Administration Of Carfilzomib Research Articles

Abstract LB-95: Estrogen receptor mRNA-directed therapy for triple-negative breast cancer

Abstract Introduction: A significant number of histologically triple negative breast cancer (TNBC) cells express substantial estrogen receptor (ER) mRNA but do not express ER protein as a result of rapid proteolytic degradation. Here we assessed whether proteasome inhibitors could sensitize such cells to tamoxifen-induced growth suppression. Methods: Data from the Katzenellenbogen laboratory elegantly demonstrate that in high ER mRNA MDA-MB-468 cells, ER protein is rapidly degraded via proteosomal degradation (Bhatt et al. MCB, 2012).Utilizing established human TNBC cell lines that express high ER mRNA (BT-20, MDA-MB-468, and newly established human TNBC cell lines from our laboratory MCJB2 and MCJB11), we defined a therapeutic strategy using the proteasome inhibitor carfilzomib in combination with the antiestrogen tamoxifen that demonstrates antitumor synergy. Results: We found that administration of carfilzomib in TNBC cell lines with high ER mRNA restored ER protein expression, and that combining this treatment with tamoxifen treatment led to enhanced growth suppression. We found that the TNBC MDA-MB-231 cells, which express no ER mRNA, show some growth suppression, but that tamoxifen provided no further growth suppression with these cells. We have previously shown that combined epigenetic therapy [methyltransferase inhibitor (decitabine) plus a histone deacetylase inhibitor (HDACi; romidepsin)] provided antitumor synergy in MDA-MB-231 cells via upregulation of the soluble Wnt suppressor, secreted frizzled protein 1 (sFRP1; Cooper et al. CCR, 2012). In addition, we show that this epigenetic therapy restores silenced ER mRNA and protein levels in MDA-MB-231 cells, and that combination of this epigenetic therapy with tamoxifen in MDA-MB-231 cells led to enhanced growth suppression by tamoxifen. We are currently testing these therapeutic strategies in vivo using established TNBC cell lines and our newly developed patient derived xenograft (PDX) mouse models that express either ERhigh or ERlow mRNA. Conclusions: If validated in animal models, we propose that ER mRNA expression level in TNBC patients could guide therapeutic choice, whereby, ERlow mRNA TNBC tumors could respond to epigenetic therapy followed by antiestrogen while ERhigh mRNA TNBC tumors could respond to a proteosome inhibitor followed by antiestrogen therapy. These results identify a novel epigenetic biomarker as predictive of therapeutic response. Citation Format: Christina A. von Roemeling, Laura A. Marlow, James Miller, Adam Mathias, Louis Dawson, Steadman Harrison, Beth A. Hollister, Derek C. Radisky, John Alton Copland. Estrogen receptor mRNA-directed therapy for triple-negative breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-95. doi:10.1158/1538-7445.AM2014-LB-95

Carfilzomib‐Associated Tumor Lysis Syndrome

Multiple myeloma is the second most common type of hematologic malignancy. It is a B-cell malignancy that affects the bone marrow and often results in thrombocytopenia as well as renal dysfunction. Treatment options range from oral and intravenous chemotherapy to bone marrow transplantation and supportive care. Carfilzomib was approved by the U.S. Food and Drug Administration in 2012 as a treatment option for patients with refractory multiple myeloma who have received at least two previous therapies and have demonstrated recent disease progression. According to the product labeling, the frequency of tumor lysis syndrome (TLS) is less than 1% in patients treated with carfilzomib. To our knowledge, no postmarketing events of TLS have been reported or published. We describe a 55-year-old man with relapsed multiple myeloma who developed a case of TLS that occurred after he received his first two doses of carfilzomib therapy on days 1 and 2; he also had chronic kidney disease secondary to his neoplastic disease. Beginning on day 4, his uric acid levels spiked to critical levels, prompting the use of rasburicase, which returned the levels to within normal limits. His phosphorus and creatinine levels increased during days 5 and 6. On day 8, the patient died, likely due to a combination of disease progression and the adverse effects of treatment. Use of the Naranjo adverse drug reaction probability scale indicated a probable relationship (score of 6) between the patient's development of TLS and carfilzomib therapy. The Hill criteria were used as a secondary measure to ensure causality, which also suggested a link between the patient's development of TLS and the administration of carfilzomib. This case report shows that even the most unlikely of adverse events may occur with medications, especially in the case of a new or recently approved medication. Caution must be taken when deciding to treat and when choosing hydration and premedications with regard to biologic and chemotherapeutic medications. In this case, additional hydration may have been considered. Although given the extent of the adverse reaction combined with the patient's underlying renal dysfunction, extra fluid may or may not have proven beneficial. The use of prophylactic rasburicase or allopurinol could have been considered, but these measures are not typically used with multiple myeloma due to the low incidence of TLS. All things considered, this unlikely adverse reaction may occur in certain patients. If other cases such as this occur, it may be advisable to use TLS prophylaxis in the future in certain patient populations, including those with renal dysfunction or worsening disease states.

Cell Non-Autonomous Modulation Of Tumor Cell Responses To Pharmacological Inhibition Of The Proteasome

