Epoxyketone Proteasome Inhibitor Research Articles

Heterologous Expression of Epoxomicin in Escherichia coli.

Epoxomicin is an epoxyketone proteasome inhibitor with synthetic derivatives approved or under investigation for treatment of multiple myeloma. To leverage the advantages of Escherichia coli as a rapidly growing and readily engineered host for the production of epoxomicin and analogues, we expressed codon-optimized versions of the epoxomicin biosynthetic genes, epxD, epxE, and epxF. Epoxomicin was detected, but the major product was a ketone resulting from α,β-keto acid precursor decarboxylation. Epoxomicin yield was improved by altering the copy numbers of each gene and creating a fusion of epxE and epxF. Our optimized system offers promise for efficient engineering and biosynthesis of improved epoxomicin analogues.

Cardiovascular Toxicity after Carfilzomib, Lenalidomide, and Dexamethasone Combination Chemotherapy in the Real-World 364 Asian Multiple Myeloma Patients on Behalf of the Korean Multiple Myeloma Working Party (KMM2201 study)

Background: Carfilzomib, a selective and irreversible epoxyketone proteasome inhibitor (PI), in combination with lenalidomide and dexamethasone (KRd) significantly improved the survival of relapsed and/or refractory multiple myeloma (RRMM) patients as well as newly diagnosed MM patients. The major dose-limiting toxicity of carfilzomib, cardiovascular adverse events (CVAE), have been reported in a variety of studies, which necessitates cautious risk management for patients who are treated with carfilzomib-based combination regimen. By far, the incidence and risk factors, as well as the outcome of CVAE in a large cohort of Asian patients after KRd regimen is lacking. Patient and method: Retrospective data of 364 RRMM patients who have received KRd regimen for RRMM in 21 centers participating in the Korean multiple myeloma working party (KMMWP) between February, 2018 and October, 2020, was analyzed. 41% of the patient was trial-ineligible, which was defined by patient-, disease-, and treatment-related features that were insufficient to meet the eligibility criteria for ASPIRE trial. CVAE was defined by dyspnea, hypertension (HTN), congestive heart failure (CHF), ischemic heart disease (IHD), arrhythmias, and venous thromboembolism (VTE). Time to onset of CVAE was defined by the first date of KRd therapy to the onset date of first CVAE. Results: The median age was 63 years (range, 28-85). CVAEs were observed in 16% of total analyzed patients after a median 9 months (range, 0-36 months) of KRd treatment, among which 3 patients (1%) showed fatal CVAEs. More specifically, dyspnea, HTN, CHF, IHD, arrhythmias, and VTE occurred in 11%, 3%, 3%, 1%, 2%, and 3% of the patients after a median of 9, 21, 16, 31, 8, and 16 months after KRd therapy. CVAE resulted in dose reduction and discontinuation of carfilzomib in 13 (4%) and 14 patients (4%), respectively. 5 patients (1%) discontinued KRd treatment due to any CVAE. By univariate analysis, among the factors affecting the cardiovascular events, such as age ≥75 years, ECOG performance status ≥ 3, creatinine clearance < 30 ml/min, Hb < 8g/dL, diabetes mellitus, HTN, underlying heart disease, cerebrovascular disease, prior bortezomib, thalidomide, doxorubicin, and cyclophosphamide therapy, and autologous stem cell transplantation, HTN was the only factor that significantly affected the occurrence of CVAEs after KRd therapy (odds ratio 1.877, 95% confidence interval, 1.025-3.440, P=0.041). CVAEs after KRd regimen did not affect progression-free or overall survival in the study cohort (Figure1A, and 1B). Conclusion: The incidence of CVAE after KRd therapy in this large cohort of real-world Asian RRMM patients was comparable with previous prospective and retrospective studies. Cardiovascular event after carfilzomib-based regimen did not affect progression-free or overall survival in RRMM patients. The recognition and management of underlying HTN has the utmost importance amongst the CV risk factors to mitigate CVAE in the Asian RRMM patients treated with KRd regimen. Acknowledgement This study would not have been possible without the collaboration of the Korean multiple myeloma working party. We especially thank the patients, families, caregivers who participated in this study. This work was supported by Amgen.

Parallelized gene cluster editing illuminates mechanisms of epoxyketone proteasome inhibitor biosynthesis.

Advances in DNA sequencing technology and bioinformatics have revealed the enormous potential of microbes to produce structurally complex specialized metabolites with diverse uses in medicine and agriculture. However, these molecules typically require structural modification to optimize them for application, which can be difficult using synthetic chemistry. Bioengineering offers a complementary approach to structural modification but is often hampered by genetic intractability and requires a thorough understanding of biosynthetic gene function. Expression of specialized metabolite biosynthetic gene clusters (BGCs) in heterologous hosts can surmount these problems. However, current approaches to BGC cloning and manipulation are inefficient, lack fidelity, and can be prohibitively expensive. Here, we report a yeast-based platform that exploits transformation-associated recombination (TAR) for high efficiency capture and parallelized manipulation of BGCs. As a proof of concept, we clone, heterologously express and genetically analyze BGCs for the structurally related nonribosomal peptides eponemycin and TMC-86A, clarifying remaining ambiguities in the biosynthesis of these important proteasome inhibitors. Our results show that the eponemycin BGC also directs the production of TMC-86A and reveal contrasting mechanisms for initiating the assembly of these two metabolites. Moreover, our data shed light on the mechanisms for biosynthesis and incorporation of 4,5-dehydro-l-leucine (dhL), an unusual nonproteinogenic amino acid incorporated into both TMC-86A and eponemycin.

