Clet Niyikiza

Phase III Study of Pemetrexed in Combination With Cisplatin Versus Cisplatin Alone in Patients With Malignant Pleural Mesothelioma

PURPOSE Patients with malignant pleural mesothelioma, a rapidly progressing malignancy with a median survival time of 6 to 9 months, have previously responded poorly to chemotherapy. We conducted a phase III trial to determine whether treatment with pemetrexed and cisplatin results in survival time superior to that achieved with cisplatin alone. PATIENTS AND METHODS Chemotherapy-naive patients who were not eligible for curative surgery were randomly assigned to receive pemetrexed 500 mg/m2 and cisplatin 75 mg/m2 on day 1, or cisplatin 75 mg/m2 on day 1. Both regimens were given intravenously every 21 days. RESULTS A total of 456 patients were assigned: 226 received pemetrexed and cisplatin, 222 received cisplatin alone, and eight never received therapy. Median survival time in the pemetrexed/cisplatin arm was 12.1 months versus 9.3 months in the control arm (P =.020, two-sided log-rank test). The hazard ratio for death of patients in the pemetrexed/cisplatin arm versus those in the control arm was 0.77. Median time to progression was significantly longer in the pemetrexed/cisplatin arm: 5.7 months versus 3.9 months (P =.001). Response rates were 41.3% in the pemetrexed/cisplatin arm versus 16.7% in the control arm (P <.0001). After 117 patients had enrolled, folic acid and vitamin B12 were added to reduce toxicity, resulting in a significant reduction in toxicities in the pemetrexed/cisplatin arm. CONCLUSION Treatment with pemetrexed plus cisplatin and vitamin supplementation resulted in superior survival time, time to progression, and response rates compared with treatment with cisplatin alone in patients with malignant pleural mesothelioma. Addition of folic acid and vitamin B12 significantly reduced toxicity without adversely affecting survival time.

In vitro chemosensitivity of freshly explanted tumor cells to pemetrexed is correlated with target gene expression

SummaryAim of the studymRNA expression of genes involved in the mechanism of action of pemetrexed was correlated with in vitro chemosensitivity of freshly explanted human tumor specimens.Experimental designChemosensitivity to pemetrexed was studied in soft-agar. Multiplex rtPCR experiments for reduced folate carrier (RFC), folate receptor-α (FR-α), folylpolyglutamate synthetase (FPGS), thymidylate synthase (TS), dihydrofolate reductase (DHFR), glycinamide ribonucleotide formyl transferase (GARFT), mrp4, and mrp5 were performed in parallel. Correlations, threshold optimization, sensitivity, specificity, and efficiency were analyzed using the appropriate statistical methodologies.ResultsIn 61 samples, low levels of TS, GARFT, DHFR, and mrp4 gene expression significantly correlated with chemosensitivity to pemetrexed. Optimization analyses demonstrated threshold values of 144 copies for TS and six copies for mrp4 relative to 104 copies of β-actin.ConclusionsThese results form a rational basis for the design of clinical trials to evaluate the expression of these enzymes as predictors for treatment outcome.

HER2-targeted liposomal doxorubicin displays enhanced anti-tumorigenic effects without associated cardiotoxicity

Anthracycline-based regimens are a mainstay of early breast cancer therapy, however their use is limited by cardiac toxicity. The potential for cardiotoxicity is a major consideration in the design and development of combinatorial therapies incorporating anthracyclines and agents that target the HER2-mediated signaling pathway, such as trastuzumab. In this regard, HER2-targeted liposomal doxorubicin was developed to provide clinical benefit by both reducing the cardiotoxicity observed with anthracyclines and enhancing the therapeutic potential of HER2-based therapies that are currently available for HER2-overexpressing cancers. While documenting the enhanced therapeutic potential of HER2-targeted liposomal doxorubicin can be done with existing models, there has been no validated human cardiac cell-based assay system to rigorously assess the cardiotoxicity of anthracyclines. To understand if HER2-targeting of liposomal doxorubicin is possible with a favorable cardiac safety profile, we applied a human stem cell-derived cardiomyocyte platform to evaluate the doxorubicin exposure of human cardiac cells to HER2-targeted liposomal doxorubicin. To the best of our knowledge, this is the first known application of a stem cell-derived system for evaluating preclinical cardiotoxicity of an investigational agent. We demonstrate that HER2-targeted liposomal doxorubicin has little or no uptake into human cardiomyocytes, does not inhibit HER2-mediated signaling, results in little or no evidence of cardiomyocyte cell death or dysfunction, and retains the low penetration into heart tissue of liposomal doxorubicin. Taken together, this data ultimately led to the clinical decision to advance this drug to Phase I clinical testing, which is now ongoing as a single agent in HER2-expressing cancers.