Extensive preclinical studies of several groups using tumor cells co-cultured with bone marrow stromal cells (BMSCs) has documented that the potent anti-MM activity of the proteasome inhibitor bortezomib is not suppressed by BMSCs (e.g. primary and immortalized BMSCs). Using our compartment-specific bioluminescence imaging (CS-BLI) assays, we extended these observations to larger panels of MM cell lines. We observed, however, a recurrent pattern that primary CD138+ MM tumor cells from bortezomib-refractory patients recurrently exhibited substantial in vitro response to clinically-achievable concentrations and durations of bortezomib treatment. To simulate this clinicopathological observation, MM.1R-Luc+ cells were injected i.v. in SCID-beige mice and treated with bortezomib (0.5 mg/kg s.c. twice weekly for 5 weeks): diffuse MM tumors initially responded to bortezomib, but eventually became refractory. These in vivo-resistant MM cells were isolated from the mice and were treated in vitro with bortezomib, exhibiteing similar responsinveness to this agent as their isogenic bortezomib-naive MM cells, To further address the possibility that this represents a previously underexplored form of a microenvironment-induced alteration in bortezomib responsiveness, we studied how MM cells respond to pharmacological proteasome inhibition after variable times of co-culture with BMSCs prior to bortezomib exposure. We observed that prolonged tumor-stromal co-culture (48-96hrs) prior to initiation of bortezomib treatment did not affect drug sensitivity for many of the MM cell lines (OPM2, H929, UM9, KMS11, KMS18 and RPMI-8226) tested. Notably, prolonged co-cultures with BMSCs prior to bortezomib treatment enhanced the activity of this agent for other MM cell lines (e.g. OPM1, Dox40, OCI-My5, KMS12BM or KMS18). However, MM.1S and MM.1R cells, when exposed to extended co-cultures with BMSCs prior to initiation of drug exposure, exhibited significant attenuation (2-3 fold increase of IC50 values) of their response to bortezomib in several independent replicate experiments. In support of these in vitro results, heterotypic s.c. xenografts of Luc+ MM.1S cells co-implanted with Luc-negative BMSCs did not show significant reduction in MM tumor growth with bortezomib treatment (0.5 mg/kg s.c. twice weekly for 5 weeks) compared to vehicle-treated controls (p=0.13), as quantified by bioluminescence imaging. In co-cultures with BMSCs, MM.1S and MM.1R cells also exhibited suppression of their response to carfilzomib (the degree of this stroma-induced resistance was more pronounced that in the case of bortezomib for these 2 cell lines). Consistent with these observations, in vivo administration of carfilzomib in the orthotopic model of diffuse bone lesions of MM.1R-Luc+ cells was associated with less pronounced reduction in tumor growth, compared to bortezomib treatment (p<0.03). These results suggest that the stroma-induced attenuation of activity against a subset of MM cells represents a class-effect for this group of therapeutics, despite quantitative differences between different proteasome inhibitors. Mechanistically, we determined a distinct transcriptional signature of stroma-induced transcripts which are overexpressed in refractory myeloma patients with significantly shorter overall survival (p<0.03, log-rank tests) after bortezomib treatment. Our results in vitro and in vivo support the notion that the responses of MM cells to proteasome inhibition can exhibit substantial plasticity depending on the specific microenvironmental context with which these MM cells interact. We also identify prognostically-relevant candidate molecular mediators of stroma-induced resistance to proteasome-inhibitor based therapy in MM. Disclosures: No relevant conflicts of interest to declare.

Retrospective analysis of cardiovascular (CV) events following compassionate use of carfilzomib (CFZ) in patients (Pts) with relapsed and refractory multiple myeloma (RRMM).

8595 Background: Herein, we report CV events observed in late-stage, heavily pretreated pts who received salvage CFZ under a compassionate use protocol. Methods: Pts received IV single-agent CFZ (20-45 mg/m2) over 2-30 min on days 1, 2, 8, 9, 15, and 16 of a 28-day cycle (C) or CFZ with weekly dexamethasone (4-40 mg) during C1, along with other agents (eg, lenalidomide, thalidomide, cyclophosphamide, doxorubicin, cisplatin, vorinostat) during C2 and beyond. CV events were grouped according to preferred terms. Serious CV events were defined as those that required hospitalization. B-type natriuretic peptide (BNP) values were measured at baseline; peak values were recorded during C1. Results: 143 pts (median age, 61 years old; 70% were male) received treatment. Prior to enrollment, pts received a median of 7 prior lines of therapy (range, 2-15), including doxorubicin (range, 0-360 mg/m2). Most pts (92%) had prior autologous stem cell transplants (ASCT), with 74% receiving ≥2 ASCT (range, 0-5). Pts received CFZ for a median of 2C (range, 1-36). Of pts with available BNP values at baseline and during C1 (n=69; 48.3%), the median peak BNP increase from baseline was 407 pg/ml (P<0.001). 27 pts (18.9%) experienced a serious CV event, 21 (77.7%) of which had preexisting CV risk factors. Of these 27 pts, 11 pts (7.7%) developed CHF or worsening of existing CHF (confirmed by echocardiogram [ECHO] ≤1 month from diagnosis), and 13 (9.1%) required hospitalization for hypotension (n=6), arrhythmia (n=2), hypertension (n=2), pulmonary edema (n=1), pulmonary embolism (n=1), or pulmonary hypertension (n=1). Additionally, 3 pts (2.1%) experienced cardiopulmonary arrest. Of the 11 CHF pts, 10 had a baseline ECHO (recorded ≤6 months before study); left ventricular ejection fraction decreased from a median of 55% (pretreatment) to 33% (posttreatment). Conclusions: Late-line, heavily pretreated pts with RRMM occasionally experienced CV events following administration of CFZ with or without other antimyeloma agents. Given the number of confounding factors and the uncontrolled nature of these data, causality for these CV events could not be definitively determined.

Carfilzomib: a novel agent for multiple myeloma

Carfilzomib is a new agent for the treatment of relapsed and refractory multiple myeloma (MM). This article presents a comprehensive overview of the pharmacokinetics, pharmacodynamics, dosing schedule, safety, efficacy, preparation and administration of carfilzomib, and its role in treating MM patients. Carfilzomib is a selective proteasome inhibitor that differs structurally and mechanistically from bortezomib. In patients' whole-blood and peripheral-blood mononuclear cells, carfilzomib inhibited proteasomal and immunoproteasomal activity by 70-80%. Approved carfilzomib dosing is based on body surface area, and is given on days 1, 2, 8, 9, 15 and 16 of a 28-day cycle (20 mg/m(2) in cycle 1; 27 mg/m(2) in cycle 2+). Premedication with dexamethasone and adequate hydration are recommended to reduce the risk of adverse events. The median t1/2 of carfilzomib is short (0.29-0.48 h), with no accumulation detected between doses. In clinical studies in relapsed and refractory MM. and in combinations in newly diagnosed MM, single-agent carfilzomib demonstrated significant durable activity, good tolerability and a favourable safety profile, supporting its extended use. Carfilzomib represents an important addition to the treatment armamentarium for patients with relapsed and/or refractory MM, and studies are underway evaluating the role of single-agent carfilzomib in additional clinical settings as well as in different combinations.