Stability and potential degradation of the α',β'-epoxyketone pharmacophore on ZnO nanocarriers: insights from reactive molecular dynamics and density functional theory calculations.

We investigate the structure and dynamics of a zinc oxide nanocarrier loaded with Carfilzomib, an epoxyketone proteasome inhibitor developed for treating multiple myeloma. We demonstrate that, even though both bare and functionalized zinc oxide supports have been used for drug delivery, their interactions with the reactive functional groups of the ligands could be detrimental. This is because pharmacophores like α',β'-epoxyketones should preserve the groups required for the drug activity and be capable of leaving the vehicle at the target site. Earlier studies showed that even when ZnO is functionalized with oleic acid surfactants, the drug could reach parts of the surface and remain stably adsorbed. Herein, we have used reactive molecular dynamics simulations and quantum chemistry calculations to explore the potential interactions of the Carfilzomib functional groups with the typical surfaces of ZnO supports. We have found that Carfilzomib can adsorb on the (0001)Zn-terminated polar surface through the carbonyl oxygens and the epoxyketone moiety. These strong connections could prevent the drug release and induce the epoxy ring opening with its consequential inactivation. Therefore, regulating the dosage to maintain the desired level of drug bioavailability is paramount. These findings emphasize the need for appropriate carrier functionalizations to efficiently entrap, transport, and release the cargo at the target sites and the crucial role played by predictive/descriptive computational techniques to complement and drive experiments to the most appropriate selections of the materials to optimize drug delivery.

Bortezomib is an effective enhancer for chemical probe-dependent superoxide detection.

Various chemical probes for the detection of reactive oxygen species have been developed to examine oxidative stress associated with different pathologies. L-012, a luminol-based chemiluminescent probe, is widely used to detect extracellular superoxide because of its high sensitivity. We herein demonstrated that the co-application of the peptide boronic acid proteasome inhibitor, bortezomib, with L-012 significantly increased its luminescence without affecting the background. More than a 5-fold increase was detected in the total luminescence of L-012 in both NADPH oxidase-expressing cells and the xanthine oxidase-dependent cell-free superoxide generation system, but not in their background. Therefore, bortezomib increased the signal-to-background ratio and improved the detection of low levels of superoxide. The application of MLN2238, another peptide boronic acid proteasome inhibitor, also enhanced the luminescence of L-012. In contrast, carfilzomib, an epoxyketone proteasome inhibitor, did not increase luminescence, suggesting that the effects of bortezomib depend on the chemical structure of the peptide boronic acid, but not on its pharmacological effects. Bortezomib-induced enhancements appeared to be specific to the detection of superoxide because the detection of H2O2 by Amplex Red/HRP was not affected by the application of bortezomib. In the quantitative detection of the superoxide-specific oxidative product 2-hydroxyethidium (2-OH-E+), the application of bortezomib resulted in a 2-fold increase in the level of 2-OH-E+. Therefore, bortezomib sensitizes the detection of superoxide in both cell-based and cell-free systems, highlighting a novel feature of compounds containing the peptide boronic acid as powerful enhancers for the detection of superoxide.

The pharmaceutical potential of AR‐65, a novel Alzheimer’s disease drug targeting the immunoproteasome

AbstractBackgroundPreviously, we reported that inhibition of the immunoproteasome (iP), which is upregulated in reactive glial cells near amyloid‐β (Aβ) deposits in the brains of patients with Alzheimer’s disease (AD), leads to improved cognitive function in an AD mouse model of Aβ amyloidosis (Tg2576) via suppression of microglia‐mediated inflammation, independent of Aβ accumulation.MethodHere, we investigated the pharmaceutical properties of our lead iP inhibitor (AR‐65), a macrocyclic peptide epoxyketone, selected based on in vitro and cellulo iP inhibitory activities and cell‐based anti‐inflammatory efficacy. Specifically, the metabolic stability of AR‐65 was assessed using pooled human liver microsomes. In addition, the flux rate of AR‐65 was measured to evaluate the permeability across Caco‐2 monolayers over‐expressing ABCB1 and ABCG2, two major efflux drug transporters present in the human brain.ResultThe macrocyclic AR‐65 displayed improved metabolic stability (6‐fold increased half‐life), permeability, and solubility (in an injectable pharmaceutical buffer system with no cyclodextrin) compared to the FDA‐approved linear peptide epoxyketone proteasome inhibitor, carfilzomib (Kyprolis®). Finally, pilot PK studies performed using ICR mice confirmed that AR‐65 is metabolically more stable than carfilzomib and can cross the blood‐brain barrier to inhibit the brain iP.ConclusionTaken together, the results suggest that AR‐65 may hold great potential to be further investigated in clinical settings.