A phase II trial of pemetrexed in advanced breast cancer: clinical response and association with molecular target expression.

Purpose: This phase II trial of pemetrexed explored potential correlations between treatment outcome (antitumor activity) and molecular target expression. Experimental Design: Chemonaïve patients with advanced breast cancer received up to three cycles of pemetrexed 500 mg/m2 (10-minute i.v. infusion) on day 1 of a 21-day cycle, with folic acid and vitamin B12 supplementation. Tumors were surgically removed after the last cycle of pemetrexed as clinically indicated. Biopsies were taken at baseline, 24 hours after infusion in cycle 1, and after cycle 3. Results: Sixty-one women (median age, 46 years; range, 32-72 years) were treated and were evaluable for response. Objective response rate was 31%. Simple logistic regression suggested a potential relationship between mRNA expression of thymidylate synthase (TS) and pemetrexed response (P = 0.103). Based on threshold analysis, patients with “low” baseline TS (≤71) were more likely to respond to pemetrexed than patients with “high” baseline TS (>71). Expression of baseline dihydrofolate reductase and glycinamide ribonucleotide formyl transferase tended to be higher in responders but this association was not significant (P > 0.311). TS expression increased significantly between baseline and biopsy 2 (P = 0.004) and dropped to near baseline levels at biopsy 3. Conversely, dihydrofolate reductase and glycinamide ribonucleotide formyl transferase decreased after pemetrexed chemotherapy. Conclusions: Our results suggest a potential association between “low” pretreatment TS expression levels and response to pemetrexed chemotherapy. Future trials examining expression levels of other genes important to the folate pathway and/or breast cancer may identify a more robust multigene profile that can better predict response to this novel antifolate.

Pemetrexed safety and dosing strategy

Pemetrexed is a novel antifolate/antimetabolite that inhibits several folate-dependent enzymes, including thymidylate synthase, dihydrofolate reductase, and glycinamide ribonucleotide transformylase. As a class, antifolates have been associated with sporadic severe myelosuppression with gastrointestinal toxicity. Although infrequent, a combination of such toxicities carries a high risk of potentially life-threatening complications. Severe toxicity from pemetrexed-based therapy has become more predictable using the vitamin deficiency marker homocysteine and, to a lesser extent, methylmalonic acid. Evidence now suggests that reducing total plasma homocysteine levels by supplementation with folic acid and vitamin B(12) leads to a better safety profile for pemetrexed, while not adversely affecting its efficacy.

Abstract C90: HER2-targeted liposomal doxorubicin MM-302 has a favorable cardiosafety profile in preclinical models.

Introduction: Anthracyclines have historically been the backbone of anticancer therapy for decades; however, the clinical observation of permanent cardiotoxicity has limited their broader use. In the HER2 positive breast cancer setting, the combination of trastuzumab with anthracyclines was associated with an unacceptable risk of clinical cardiotoxicity. This appears to be related to 1) doxorubicin-induced cardiomyocyte stress and 2) inability of the cardiomyocytes to respond to the stress due to the trastuzumab-related impairment of HER2 signaling. Encapsulation of doxorubicin into liposomes (Doxil®) has been associated with a reduced risk of doxorubicin-associated cardiotoxicity relative to free doxorubicin. MM-302 is a HER2-targeted liposomal doxorubicin-based agent designed to target doxorubicin to HER2-overexpressing cancer cells, while limiting uptake into non-target cells. Since HER2-mediated signaling plays an important role in cardiac repair, the purpose of this work was to support clinical development of MM-302 by investigating whether HER2-targeting of liposomal doxorubicin would adversely affect the cardiac safety profile relative to untargeted liposomal doxorubicin. Methods: MM-302, untargeted liposomal doxorubicin and free doxorubicin were compared for their uptake into HER2-overexpressing cancer cells and two models of human stem cell-derived cardiomyocytes. High-content microscopy was used to determine the effects of exposure to specific cellular targets in cardiomyocytes. Mouse biodistribution studies were used to assess the total and nuclear accumulation of doxorubicin in mouse cardiac tissue upon treatment with either MM-302, untargeted liposomes or free doxorubicin. Quantitative immunofluorescence was used to quantify the HER2 expression on human normal and diseased heart specimens. Kinetic computational modeling was applied to interpret study results and to make predictions on human heart exposure to doxorubicin based on the experimental mouse data. Results: Human stem cell-derived cardiomyocytes showed nuclear accumulation of doxorubicin followed by cell death upon free doxorubicin treatment but not upon MM-302 or untargeted liposomes treatment. Similarly, MM-302 resulted in a significantly lower nuclear accumulation of doxorubicin compared to free doxorubicin treatment in mouse cardiac tissue. HER2 expression levels on normal and diseased human heart tissue were shown to be ≤100,000 receptors/cell, in concordance with the HER2 levels on the stem cell-derived cardiomyocytes found in vitro. Computational modeling predicts liposomal encapsulation to significantly protect the human heart from exposure to doxorubicin, consistent with clinical findings. Conclusions: MM-302 is not taken up by human cardiomyocytes via HER2, but selectively increases doxorubicin delivery to human HER2-overexpressing tumor cells. MM-302 can potentially improve the clinical efficacy demonstrated by conventional anthracyclines and maintain the cardiac safety profile of untargeted liposomal doxorubicin in patients with HER2 overexpressing cancers. Clinical evaluation of this patient population is currently in Phase I. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C90.