Phase II Study of Infusional Carfilzomib in Patients with Relapsed or Refractory Multiple Myeloma

AbstractAbstract 947 Background:Carfilzomib (CFZ) is a selective, irreversible epoxyketone inhibitor of the chymotrypsin-like activity of both the constitutive proteasome and the immunoproteasome. In patients with multiple myeloma single-agent CFZ is active and well tolerated at doses up to 27 mg/m2 when administered intravenously over 2–10 minutes. Based on preclinical safety data showing that a slower infusion was better tolerated and permitted administration of higher doses than a 2–10 minute infusion, the phase1b/2 PX-171-007 (NCT00531284) study evaluated the administration of CFZ as a 30-minute infusion. That study found the maximally tolerated dose of CFZ given as a 30 minutes infusion to be 56mg/m2. We designed a single institution, phase 2 study of high dose, infusional CFZ in patients with relapsed and/or refractory multiple myeloma. Methods:CFZ was given as a 30-minute IV infusion on days 1, 2, 8, 9, 15, and 16 of a 28-day cycle. Cycle1 Day1–2 doses were 20 mg/m2, followed by escalation to 56mg/m2. Dexamethasone 8 mg was given prior to infusion as premedication to mitigate infusion-related reactions. Patients who did not achieve a partial response (PR) after two cycles of CFZ or initially responded to single agent CFZ, but later showed evidence of progression of disease (POD) had dexamethasone (40mg/week) added to their regimen. Overall response rate (ORR; [sCR + CR + VGPR + PR]) was determined according to International Myeloma Working Group Uniform Response Criteria. Subjects were evaluated for adverse events according to the Common Terminology Criteria for Adverse Events v 4.0. Results:Thirty-four patients have been enrolled. Patients had received a median of 5 (range 1–11) prior treatment regimens and included 7 patients who relapsed following allogeneic stem cell transplant. Seventy eight percent of patients were bortezomib-refractory. ORR among patients who completed 4 cycles of therapy or experienced POD prior to completing 4 cycles of therapy was 58% with 1 CR, 7 VGPRs, and 6 PRs. ORR after 4 cycles of therapy was 57% in bortezomib-refractory patients. By intention to treat analysis the ORR in all comers was 50%. Median progression free survival was 4.6 months, median overall survival has not been reached with a median follow-up among survivors of 9.6 months (range: 0.3–14.3 months). The average time to best response was two cycles. Three out of the 11 patients that had dexamethasone 40mg/week added to their regimen obtained an improved response. Twelve patients (35%) were dose reduced. Treatment emergent, non-hematologic Grade 3/4 adverse events for which contribution of CFZ cannot be excluded were: HTN (n = 7), lung infection (n = 6), pulmonary edema (n = 3), reduced ejection fraction (n=1), sepsis (n=2), febrile neutropenia (n=1), bacteremia (n = 1), protothecosis (n = 1), fatigue (n=1), neuropathy (n=1), microangiopathic hemolytic anemia(n=1), nausea/vomiting/+/− diarrhea (n = 2), gastrointestinal bleed in the setting of Grade 4 thrombocytopenia (n = 1), hyperkalemia (n = 1). Conclusions:The 20/56 mg/m2 dose for CFZ administered as a 30-minute IV infusion was associated with 58% ORR, in a heavily pretreated patient population, including 21% of patients w/ prior allogeneic transplant, where the majority of patients were bortezomib-refractory. The ORR from this study is consistent with the one previously reported at the similar dose. Higher doses of carfilzomib continue to be explored in ongoing Phase 2 and Phase 3 studies. Disclosures:Landau:Onyx: Research Funding; Milleneum: Membership on an entity's Board of Directors or advisory committees, Research Funding. Hassoun:Millenium: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Research Funding. Giralt:Onyx: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Milleneum: Membership on an entity's Board of Directors or advisory committees.

Clinical Pharmacokinetics, Metabolism, and Drug-Drug Interaction of Carfilzomib

Carfilzomib, an irreversible proteasome inhibitor, has a favorable safety profile and significant antitumor activity in patients with relapsed and refractory multiple myeloma (MM). Here we summarize the clinical pharmacokinetics (PK), metabolism, and drug-drug interaction (DDI) profile of carfilzomib. The PK of carfilzomib, infused over 2-10 minutes, was evaluated in patients with solid tumors or MM. Metabolites of carfilzomib were characterized in patient plasma and urine samples. In vitro drug metabolism and DDI studies were conducted in human liver microsomes and hepatocytes. A clinical DDI study was conducted in patients with solid tumors to evaluate the effect of carfilzomib on CYP3A activity. Plasma concentrations of carfilzomib declined rapidly and in a biphasic manner after intravenous administration. The systemic half-life was short and the systemic clearance rate was higher than hepatic blood flow. Carfilzomib was cleared largely extrahepatically via peptidase cleavage and epoxide hydrolysis. Cytochrome P450-mediated metabolism played a minor role, suggesting that coadministration of P450 inhibitors or inducers is unlikely to change its PK profile. Carfilzomib showed direct and time-dependent inhibition of CYP3A in human liver microsome preparations and exposure to carfilzomib resulted in reductions in CYP3A and 1A2 gene expression in cultured human hepatocytes. However, administration of carfilzomib did not affect the PK of midazolam in patients with solid tumors, and there were no safety signals indicative of potential drug interactions. We conclude that the rapid systemic clearance and short half-life of carfilzomib limit clinically significant DDI.