Addition of carfilzomib as a third agent in lenalidomide-refractory multiple myeloma: Switching from doublet to triplet.

e20002 Background: Carfilzomib is an epoxyketone proteasome inhibitor of second generation, proved to be effective and safe in relapsed and refractory Multiple Myeloma (rrMM), in combination with dexamethasone or lenalidomide and dexamethasone. In this retrospective observational trial, it has been evaluated efficacy and safety of carfilzomib, in combination with lenalidomide-dexamethasone (KRD) as salvage regimen in patients with rrMM, refractory to lenalidomide, where lenalidomide-based regimens have no proven efficacy. Methods: 41 patients (23 M/18 F), with rrMM, median age at diagnosis 63.7 years (r. 43-82), median age at start of treatment 67 years (r. 48-84) previously treated with several lines of treatments (median 3, r. 2-11), underwent to KRD regimen (ASPIRE trial schedule) for a median treatment cycles of 8 (r 2-18). ISS was equally distributed, and all patients had previously been treated with bortezomib and IMIDs, and were refractory to this agents. 61% (19/31) of them had undergone at least to a single ASCT. Results: According to IMWG criteria, after a median follow-up of 9 months (r. 2-18), ORR was 68,2% (28/41: 9 CR, 12 VGPR, 7 PR) with 5 progressive diseases (PD) and 8 patients in stable disease (SD): this can be considered as an impressive result in this subset of rrMM patients, refractory to lenalidomide. In particular, for 11 patients, KRD was, after having achieved at least a PR, a bridge to second/third autologous SCT. Median time to response was 1.3 months (r.1-4), median OS from diagnosis was 62 months (r. 9-170), median OS from start of Carfilzomib was 11 months (r. 2-18). Carfilzomib was well tolerated, with grade 2 anemia in 39%(16/41) of patients, successfully managed by ESAs, without necessity of blood transfusions; 29% (12/41) grade 3-4 neutropenia (pegfilgrastim in primary prophylaxis was given, no ospedalization was required, no septic shocks were observed); 34% (14/41) grade 2, 21% (9/41) grade 3 and 12% (5/41) grade 4 thrombocytopenia, without hemorrhagic events and transfusion-dependency. Moreover, it was observed pneumonia in 39% (16/41) of patients, treated by common antibiotic drugs and always solved. A cardiac monitoring was performed for all patients: hypertension (grade 2-3) in 34% (14/41) of patients; fatigue in 39% (16/31) of patients. Conclusions: Carfilzomib-Lenalidomide-Dexamethasone has shown significant efficacy in a particularly severe setting of patients, relapsed and refractory to all available therapeutic resources, also lenalidomide, and it could be considered as a bridge to a second autologous or allogenic SCT. Clinical trial information: A108.

Phase 2 Study of Carfilzomib and Bone Metabolism in Patients with Relapsed Multiple Myeloma

Background: Carfilzomib is an epoxyketone proteasome inhibitor (PI) that binds selectively and irreversibly to the constitutive proteasome and immunoproteasome. In addition to its direct cytotoxicity, PIs possess bone anabolic effect, by facilitating osteoblast differentiation, which in turn, inhibits multiple myeloma (MM) growth. However, the relative contribution of carfilzomib on direct tumor killing and bone formation has been difficult to discern when it is used in combination with other agents such as steroids, chemotherapeutics, or immunomodulatory agents (iMiDs); most of which increase bone loss.Objective: We explored the effect of single agent carfilzomib on bone metabolism and disease response in patients with relapsed MM.Methods: We conducted a phase II study of single-agent carfilzomib at 56 mg/m2 day 1, 2, 8, 9, 15, 16 of a 28 day cycle in patients with relapsed MM. Serum markers of bone resorption including carboxyterminal cross-linked telopeptide of type I collagen (ICTP) and tartrate resistant acid phosphatase 5b (TRACP 5b) and bone formation markers including N-terminal type 1 procollagen (P1NP) and osteocalcin (OC) were analyzed at baseline and 6 months after therapy, and compared using paired t-tests. Imaging using 18F-NaF PET-CT scans to monitor bone response and 8F-FDG PET-CT to measure localized tumor burden were done at baseline and 6 months post treatment. For each patient, the three sites with the highest baseline SUVs and an iliac crest (representing an uninvolved site) were selected for quantitative comparisons using paired t-test.Results: Ten patient were accrued. Average age was 70.1 +/- 6.79 (mean +/- SD) years. Median prior lines of therapy was 2. 9 were evaluable for response. Overall response rate was 77.8%: VGPR (1/9: 11%), partial response (6/9: 67%), stable disease (2/9: 22%). Median PFS was 11.5 months. Overall survival was 21.9 months. Common treatment adverse events (AE) were comparable to previous reports including diarrhea (40%), fever (40%), dyspnea (30%) and fatigue (30%). Grade 3/4 AEs, occurring in 10% each included neutropenia, thrombocytopenia, dyspnea, fatigue, vomiting, ataxia, heart failure and pulmonary edema.Serum ICTP and TRACP were elevated at baseline in 80% of subjects; means (+/- SD) of serum ICTP (14.4 +/- 3.1 ug/L) and TRACP (8.2 +/- 4.2 U/L), respectively. Both markers declined significantly after therapy (means +/- SD of serum ICTP 8.4 +/- 3.1 ug/L and TRACP (5.2 +/- 4.2 U/L); p<0.01), suggesting osteoclast suppression. Serum PINP and OC varied among subjects; 5 patients with low levels and 4 normal levels and 1 with elevated level: mean (+/- SD) of PINP 25 +/- 23 ug/L and OC (18 +/- 12 ug/L). Neither post treatment mean PINP nor OC differed significantly from baseline overall (p=0.16). However, all subjects who achieve PR or better had a more than 25% increase in PINP and OC, suggesting increased new bone formation.8F-FDG and 18F-NaF were positive in all subjects. Number of active sites and SUVs vary largely among subjects. 18F-NaF images mirrored the 8F-FDG signals, appearing as rings around the tumor sites, suggesting a bone reaction to the localized tumors. 6 of 9 subjects had post treatment PET/CTs for paired comparison. Post treatment 8F-FDG SUV of representative sites decreased significantly, while 18F-NaF SUVs varied; 58% of lesions had a decline in SUV, while 32% were unchanged and 10% were brighter, suggesting heterogeneous bone response. 18F-NaF SUVs of iliac sites decreased post treatment, suggesting a lower bone turnover overall. CT scans showed increased sclerosis at the rim of lytic lesions without significant change in lesion size, at this early time point.Conclusion: Carfilzomib at 56 mg/m2 dosing strongly inhibits bone resorption and demonstrates bone anabolic effects, in addition to cytotoxicity, in relapsed MM. Single agent use allows bone response analysis without the confounding effects of other anti-MM agents used in combination therapy. Response rates and side effects are favorable to prior reports. Serum markers of bone remodeling are useful for an assessment of overall bone metabolism while the 18F-NaF PET/CT allows assessment of both local and systemic changes but lacks specificity. Studies of osteoblast differentiation from subject's bone marrow stromal cells pre and post treatment are ongoing. Whether carfilzomib can heal bone lesions requires longer follow up. DisclosuresSuvannasankha:Celgene: Research Funding; Bristol-Myers Squibb: Research Funding; Takeda: Research Funding; Janssen Pharmaceuticals: Research Funding. Abonour:Celgene: Other: Steering committee, Research Funding; Prothena: Research Funding; Takeda: Other: Steering committee, Research Funding. Roodman:PharmaMar: Research Funding.

