutic lines, demonstrate the possibility of achieving molecular remission in most of the patients. The deeper responses obtained with new drugcombinations questioned the role of ASCT, and large, ongoing, phase 3 trials will shed light on the role and the timing of ASCT.