Carfilzomib: Second proteasome inhibitor approved for multiple myeloma

Carfilzomib: Second proteasome inhibitor approved for multiple myeloma

Obatoclax Interacts Synergistically with the Irreversible Proteasome Inhibitor Carfilzomib in GC- and ABC-DLBCL Cells In Vitro and In Vivo

Interactions between the irreversible proteasome inhibitor carfilzomib and the pan-BH3 mimetic obatoclax were examined in germinal center (GC)- and activated B-cell-diffuse large B-cell lymphoma (ABC-DLBCL) cells. Cotreatment with minimally toxic concentrations of carfilzomib (i.e., 2-6 nmol/L) and subtoxic concentrations of obatoclax (0.05-2.0 μmol/L) synergistically increased apoptosis in multiple DLBCL cell lines and increased lethality toward primary human DLBCL but not normal CD34(+) cells. Synergistic interactions were associated with sharp increases in caspase-3 activation, PARP cleavage, p-JNK induction, upregulation of Noxa, and AKT dephosphorylation. Combined treatment also diminished carfilzomib-mediated Mcl-1 upregulation whereas immunoprecipitation analysis revealed reduced associations between Bak and Mcl-1/Bcl-xL and Bim and Mcl-1. The carfilzomib/obatoclax regimen triggered translocation, conformational change, and dimerization of Bax and activation of Bak. Genetic interruption of c-jun-NH(2)-kinase (JNK) and Noxa by short hairpin RNA knockdown, ectopic Mcl-1 expression, or enforced activation of AKT significantly attenuated carfilzomib/obatoclax-mediated apoptosis. Notably, coadministration of carfilzomib/obatoclax sharply increased apoptosis in multiple bortezomib-resistant DLBCL models. Finally, in vivo administration of carfilzomib and obatoclax to mice inoculated with SUDHL4 cells substantially suppressed tumor growth, activated JNK, inactivated AKT, and increased survival compared with the effects of single-agent treatment. Together, these findings argue that a strategy combining carfilzomib and obatoclax warrants attention in DLBCL.

Abstract 2607: The benefits of irreversibility: Infusion administration of carfilzomib results in potent proteasome inhibition and improved safety in animals

Abstract Carfilzomib (CFZ) is a tetrapeptide epoxyketone-based inhibitor whose primary target is the chymotrypsin-like (CT-L) activity of the 20S proteasome. In clinical trials, intravenous (IV) bolus administration of carfilzomib has shown promising anti-tumor activity in multiple myeloma (MM), including patients relapsed and/or refractory to bortezomib. CFZ is a time-dependent and irreversible inhibitor, suggesting that target inhibition in vivo is likely a result of total dose administered and not steady state or maximum plasma concentration (Cmax). In order to determine if the method of delivery would affect the pharmacodynamics (PD) and toxicity of carfilzomib, we performed a series of experiments in rats. When delivered as an IV bolus or 30-minute infusion, a dose of 8 mg/kg resulted in ≥90% inhibition of the CT-L activity of the proteasome in whole blood and in all analyzed tissues (adrenal, lung, heart, and liver). Inhibition of the trypsin-like (T-L) and caspase-like (C-L) activity of the proteasome was ≥80% and ≥60%, respectively, was indistinguishable between the 2 groups. Despite an identical PD profile, infusion administration was significantly better tolerated than bolus. In the bolus group mortality was 44% (14 of 32) and the remaining animals displayed clinical signs of dyspnea, lethargy and poor blood perfusion to the extremities. None of the 24 animals receiving an infusion administration of CFZ died or were euthanized for humane reasons. A dose of 0.5 mg/kg bortezomib (BTZ), delivered as an IV bolus, produced similar clinical signs and mortality rates as bolus CFZ in rats. This result enabled us to determine which toxicity findings were target mediated. Interestingly, this dose of BTZ resulted in similar levels of inhibition of the CT-L (≥90%) but substantially less inhibition of T-L activity (≤20%) and slightly greater inhibition in C-L activity (≥80%) in blood and tissues. Bolus administration of either CFZ or BTZ resulted in 3 – 6-fold increases in blood urea nitrogen (BUN) and serum creatinine, while infusion administration of CFZ resulted in only a modest increase in BUN and no changes in creatinine levels. Other changes, such as a transient decrease in platelets, were not affected by the method of delivery of carfilzomib. These data suggest that bolus administration of either CFZ or BTZ in rats produced sequelae including pulmonary and cardiovascular function changes, renal azotemia and death and these toxicities are likely Cmax-related. However, because of its irreversible mechanism of proteasome inhibition, CFZ can be safely administered as an infusion resulting in profound proteasome inhibition in blood and tissues. The improved safety of infusion administration seen in these studies provides rationale for clinical application of infusion administration of CFZ in patients with MM. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2607. doi:10.1158/1538-7445.AM2011-2607

Abstract 5366: The small molecule Bcl-2 antagonist obatoclax interacts synergistically with the irreversible proteasome inhibitor carfilzomib in GC and ABC- sub types of DLBCL cells both in vitro and in vivo