An Open-Label Phase I/II Study of Carfilzomib, Bendamustine and Dexamethasone (CBD) in Newly Diagnosed Multiple Myeloma Patients

BackgroundCarfilzomib is an irreversible epoxyketone proteasome inhibitor which has demonstrated superior disease control and PFS for myeloma compared to bortezomib. Bendamustine is a bifunctional alkylating agent with demonstrated activity in relapsed/refractory myeloma. We hypothesized that the combination of carfilzomib and bendamustine may result in additive and synergistic effects, without excessive toxicity. In this phase I/II study, we investigate the safety and efficacy of carfilzomib, bendamustine and dexamethasone (CBD) for newly diagnosed myeloma.ObjectivesThe primary objective is to determine the maximum tolerated dose (MTD) of carfilzomib in combination with bendamustine and dexamethasone. Secondary endpoints include overall response rate (ORR), duration of response (DOR), progression free survival (PFS), overall survival (OS), and assessment of stem cell collection.MethodsPatients with newly diagnosed myeloma were enrolled. Carfilzomib was administered intravenously on days 1, 2, 8, 9, 15, 16 of a 28-day cycle. Bendamustine was given intravenously on days 1 and 2. Dexamethasone was administered orally or intravenously on days of carfilzomib and on day 22 of each cycle. The dose escalation portion of the study used an Up-and-Down dose escalation scheme (Storer's schema D), with one patient per dose level run in. Transplant eligible patients received 4 cycles of treatment and then underwent peripheral blood stem cell harvest (SCH) after mobilization with granulocyte-stimulating factor +/- plerixafor. They then received an additional 4 cycles and underwent stem cell transplant (SCT) after 8 total cycles of CBD. Transplant ineligible patients also received 8 total cycles of treatment. Both groups then received maintenance therapy with carfilzomib 36 mg/m2 on days 1, 2, 15, 16 of each 28-day cycle for up to 2 years. Adverse events were assessed according to CTCAE V4 and responses were assessed using IMWG criteria.ResultsAt 38 months into the study, 15 patients have been treated, amongst which 14 were evaluable for response. Median age was 65 (range: 51-74); 10/15 (67%) were male; 6 (40%) were Hispanic, 6 Caucasian, 2 (13%) Asian and 1 (7%) African American. Four patients were allocated to lower level doses and 11 patients thus far have been given the maximum planned dose - carfilzomib 56 mg/m2, bendamustine 90 mg/m2, dexamethasone 20 mg. No dose-limiting toxicities have been noted and the most common adverse events (AEs) are shown in Table 1. Hematologic AEs were most common, occurring in 9 (64%) patients, and Grade 3 or 4 events occurring in 7 (50%). Other notable AEs included Grade 1 or 2 fever (50%), Grade 1 or 2 headache (43%) and thromboembolic events (29%). There were no deaths. No congestive heart failure or arrhythmias were noted. 11 patients underwent stem cell collection, with data available for 10. Median total stem cell yield, in million CD34/kg, was 6.85 (range 5.44-13.83). Median day 1 stem cell yield was 4.43 (range 1.28-13.83). 5 patients received plerixafor. 5 patients had 2 days of collection and 1 had a third.ResponseThe ORR was 14/14 (100%). 10 (71%) had a complete response (CR) / very good partial response (VGPR): 1 (7%) MRD-negative CR by flow, 1 stringent CR, 2 (14%) CR / near CR, 6 (43%) VGPR. 4 (29%) had partial response (PR). 3 of the 6 patients with VGPR and all 4 patients with PR are still on induction therapy and anticipated to have deeper responses. 3 of 4 patients with ISS stage III disease had CR with one being MRD-negative by flow. By time of SCH (after 4 cycles), 1/11 (9%) had attained CR, 7 (64%) VGPR and 3 (27%) PR. To date, 4 patients underwent SCT, and all have attained CR with 1 being MRD-negative by flow. Amongst other patients who underwent SCH, 3 were deemed ineligible for SCT, 1 declined, and 4 are eligible. Median time to best response was 3 months (range 1-11) amongst patients whose response has plateaued, and median time to CR was 4 months (range 1-11). Median follow-up was 8.8 months (range 2-37.3). To date, only 1 patient has progressed, at 18 months after start of therapy, and median PFS has not been reached. No patients have died.ConclusionsCurrent preliminary evidence suggests that CBD is a potent induction regimen. CBD does not appear to impact stem cell collection, and toxicities were acceptable, although increased attention to thromboembolic events is warranted. Further evaluation of this regimen at our maximum dose level is planned. [Display omitted] DisclosuresLentzsch:Caelum Biosciences: Other: leadership position and stock; BMS: Consultancy; Amgen: Consultancy.