Abstract A6: Sustained intratumoral activation of MM-398 results in superior activity over irinotecan demonstrated by using a systems pharmacology approach

MM-398 is a stable nanotherapeutic encapsulation of the prodrug irinotecan with an extended plasma half-life and higher intratumoral deposition compared with free-irinotecan. MM-398 is currently in multiple clinical trials, including a phase 3 trial for patients with advanced gemcitabine-resistant pancreatic cancer (NAPOLI-1). Pancreatic cancer has been described as being notoriously difficult to treat, potentially due to inadequate drug penetration through the dense stroma, or because the hypoxic tumor microenvironment suppresses cytotoxic activity. We sought to better understand how MM-398, a relatively large (100nm) liposomal nanotherapeutic, could potentially treat pancreatic cancer by determining the relative roles of systemic vs. local tumor activation of irinotecan in contributing to the activity of MM-398. Using a systems pharmacology approach, we developed a mechanistic pharmacokinetic (PK) model of MM-398 and free-irinotecan to predict both plasma and intratumoral levels of irinotecan and SN-38. The model was trained with PK and biodistribution data from mice bearing HT-29 xenografts, which were administered intravenously with varying doses of MM-398 or free-irinotecan. Model simulations predicted that MM-398 resulted in equivalent SN-38 exposure (area under curve, AUC) in tumor at a fivefold lower dose than free-irinotecan. However, an in vivo animal activity study showed that 15-fold lower dose of MM-398 was sufficient to yield equal growth inhibition of HT-29 xenografts, which reveals the limit of relating simple AUC-based exposure to in vivo tumor response. While intratumoral SN-38 exposure from free-irinotecan was limited to the first 48 hours after dosing, MM-398 maintained high levels of SN-38 throughout the week-long time window. Further analysis of the exposure-response identified that the duration of intratumoral SN-38 levels above the threshold was a valid predictive marker for xenograft tumor response. Identifying the source of intratumoral SN38 is confounded by the fact that the mouse species has an additional carboxylesterase (CES) that can convert irinotecan to SN-38 in serum. The serum SN-38/irinotecan ratio in mice is tenfold higher than that observed in humans. In order to translate this preclinical observation into the clinic, it is critical to identify the role of mouse-specific serum CES on intratumoral SN-38 exposure. Thus, we performed a PK study with knockout mice lacking the Ces1c gene, which encodes serum CES, and then retrained our mechanistic PK model. Serum SN-38 levels in the Ces1c knockout mice were measurably decreased by ˜85% in the central compartment. In contrast, simulating the effect of knock-out of either serum CES or tumor CES, predicts that the duration of intratumoral residence of SN-38 is significantly affected by tumor CES, rather than serum CES. This suggests that local activation to SN-38 by tumor CES as the main driver for SN-38 tumor residence, which in turn drives response. In summary, we applied a systems pharmacology approach to identify the importance of tumor CES (local SN-38 generation) as one of the determinants of MM-398 response. Liposomal encapsulation of irinotecan dramatically alters the pharmacokinetic profile of SN-38 in the tumor, as well as tumor response, by maintaining SN-38 levels above the response threshold. Local, sustained activity of this active irinotecan metabolite could result in prolonged cytotoxic and tumor microenvironment modifications with beneficial effects on treatment of pancreatic cancer and other solid tumors. Citation Format: Jaeyeon Kim, Eliel Bayever, Peter Laivins, Clet Niyikiza, Ulrik Nielsen, Jonathan Fitzgerald, Ashish Kalra, Milind Chalishazar, Stephan Klinz, Nancy Paz, Bart Hendriks, Daryl Drummond, Dmitri Kirpotin, Victor Moyo. Sustained intratumoral activation of MM-398 results in superior activity over irinotecan demonstrated by using a systems pharmacology approach [abstract]. In: Proceedings of the AACR Special Conference on Chemical Systems Biology: Assembling and Interrogating Computational Models of the Cancer Cell by Chemical Perturbations; 2012 Jun 27-30; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2012;72(13 Suppl):Abstract nr A6.