Abstract Introduction: Based on previous studies showing synergistic interactions between proteasome inhibitors (e.g., bortezomib) and Bcl-2 antagonists, we have investigated whether the activity of the irreversible proteasome inhibitor carfilzomib (CFZ) could be enhanced in DLBCL cells by a clinically relevant Bcl-2 antagonist such as obatoclax (OBTX). Experimental Procedures: SUDHL16, SUDHL4 (GC -subtype) and OCI-LY10, OCI-LY-3 (ABC -subtype) cell lines were used as model systems. CFZ and OBTX were provided by Onyx Pharmaceuticals and GeminX Inc, respectively. Apoptosis was monitored by Annexin V/PI following drug treatment with OBTX ± CFZ. Synergism was assessed by CalcuSyn software. Effects of drug treatment on protein expression was monitored by western blotting. Beige-nude-XID (NIH-III) mice were used for in vivo studies. Results : Co-treatment (24- 48 hrs) with minimally toxic CFZ concentrations (i.e., 2-6 nM) and sub-toxic concentrations of either OBTX (0.05-2.0µM) or HA14-1 (2-10 µM) resulted in a striking increase in apoptosis in diverse DLBCL cells. Median Dose Effect analysis demonstrated that interactions were highly synergistic, with Combination Index values less than 1.0. The CFZ/OBTX regimen also strikingly induced apoptosis in 2 primary human DLBCL (1-GC and 1-ABC types) samples but exhibited minimal toxicity toward normal hematopoietic cells (i.e., CD34+ cells). Synergistic interactions were associated with a sharp increase in caspase-3 activation, PARP cleavage, induction of p-JNK, up-regulation of Noxa, and de-phosphorylation of AKT. Additionally, combined exposure diminished up-regulation of Mcl-1 induced by CFZ. Enhanced activity of this drug regimen was associated with Bax translocation, as well as Bax conformational change and dimerization. Notably, genetic interruption of JNK signaling (e.g., by shRNA knockdown) significantly attenuated CFZ/OBTX-mediated apoptosis, demonstrating a significant role for JNK activation in enhanced lethality. Additionally, shRNA knock down of Noxa or enforced activation of AKT significantly attenuated the lethality of the CFZ/ OBTX regimen in DLBCL cells. Notably, co-administration of CFZ/ OBTX resulted in sharp increases in apoptosis in 3 separate bortezomib-resistant DLBCL models. Finally, in vivo administration of CFZ and OBTX to mice inoculated with SUDHL4 cells substantially suppressed tumor growth compared to the effects of single agent treatment. Conclusions: A strategy combining the carfilzomib with obatoclax is active against parental DLBCL cells, including both GC- and ABC-subtypes, their bortezomib-resistant DLBCL counterparts, as well as primary DLBCL cells. It also displays significant in vivo activity in a xenograft DLBCL model. This approach warrants further exploration as therapeutic strategy in DLBCL. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5366. doi:10.1158/1538-7445.AM2011-5366

Phase 1b Evaluation of the Safety and Efficacy of a 30-Minute IV Infusion of Carfilzomib In Patients with Relapsed and/or Refractory Multiple Myeloma

AbstractAbstract 3024 Background:Carfilzomib (CFZ) is a novel, highly selective, epoxyketone proteasome inhibitor. In two separate Phase 2 trials in patients (pts) with relapsed and/or refractory (R/R) multiple myeloma (MM), single-agent CFZ administered as an IV bolus over 1–10 minutes has demonstrated durable activity at 20/27 mg/m2 and is well-tolerated with no clinically significant cumulative toxicity. In rats, significantly improved tolerability of CFZ was obtained following administration as a 30 min infusion as compared to a rapid IV bolus. Notably, a dose of 48 mg/m2 via IV bolus resulted in 50% lethality, compared to minimal toxicity without lethality at the same dose via a 30 min infusion. The reduced toxicity with 30-min infusion may reflect the role of Cmax (45 μM for bolus vs. 1.5 μM for infusion), since proteasome inhibition in blood and tissue was equivalent in both groups. Here we report on the results of administration of CFZ as a 30-minute IV infusion in a Phase 1b study in pts with R/R MM. The goals of this study are to determine the maximum recommended dose for infusion, safety, efficacy, pharmacokinetics (PK), and pharmacodynamic (PD) parameters. Methods:This Phase 1b trial is enrolling pts with R/R MM after ≥2 prior treatment failures. CFZ is given as a 30-minute IV infusion on days (D) 1, 2, 8, 9, 15, and 16 of a 28-day cycle (C) until progression. Dosing in all cohorts is initiated at 20 mg/m2 for the first two doses, with subsequent escalation to 36, 45, 56, or 70 mg/m2. Dose escalation follows standard 3+3 rules. Dexamethasone (4 mg for doses up to 45 mg/m2) is given prior to each infusion, with 8 mg given at higher doses. Responses by IMWG Uniform Response Criteria are measured at every C. Plasma samples for PK analysis and peripheral blood samples for PD analysis were obtained from pts at C1D1 (20 mg/m2) and C2D1 (all dose cohorts). Results:To date, 16 pts with R/R MM have been enrolled in the Phase 1b infusion study (4 at 36 mg/m2; 3 at 45 mg/m2; 7 at 56 mg/m2 and 2 at 70 mg/m2). Pts have remained on study for a median of 4 cycles (range 1–13+). Dose Limiting Toxicity (DLT) was observed in both pts treated at 70 mg/m2: reversible Grade (G) 3 renal failure in one pt within 24-hours following his first dose at 70 mg/m2 (C1D8); reversible G3 fatigue with fevers 4 days following four doses of 70 mg/m2 (C1 D20). Both pts were successfully rechallenged and continue on treatment. Seven patients have started dosing at 56 mg/m2; to date, one DLT (reversible G3 hypoxia with fevers) was observed. Thirteen pts are evaluable for efficacy (2 pts withdrew prior to 1st response assessment; 1 pt is too early to assess). Responses, time on study and prior regimens are detailed in the following table.Preliminary PK analysis demonstrates that the Cmax with 30-minute infusion is lower than obtained with a 5–10 minute IV bolus of the same dose. Inhibition of proteasome activity in red blood cells (RBCs) and peripheral blood mononuclear cells (PBMCs) was >80% at 20 mg/m2 and >90% at 36 mg/m2 and above. Common adverse events (AEs) with CFZ delivered as a 30-minute infusion have included fatigue, fevers, myalgias, diarrhea, nausea, thrombocytopenia, and reversible elevations in serum creatinine. There have been no episodes of worsening of baseline peripheral neuropathy or hepatotoxicity. Conclusions:In pts with R/R MM, single-agent CFZ as 30-minute IV infusion is both active and well-tolerated at doses ≥36 mg/m2; the dose level of 56 mg/m2 is being expanded as the recommended phase 2 dose on this schedule. Responses were seen in 8 out of 13 evaluable MM pts, including three VGPRs in pts who had received 5–7 prior regimens. Similar to animal studies, improved safety outcomes in MM patients can be achieved with near complete proteasome inhibition when CFZ is administered as a 30-minute infusion. An additional schedule of CFZ using weekly dosing (30-minute infusion for 5 weeks out of every 6) will be investigated in this trial.Best ResponseDose (mg/m2)Prior RegimensPrior Bortezomib RegimensTime on Study (months)VGPR56733.7+56534.8+56625.1+PR361112.4+36734.845603.056300.9+70 −> 56411.6+SD36105.145424.870 −>36/45632.6+ Disclosures:Papadopoulos:Onyx Pharmaceuticals: Consultancy, Research Funding. Siegel:Millenium: Consultancy, Honoraria; Celgene: Consultancy, Honoraria. Singhal:Celgene: Speakers Bureau; Takeda/Millenium: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Onyx: Research Funding. Gordon:Onyx Pharmaceuticals: Research Funding. Kauffman:Onyx Pharmaceuticals: Employment. Woo:Onyx Pharmaceuticals: Employment. Lee:Onyx Pharmaceuticals: Employment. Bui:Onyx Pharmaceuticals: Employment. Hannah:Onyx Pharmaceuticals: Consultancy.