Carfilzomib-Lenalidomide-Dexamethasone in the Management of Lenalidomide-Refractory Multiple Myeloma

Background Carfilzomib is an epoxyketone proteasome inhibitor of second generation, proved to be effective and safe in relapsed and refractory Multiple Myeloma (rrMM), in combination with dexamethasone or lenalidomide and dexamethasone. Aims In this retrospective observational trial, it has been evaluated efficacy and safety of carfilzomib, in combination with lenalidomide-dexamethasone (KRD) as salvage regimen in patients with rrMM, refractory to lenalidomide, where lenalidomide-based regimens have no proven efficacy. Methods 41 patients (23 M/18 F), with rrMM, median age at diagnosis 63.7 years (r. 43-82), median age at start of treatment 67 years (r. 48-84) previously treated with several lines of treatments (median 3, r. 2-11), underwent to KRD regimen (ASPIRE trial schedule) for a median treatment cycles of 8 (r 2-18). ISS was equally distributed, and all patients had previously been treated with bortezomib and IMIDs, and were refractory to this agents. 61% (19/31) of them had undergone at least to a single ASCT. Results According to IMWG criteria, after a median follow-up of 9 months (r. 2-18), ORR was 68,2% (28/41: 9 CR, 12 VGPR, 7 PR) with 5 progressive diseases (PD) and 8 patients in stable disease (SD): this can be considered as an impressive result in this subset of rrMM patients, refractory to lenalidomide. In particular, for 11 patients, KRD was, after having achieved at least a PR, a bridge to second/third autologous SCT. Median time to response was 1.3 months (r.1-4), median OS from diagnosis was 62 months (r. 9-170), median OS from start of Carfilzomib was 11 months (r. 2-18). Carfilzomib was well tolerated, with grade 2 anemia in 39%(16/41) of patients, successfully managed by ESAs, without necessity of blood transfusions; 29% (12/41) grade 3-4 neutropenia (pegfilgrastim in primary prophylaxis was given, no ospedalization was required, no septic shocks were observed); 34% (14/41) grade 2, 21% (9/41) grade 3 and 12% (5/41) grade 4 thrombocytopenia, without hemorrhagic events and transfusion-dependency. Moreover, it was observed pneumonia in 39% (16/41) of patients, treated by common antibiotic drugs and always solved. A cardiac monitoring was performed for all patients: hypertension (grade 2-3) in 34% (14/41) of patients; fatigue in 39% (16/31) of patients. Conclusions Carfilzomib-Lenalidomide-Dexamethasone has shown significant efficacy in a particularly severe setting of patients, relapsed and refractory to all available therapeutic resources, also lenalidomide, and it could be considered as a bridge to a second autologous or allogenic SCT. Disclosures Lucchesi: Incyte: Honoraria; Novartis: Honoraria; Pfizer: Honoraria. Martinelli:Amgen: Consultancy; Celgene: Consultancy, Speakers Bureau; Jazz: Consultancy; Incyte: Consultancy; Pfizer: Consultancy, Research Funding, Speakers Bureau; Roche: Consultancy; Daichii Sankyo: Consultancy, Research Funding; AbbVie: Consultancy, Research Funding; Janssen: Consultancy.

MM-385: Carfilzomib-Lenalidomide-Dexamethasone in the Management of Lenalidomide-Refractory Multiple Myeloma

Background Carfilzomib is an epoxyketone proteasome inhibitor of second generation, proved to be effective and safe in relapsed and refractory Multiple Myeloma (rrMM), in combination with dexamethasone or lenalidomide and dexamethasone. Aims In this retrospective observational trial, it has been evaluated efficacy and safety of carfilzomib, in combination with lenalidomide-dexamethasone (KRD) as salvage regimen in patients with rrMM, refractory to lenalidomide, where lenalidomide-based regimens have no proven efficacy. Methods 41 patients (23 M/18 F), with rrMM, median age at diagnosis 63.7 years (r. 43-82), median age at start of treatment 67 years (r. 48-84) previously treated with several lines of treatments (median 3, r. 2-11), underwent to KRD regimen (ASPIRE trial schedule) for a median treatment cycles of 8 (r 2-18). ISS was equally distributed, and all patients had previously been treated with bortezomib and IMIDs, and were refractory to this agents. 61% (19/31) of them had undergone at least to a single ASCT. Results According to IMWG criteria, after a median follow-up of 9 months (r. 2-18), ORR was 68,2% (28/41: 9 CR, 12 VGPR, 7 PR) with 5 progressive diseases (PD) and 8 patients in stable disease (SD): this can be considered as an impressive result in this subset of rrMM patients, refractory to lenalidomide. In particular, for 11 patients, KRD was, after having achieved at least a PR, a bridge to second/third autologous SCT. Median time to response was 1.3 months (r.1-4), median OS from diagnosis was 62 months (r. 9-170), median OS from start of Carfilzomib was 11 months (r. 2-18). Carfilzomib was well tolerated, with grade 2 anemia in 39%(16/41) of patients, successfully managed by ESAs, without necessity of blood transfusions; 29% (12/41) grade 3-4 neutropenia (pegfilgrastim in primary prophylaxis was given, no ospedalization was required, no septic shocks were observed); 34% (14/41) grade 2, 21% (9/41) grade 3 and 12% (5/41) grade 4 thrombocytopenia, without hemorrhagic events and transfusion-dependency. Moreover, it was observed pneumonia in 39% (16/41) of patients, treated by common antibiotic drugs and always solved. A cardiac monitoring was performed for all patients: hypertension (grade 2-3) in 34% (14/41) of patients; fatigue in 39% (16/31) of patients. Conclusions Carfilzomib-Lenalidomide-Dexamethasone has shown significant efficacy in a particularly severe setting of patients, relapsed and refractory to all available therapeutic resources, also lenalidomide, and it could be considered as a bridge to a second autologous or allogenic SCT. Disclosures Lucchesi: Incyte: Honoraria; Novartis: Honoraria; Pfizer: Honoraria. Martinelli: Amgen: Consultancy; Celgene: Consultancy, Speakers Bureau; Jazz: Consultancy; Incyte: Consultancy; Pfizer: Consultancy, Research Funding, Speakers Bureau; Roche: Consultancy; Daichii Sankyo: Consultancy, Research Funding; AbbVie: Consultancy, Research Funding; Janssen: Consultancy.

Carfilzomib-Lenalidomide-Dexamethasone in the Management of Lenalidomide-Refractory Multiple Myeloma

Carfilzomib is an epoxyketone proteasome inhibitor of second generation, proved to be effective and safe in relapsed and refractory Multiple Myeloma (rrMM), in combination with dexamethasone or lenalidomide and dexamethasone. In this retrospective observational trial, it has been evaluated efficacy and safety of carfilzomib, in combination with lenalidomide-dexamethasone (KRD) as salvage regimen in patients with rrMM, refractory to lenalidomide, whose prognosis is particularly severe. 41 patients (23 M/18 F), with rrMM, median age at diagnosis 63.7 years (r.

Preparation and biological evaluation of soluble tetrapeptide epoxyketone proteasome inhibitors

A series of novel tetrapeptidyl epoxyketone inhibitors of 20S proteasome was designed and synthesized. To fully understand the SAR, various groups at R1, R2, R3, R4 and R5 positions, including aromatic and aliphatic substituents were designed, synthesized and biologically assayed. Based on the enzymatic results, seven compounds were selected to evaluate their cellular activities and soluble compound 36 showed strong potency against human multiple myeloma (MM) cell lines. Microsomal stability results indicated that compound 36 was more stable in mice, rat and human microsomes than marketed carfilzomib. The in vivo activities of this compound were evaluated with the xenograft mice models of MM cell lines ARH77 and RPMI-8226 with luciferase expression and the T/C value of the two models were 49.5% and 37.6%, respectively. To evaluate the potential cardiovascular toxicity, inhibition of hERG ion channel in HEK293 cells by compound 36 and carfilzomib was carried out. The results indicated that 36 had no binding affinity for the hERG ion channel while carfilzomib could bind it with IC50 of 92.1 μM.

PB2163 CARFILZOMIB, LENALIDOMIDE AND DEXAMETHASONE “ON DEMAND” IN RELAPSED/REFRACTORY MULTIPLE MYELOMA: REAL LIFE EXPERIENCE AND NEW PERSPECTIVES ON DOSE‐DENSE CHEMOTHERAPY