Histone Deacetylase Inhibitors Potentiate the Lethality of the Irreversible Proteasome Inhibitor Carfilzomib In Mantle Cell Lymphoma Cells In Vitro and In Vivo

AbstractAbstract 3938The proteasome inhibitor (PI) bortezomib (BTZ) is approved for the treatment of refractory mantle cell lymphoma (MCL). However, BTZ achieves clinical responses in only 40% of refractory MCL patients, justifying the search for newer proteasome inhibitors and/or the development of rational combination regimens. Recently we reported that the irreversible PI carfilzomib (CFZ) interacted synergistically with the histone deacetylase inhibitor (HDACI) vorinostat (Vor) in diverse DLBCL cells including GC (germinal center) and ABC (activated B-cell) sub-types both in vitro and in vivo. (Dasmahapatra et al., Blood 2010; 115:4478-87, 2010). Moreover, regimens combining PIs with histone deacetylase inhibitors (HDACIs) have shown promising activity in multiple myeloma. Collectively, these preclinical and clinical findings raise the possibility combining CFZ with HDACIs might be an effective strategy in MCL. To test this possibility, CFZ/HDACI interactions were investigated in diverse MCL cell types, including those resistant to BTZ. Simultaneous treatment (24-48 hr) with sub-lethal CFZ concentrations (2-5 nM) and minimally toxic concentrations of vorinostat (Vor; 1–2 μM), SNDX-275 (1-2 μM) or SBHA (30-50 μM) strikingly increased apoptosis (manifested by annexin V, 7-AAD or TUNEL positivity) in various MCL lines, including Granta, HF-4B, Rec-1, JVM-2, MINO and JVM-12. Interactions were highly synergistic, as determined by Median Dose Effect analysis, with Combination Index values significantly less than 1.0. HDACIs also interacted synergistically with ONX 0912 (formerly PR-047), an orally bioavailable analog of CFZ. The activity of these regimens was associated with a sharp increase in caspase-3 activation, PARP cleavage, mitochondrial damage (loss of ΔΨm, cytoplasmic cytochrome c release), and induction of p21CIP1. Combined CFZ/Vor exposure also markedly induced phosphorylation of JNK and c-Jun, down-regulation of phospho-AKT and phospho-ERK1/2, and induction of γH2A.X, a marker of double-stranded DNA breaks. In contrast to single agent administration (48 hr), where the percentage of apoptotic cells gradually declined following drug washout over the ensuing 4–5 days, extensive apoptosis (e.g. > 80%) persisted in cells co-treated with CFZ/Vor. Combined treatment of MCL cells with CFZ/Vor induced more sustained inhibition of chymotrypsin-like (CT-L) proteasome activity than that observed following single-agent treatment. CFZ alone exhibited partial cross resistance to BTZ- resistant Granta cells (GR-25BR). However, co-administration of CFZ/Vor resulted in the highly synergistic induction of apoptosis in BTZ-resistant cells. Genetic interruption of JNK signaling (e.g., via shRNA knockdown) significantly attenuated CFZ/Vor-mediated apoptosis, indicating that JNK activation plays a functional role in the lethality of this regimen in MCL cells. Combined treatment with CFZ and HDACIs induced G2M arrest in both parental and BTZ-resistant GR-25BR cells, an effect that was not modified by genetic interruption of JNK signaling. The CFZ/Vor regimen also strikingly induced apoptosis in 3 primary human MCL specimens, in contrast to the minimal lethality exhibited toward normal CD34+ cells as previously described (Blood; 115:4478-87, 2010). Finally, in vivo administration of CFZ (IV BIW) and Vor (IP TIW) to Beige-nude-XID mice (NIH-III) inoculated in the flank with Granta cells substantially suppressed tumor growth compared to single agent treatment. Collectively, these findings indicate that combining HDACIs with CFZ synergistically induces apoptosis in MCL cells through a JNK-dependent mechanism in association with G2M arrest and the induction of DNA damage. They also suggest that this strategy, which is active against diverse MCL cell types, including BTZ-resistant and primary MCL cells, and which displays in vivo activity, warrants further examination in MCL. Accordingly, plans for a Phase I CFZ/HDACI trial in NHL, including MCL patients, are currently underway. Supported by Lymphoma SPORE 1P50 CA130805. Disclosures:Friedberg:Genentech: Honoraria.

Pharmacokinetics, Metabolism, Distribution and Excretion of Carfilzomib in Rats.

Abstract 2754Poster Board II-730 Introduction:Carfilzomib, a tetrapeptide epoxyketone that selectively and irreversibly inhibits the 20S proteasome, has demonstrated a favorable safety profile and anti-tumor efficacy in phase II trials in patients with multiple myeloma [1, 2]. In this study, we characterized the pharmacokinetics (PK), metabolism, distribution and excretion of carfilzomib in Sprague Dawley rats. Methods:In vivo studies were conducted following single intravenous (IV) administrations of carfilzomib to rats. For pharmacokinetic evaluations, plasma samples were collected pre-dose and from 0.1 to 60 minutes post-dose. Biliary excretion was evaluated in bile-duct cannulated rats with bile collection for 8 hours. Urine was collected for 24 hours post-dose for evaluation of renal excretion. Metabolism was studied in plasma, bile and urine samples collected from rats treated with carfilzomib and following in vitro incubations of carfilzomib with hepatocytes and tissue homogenates of lung, heart and kidney from rats. Liquid chromatography with tandem mass spectrometric detection was used in determination of carfilzomib and metabolites in different matrices. Noncompartmental analysis was used in calculation of pharmacokinetic parameters. Results:(1) Carfilzomib was rapidly cleared from the plasma compartment following bolus IV administration with a terminal half life of less than 20 minutes. The plasma clearance was 229, 195 and 296 mL/min/kg, the Cmax (the maximum concentration) was 16.4, 39.3 and 42.9 μM and the AUClast (area under the plasma concentration vs time curve) was 12.6, 28.5 and 37.6 min*μmol/L, at the dose levels of 2, 4 and 8 mg/kg, respectively. At all dose levels, the clearance was much higher than rat hepatic flow. When carfilzomib was given as 30-minute intravenous infusion at 8 mg/kg, the steady state concentration of 1.5 μM was reached within 15 minutes and was 28-fold lower than the Cmax with IV bolus at the equivalent dose. Other PK parameters (clearance, AUC and t1/2) were comparable between bolus and infusion dosing. Rapid and potent proteasome inhibition in blood and tissues (brain excepted) was observed following either bolus or infusion administration, demonstrating wide tissue distribution of carfilzomib. (2) Carfilzomib was rapidly metabolized not only in rat hepatocytes, but also in incubations with tissue homogenates of rat lung, kidney and liver as well as in rat blood, indicating that carfilzomib could be metabolized in tissues and in blood in vivo following IV administration to rats. These results suggested that the high clearance was due to extrahepatic metabolism of carfilzomib in vivo. In plasma, urine and bile collected from rats treated with carfilzomib, as well as in incubations of carfilzomib with rat hepatocytes and tissue homogenates, the most abundant metabolites were found to be peptide fragments and the diol of carfilzomib. These results indicated that peptidase cleavage and epoxide hydrolysis were the principal pathways of metabolism and that P450-mediated mechanisms were not significant in the overall metabolism of carfilzomib. (3) Carfilzomib was eliminated mainly in the form of metabolites following intravenous dosing to rats via renal and biliary excretions. Conclusions:Carfilzomib is rapidly cleared and widely distributed following IV administrations to rats. Metabolism of carfilzomib is mediated by plasma and tissue resident peptidases and epoxide hydrolases. The irreversible mechanism of proteasome inhibition by carfilzomib overcomes clearance mechanisms and enables equivalent proteasome inhibition with both bolus and infusion administration. These data support ongoing efforts to study the effect of infusion administration of carfilzomib in patients with myeloma.[1] S. Jagennath, et al. (2008) Blood 112(11): Abstract #864.[2] R. Vij, et al., (2008) Blood 112(11): Abstract #865. Disclosures:Yang:Proteolix Inc: Employment, Equity Ownership. Fang:Proteolix Inc: Employment, Equity Ownership. Wang:Proteolix, Inc.: Employment, Equity Ownership. Jiang:Proteolix Inc: Employment, Equity Ownership. Muchamuel:Proteolix Inc: Employment, Equity Ownership. Lee:Proteolix, Inc.: Employment, Equity Ownership. Kirk:Proteolix Inc: Employment, Equity Ownership. Zhao:Proteolix Inc: Employment, Equity Ownership. Bennett:Proteolix: Employment, Equity Ownership. Molineaux:Proteolix Inc: Employment, Equity Ownership.

Inhibition of the Proteasome in Bone Marrow-Derived CD138+ Tumor Cells Following Carfilzomib Administration in Relapsed or Refractory Myeloma Patients.