Background:The natural history of Multiple Myeloma (MM) is characterized by improved survival rates but relapses are still common, thus new therapeutic approaches are still needed.Carfilzomib (K) is a selective and irreversible epoxyketone proteasome inhibitor approved in combination with dexamethasone (d) and lenalidomide (R) for the treatment of patients with relapsed‐refractory MM (1–3 previous lines of therapy) and as a single agent for the treatment of patients with relapsed or refractory MM (1 or more previous lines of therapy).Aims:The aim of this study is to describe the real life of our patients treated with KRd and to give new perspectives on dose‐dense chemotherapy.MethodsData from patients with relapsed/refractory MM treated with KRD in the Federico II University of Naples, were retrospectively analyzed.Response was evaluated according to International Myeloma Working Group Uniform Response Criteria. Disease assessments were performed on day 1 of each cycle. In patients that had obtained a PR, VGPR, CR in at least 3 consecutive measurements carried out at monthly intervals during treatment the therapy was discontinued and the patients were reassessed every 30 days. The same treatment was reintroduced when disease progressed according to clinical and/or biochemical parameters. Finally, a different line of therapy was employed when signs of disease resistance to KRd appeared.KRd treated patients were than divided into three cohorts as per chemotherapy dose‐density: in the first group patients received ≤50% of the expected cycles (low‐dose); in the second group patients received a dose comprised between 50% and 75% of the expected cycles (int‐dose); in the third group ≥75% of the expected cycles (high‐dose) was employed.The primary endpoint was the time to next treatment (TTNT) for the entire population and among the three dose dense cohorts.Secondary endpoint was the overall response rate (ORR).Results:From January 2016 to January 2019 forty‐two patients with a diagnosis of MM treated with KRD combination, relapsed after a previous therapy (1‐7) including bortezomib and Immunomodulatory Drugs (IMiDS), were enrolled. Median age was 63.2 years; Performance status was &gt;2 in the 40% patients. Patients with high cytogenetic risk were the ones treated with more than two previous chemotherapy lines. All patients with more than two previous lines had received IMIDs, while these drugs weren’t employed in the 40% of those previously treated with only one line. Bortezomib was employed in almost all patients. TTNT was 13 (range 1‐30) months for the entire cohort while the median TTNT for low‐dose cohort was not reached; the int‐dose and the high‐dose cohort had a median TTNT of 13 and 5 months respectively (figure). The ORR of the entire cohort was 95%.Summary/Conclusion:This retrospective analysis indicates that KRd improved the outcome of patients in first relapse, of patients with ≥ 2 previous lines of therapy and of those refractory to bortezomib and IMiDS.imageAlthough therapies for relapsed MM are licensed as continuous treatment until progression, our data suggest that less dose‐density appears to have the same efficacy of continuous treatments but with less toxicity and significant economic savings.

Carfilzomib-lenalidomide-dexamethasone in the management of lenalidomide-refractory multiple myeloma.

e19522 Background: Carfilzomib is an epoxyketone proteasome inhibitor of second generation, proved to be effective and safe in relapsed and refractory Multiple Myeloma (rrMM), in combination with dexamethasone or lenalidomide and dexamethasone. In this retrospective observational trial, it has been evaluated efficacy and safety of carfilzomib, in combination with lenalidomide-dexamethasone (KRD) as salvage regimen in patients with rrMM, refractory to lenalidomide, whose prognosis is particularly severe. Methods: 41 patients (23 M/18 F), with rrMM, median age at diagnosis 63.7 years (r. 43-82), median age at start of treatment 67 years (r. 48-84) previously treated with several lines of treatments (median 3, r. 2-11), underwent to KRD regimen (ASPIRE trial schedule) for a median treatment cycles of 8 (r 2-18). ISS was equally distributed, and all patients had previously been treated with bortezomib and IMIDs, and were refractory to this agents. 61% (19/31) of them had undergone at least to a single ASCT. Results: According to IMWG criteria, after a median follow-up of 9 months (r. 2-18), ORR was 68,2% (28/41: 9 CR, 12 VGPR, 7 PR) with 5 progressive diseases (PD) and 8 patients in stable disease (SD): this can be considered as an impressive result in this subset of rrMM patients, refractory to lenalidomide. In particular, for 11 patients, KRD was, after having achieved at least a PR, a bridge to second/third autologous SCT. Median time to response was 1.3 months (r.1-4), median OS from diagnosis was 62 months (r. 9-170), median OS from start of Carfilzomib was 11 months (r. 2-18). Carfilzomib was well tolerated, with grade 2 anemia in 39% (16/41) of patients, successfully managed by ESAs, without necessity of blood transfusions; 29% (12/41) grade 3-4 neutropenia (pegfilgrastim in primary prophylaxis was given, no ospedalization was required, no septic shocks were observed); 34% (14/41) grade 2, 21% (9/41) grade 3 and 12% (5/41) grade 4 thrombocytopenia, without hemorrhagic events and transfusion-dependency. Moreover, it was observed pneumonia in 39% (16/41) of patients, treated by common antibiotic drugs and always solved. A cardiac monitoring was performed for all patients: hypertension (grade 2-3) in 34% (14/41) of patients; fatigue in 39% (16/31) of patients. Conclusions: Carfilzomib-Lenalidomide-Dexamethasone has shown significant efficacy in a particularly severe setting of patients, relapsed and refractory to all available therapeutic resources, also lenalidomide, and it could be considered as a bridge to a second autologous or allogenic SCT.