Abstract 1845Poster Board I-871Background: Proteasome inhibition is an effective strategy for the treatment of multiple myeloma. In patients, proteasome inhibition has primarily been measured in peripheral blood samples (whole blood or mononuclear cells). However, it is unknown whether myeloma cells in the bone marrow (BM) are equally sensitive to proteasome inhibitors such as bortezomib (BTZ) and carfilzomib (CFZ). Aim: To measure proteasome inhibition in purified tumor cells from BM samples taken from patients enrolled in two ongoing Phase 2 trials of single agent CFZ in relapsed or refractory myeloma: PX-171-003 (003) and PX-171-004 (004). Methods: CFZ was administered as an IV bolus of 20 mg/m2 on Days 1, 2, 8, 9, 15 and 16 of a 28-day cycle on both trials. Bone marrow samples, from an optional sub-study of both trials, were taken during screening and Day 2 (post-treatment) and sorted into CD138+ and CD138− cells. Proteasome activity was measured by an enzymatic assay using a fluorogenic substrate (LLVY-AMC) for the chymotrypsin-like (CT-L) activity and an active site ELISA (ProCISE) to quantitate levels of the CT-L subunits of the constitutive proteasome (Beta5) and immunoproteasome (LMP7) and the immunoproteasome subunit MECL1. Results: Whole blood samples from patients treated with CFZ showed inhibition of CT-L activity of ∼80+, similar to values obtained in Phase 1 studies. A total of 10 CD138+ screening samples, 6 from 004 and 4 from 003, and 9 post-dose samples, 5 from 004 and 4 from 003, were analyzed for proteasome levels and activity. In addition, 15 CD138−screening samples, 7 from 004 and 8 from 003, and 9 post-dose samples, 5 from 004 and 4 from 003, were analyzed. When compared to the average base-line activity, CFZ treatment resulted in 88% CT-L inhibition in CD-138+tumor cells from 004 patients (P = 0.0212 by unpaired t-test) and 59% CT-L inhibition in CD-138+ tumor cells from 003 patients (P = 0.25). Baseline CT-L activity in CD138+ tumor cells was 3-fold higher in 004 than 003, which includes a more heavily pre-treated patient population with greater prior exposure to BTZ. Higher specific enzymatic activity was due to increased levels of both constitutive and immunoproteasomes in tumor cells, where immunoproteasomes account for >75% of total cellular proteasomes. No differences between trials were seen in baseline CT-L activity from non-tumor (CD138−) cells. Inhibition in CD138− cells was 84% (P = 0.0380 and 42% (P = 0.38) in 004 and 003, respectively. Using ProCISE, we measured inhibition of LMP7 (66%), beta5 (48%) and MECL1 (64%) in CD138+ tumor cells from 004 patients. Three patients from 004 and one from 003 had both a screening and post-dose tumor cell samples available for analysis. Inhibition of CT-L activity was >80% in two of the 3 patients on 004; the third patient showed no proteasome inhibition by ProCISE and was unavailable for analysis by CT-L. CT-L activity in the CD138+ tumor cells in the 003 patient was not inhibited, however, inhibition was seen in non-tumor cells. Conclusions: CFZ inhibits the proteasome activity of myeloma cells in the bone marrow of relapsed and refractory myeloma patients. The levels of inhibition were similar to those measured in whole blood samples, supporting the use of the blood-based assay as a surrogate marker for proteasome inhibition in tumor cells. CFZ treatment resulted in inhibition of both CT-L subunits as well as additional subunits of the immunoproteasome in tumor cells. Reduced baseline activity in the more heavily pretreated 003 patients may reflect reduced tumor-dependency on the proteasome and may be related to prior treatment with BTZ in these patients. More samples are needed in order to make correlations between levels of proteasome inhibition in bone marrow tumor cells and prior therapies or response. These observations support further evaluation of proteasome activity and the effects of this promising new agent in primary tumors cells from myeloma patients. Disclosures:Trudel:Celgene: Honoraria, Speakers Bureau; Ortho Biotech: Honoraria. Lee:Proteolix, Inc.: Employment. Kirk:Proteolix, Inc.: Employment. Lonial:Celgene: Consultancy; Millennium: Consultancy, Research Funding; BMS: Consultancy; Novartis: Consultancy; Gloucester: Research Funding. Wang:Proteolix, Inc.: Research Funding. Kukreti:Celgene: Honoraria. Stewart:Genzyme, Celgene, Millenium, Proteolix: Honoraria; Takeda, Millenium: Research Funding; Takeda-Millenium, Celgene, Novartis, Amgen: Consultancy. Jagannath:Millennium: Honoraria, Membership on an entity's Board of Directors or advisory committees; Merck: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. McDonagh:Proteolix: Research Funding. Zonder:Celgene: Speakers Bureau; Pfizer: Consultancy; Seattle Genetics, Inc.: Research Funding; Amgen: Consultancy; Millennium: Research Funding. Bennett:Proteolix: Employment.

The Selective Proteasome Inhibitor Carfilzomib Is Well Tolerated in Experimental Animals with Dose Intensive Administration

Carfilzomib (CFZ) is a selective inhibitor of the chymotrypsin-like (CT-L) activity of the proteasome. Phase I trials have demonstrated that doses of carfilzomib from 11 – 20 mg/m2 were well tolerated, inhibited blood CT-L activity by ≥80%, and resulted in objective anti-tumor responses in patients with multiple myeloma (MM) and non-Hodgkin's lymphoma (NHL) (Blood 110:409 & 411). In order to further characterize the safety profile of CFZ, studies in mice, rats and monkeys were conducted to assess the toxicologic consequences of acute and chronic administration. In both rats and monkeys, a single dose of CFZ (equivalent to 24 mg/m2) resulted in > 80% inhibition of CT-L activity in blood and tissues with recovery occurring in most tissues with a t1/2 of 24 hr. At this dose, monkeys developed a pre-renal azotemia within 24 hr, which was characterized by increases in serum creatinine, blood urea nitrogen (BUN) levels and urine specific gravity. These changes resolved in 72 hr, were not associated with histologic changes in the kidney, and were not exacerbated by consecutive daily administrations, suggesting that CFZ may not have a direct nephrotoxic effect. A single dose in either rats or monkeys induced a transient drop in platelets within 48 hr that resolved within 7 days and neither the level of nor recovery from this thrombocytopenia were affected by consecutive daily administration. This phenomenon has also been noted with bortezomib (BTZ), and is observed clinically with both compounds, suggesting that thrombocytopenia is a hallmark of inhibition of proteasome CT-L activity. However, in contrast to BTZ, which has been reported to induce both lymphopenia and neutropenia in animals, CFZ treatment in both rats and monkeys resulted in acute increases in circulating neutrophils and monocytes which rose within 24 hr of a single dose and returned to baseline in 3 – 4 days. We were unable to detect a significant induction of cytokines or chemokines capable of mediating leukocyte fluxes. However, since the acute phase proteins fibrinogen and C-reactive protein were elevated during the same time period of leukocyte flux, cytokines may play a role in this response. In chronic studies, CFZ was administered on Days 1, 2, 8, 9, 15, and 16 of a 28-day cycle to rats and monkeys for 6 and 9 cycles, respectively. This dosing schedule results in anti-tumor activity in mouse models of MM, NHL and solid tumors and is currently being utilized in Phase 2 studies in MM and solid tumors. In these chronic studies, transient pre-renal azotemia and increases in circulating neutrophils were manifest in monkeys at 24 mg/m2 following the first dose of both the first and last cycles of treatment. However, no histologic signs of nephropathy were seen in the kidneys of rats receiving 6 cycles of treatment. In addition, chronic administration of CFZ at doses equivalent to those currently administered in the clinic did not impact on the neurobehavioral function of rats and monkeys. There was no severe degeneration in the dorsal root ganglion or in the sciatic, sural, and peroneal nerves of CFZ treated rats. The data with CFZ are in distinct contrast to the axonal swelling, demyelination and severe neural degeneration that is induced by BTZ in rodents and monkeys. In the clinic, peripheral neuropathy associated with CFZ treatment appears to be less severe and possibly less frequent than what is seen with BTZ. Overall, studies in rodents and monkeys have been largely predictive of the clinical experience to date with CFZ. Together with anti-tumor activity in human tumor xenograft models, the toxicologic data support that CFZ administered at therapeutic doses is well tolerated and prolonged treatment of patients with this agent will be feasible.