Covalent docking modelling-based discovery of tripeptidyl epoxyketone proteasome inhibitors composed of aliphatic-heterocycles

The potential of specific proteasome inhibitors to act as anti-cancer agents has attracted intensive investigations. The proteasome can be covalently inhibited by epoxyketone derivatives via a two-step reaction. Several computational approaches have been developed to mimic the covalent binding event. Compound 1 composed of a six-membered heterocyclic ring was designed by using covalent docking. With a possible different binding mode from the clinical compound Carfilzomib, it occupied the S5 pocket of 20S proteasome and showed favorable inhibitory activity. Subsequently optimization and evaluation were taken place. Among these compounds, 11h demonstrated extraordinary in vitro inhibitory activity and selectivity, and good in vivo proteasome inhibitory activity, a favorable pharmacokinetic profile and xenograft tumor inhibition. The possible binding pattern of compound 11h against proteasome was further fully explored via calculations, providing a theoretical basis for finding potent proteasome inhibitors.

Carfilzomib-Lenalidomide-Dexamethasone in the Management of Lenalidomide-Refractory Multiple Myeloma

Carfilzomib-Lenalidomide-Dexamethasone in heavily pretreated Multiple Myeloma, refractory to lenalidomide.

Novel Polymer Micelle and Nanocrystal Formulations for the Proteasome Inhibitor Drug Carfilzomib: Pharmacokinetic and Pharmacodynamic Studies in Human Lung and Breast Cancer Models

Carfilzomib (CFZ) is a peptide epoxyketone proteasome inhibitor with much improved efficacy and safety profiles over bortezomib and has become a mainstay of multiple myeloma (MM) therapy. Despite the promising preclinical data of CFZ against solid cancers, the results from clinical trials in patients with solid cancers were rather disappointing. Rapid degradation of CFZ and its poor penetration to solid tumor sites have been considered to be the main culprits for the lack of efficacy. Thus, we explored nanoparticle‐based systems, in particular, polymer micelle and nanocrystal formulations, as new CFZ delivery strategies for the treatment of human lung and breast cancer. We first developed CFZ‐loaded polymer micelle (CFZ‐PM) composed of block copolymers poly‐(ethylene glycol) and poly‐(caprolactone). While CFZ‐PM improved the metabolic stability of CFZ in vitro, CFZ‐PM did not improve in vivo performances (in terms of anticancer efficacy and pharmacokinetic profiles) over the clinically used cyclodextrin‐based CFZ formulation. The proteasome activities in excised human lung cancer xenograft tissues from mice treated with CFZ‐PM displayed an incomplete inhibition, suggesting that CFZ‐PM did not effectively enhance the penetration of active CFZ to solid cancer tissues. As an alternative strategy to further improve in vivo stability and active targeting of CFZ to solid cancer cells, we designed albumin‐coated nanocrystal formulations containing CFZ (Alb‐CFZ‐NC). With optimized processing methods and surface stabilizers, Alb‐CFZ‐NC displayed the average particle size of ~260 nm and ~70% drug content as well as albumin conformation close to the native protein (with favorable uptake profiles by cancer cells). Ongoing investigations are focused on the assessment of Alb‐CFZ‐NC in terms of pharmacokinetic/biodistribution profiles and anticancer efficacy in human breast cancer models. Our current efforts may provide effective CFZ formulations to validate and harness the potential of CFZ‐based therapy for solid cancer patients.

Polymer micelle formulation for the proteasome inhibitor drug carfilzomib: Anticancer efficacy and pharmacokinetic studies in mice

Carfilzomib (CFZ) is a peptide epoxyketone proteasome inhibitor approved for the treatment of multiple myeloma (MM). Despite the remarkable efficacy of CFZ against MM, the clinical trials in patients with solid cancers yielded rather disappointing results with minimal clinical benefits. Rapid degradation of CFZ in vivo and its poor penetration to tumor sites are considered to be major factors limiting its efficacy against solid cancers. We previously reported that polymer micelles (PMs) composed of biodegradable block copolymers poly(ethylene glycol) (PEG) and poly(caprolactone) (PCL) can improve the metabolic stability of CFZ in vitro. Here, we prepared the CFZ-loaded PM, PEG-PCL-deoxycholic acid (CFZ-PM) and assessed its in vivo anticancer efficacy and pharmacokinetic profiles. Despite in vitro metabolic protection of CFZ, CFZ-PM did not display in vivo anticancer efficacy in mice bearing human lung cancer xenograft (H460) superior to that of the clinically used cyclodextrin-based CFZ (CFZ-CD) formulation. The plasma pharmacokinetic profiles of CFZ-PM were also comparable to those of CFZ-CD and the residual tumors that persisted in xenograft mice receiving CFZ-PM displayed an incomplete proteasome inhibition. In summary, our results showed that despite its favorable in vitro performances, the current CFZ-PM formulation did not improve in vivo anticancer efficacy and accessibility of active CFZ to solid cancer tissues over CFZ-CD. Careful consideration of the current results and potential confounding factors may provide valuable insights into the future efforts to validate the potential of CFZ-based therapy for solid cancer and to develop effective CFZ delivery strategies that can be used to treat solid cancers.

An Open-Label, Single-Arm, Phase 1 Study of the Pharmacokinetics (PK) and Safety of Carfilzomib in Patients with Relapsed Multiple Myeloma and End-Stage Renal Disease (ESRD)

An Open-Label, Single-Arm, Phase 1 Study of the Pharmacokinetics (PK) and Safety of Carfilzomib in Patients with Relapsed Multiple Myeloma and End-Stage Renal Disease (ESRD)