Abstract A63: MM-398/PEP02, a novel liposomal formulation of irinotecan, demonstrates stromal-modifying anticancer properties.

MM-398 is a stable nanotherapeutic encapsulation of the prodrug irinotecan (CPT-11) with longer plasma half-life and higher tumor deposition due to an enhanced permeability and retention effect. Pancreatic cancer has responded poorly to many therapeutics, largely because of inadequate drug penetration due to poor vascularization and the highly aggressive, hypoxic nature of the disease. We sought to better understand how MM-398, a relatively large (100nm) liposomal nanotherapeutic, could be used treat pancreatic cancer. We have tested MM-398 in several pancreatic xenograft models: BxPC3 (KRAS wild type), AsPC-1(KRAS G12D) , Panc-1 (KRAS G12D) and MiaPaCa (KRAS G12C). All models demonstrated complete tumor regression at 20 mg/kg or a human equivalent dose of 60-120 mg/m2. At this same dose, MM-398 suppresses tumor growth in a gemcitabine insensitive AsPC-1 xenograft. MM-398 functionally blocked AsPC-1 tumor cell proliferation as measured by ki-67 staining; however, gemcitabine administered at its maximum tolerated dose did not impact proliferation. MM-398 is currently in multiple clinical trials, including a phase 3 trial for patients with advanced gemcitabine-resistant pancreatic cancer (NAPOLI-1). In order to further understand mechanisms driving response to MM-398, we screened and ranked several cell lines for their ability to convert irinotecan into the active metabolite, SN38. BxPC3 and HT-29 tumors ranked highest in ability to convert irinotecan to SN-38, as measured by HPLC. In a BxPC3 pancreatic orthotopic model which spontaneously metastasizes, 10 mg/kg MM-398 significantly reduced both primary and metastatic tumor load as measured by ex vivo biophotonic imaging of BxPC3luc cells to spleen, lung, liver, diaphragm and GI associated lymph nodes (p In summary, MM-398 induces tumor regression in multiple mouse models of pancreatic cancer, including an orthotopic metastatic model. MM-398 activity may be driven in part by the ability to modify tumor microenvironment parameters, such as hypoxia and vascularization, both of which limit efficacy of chemotherapeutic agents in the treatment of pancreatic cancer. These data support the continued investigation of MM-398 in pancreatic cancer. Citation Format: Nancy Paz, Peter Laivins, Clet Niyikiza, Ulrik Nielsen, Jonathan Fitzgerald, Ashish Kalra, Milind Chalishazar, Stephan Klinz, Jaeyeon Kim, Daryl Drummond, Dmitri Kirpotin, Victor Moyo, Eliel Bayever. MM-398/PEP02, a novel liposomal formulation of irinotecan, demonstrates stromal-modifying anticancer properties. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Progress and Challenges; Jun 18-21, 2012; Lake Tahoe, NV. Philadelphia (PA): AACR; Cancer Res 2012;72(12 Suppl):Abstract nr A63.

Abstract C207: Identifying differential mechanisms of action for MM-398/PEP02, a novel nanotherapeutic encapsulation of irinotecan.

MM-398 (aka PEP02) is a stable, nanotherapeutic encapsulation of the pro-drug CPT-11 (irinotecan) that is currently in clinical development. In preclinical experiments, treatment with MM-398 resulted in significantly higher intratumoral concentrations of both irinotecan (142-fold) and its major metabolite, SN-38 (9-fold) and exhibited superior anti-tumor activity compared to free irinotecan in multiple tumor xenografts. Subsequently, multiple phase 1 and 2 studies have established a pharmacokinetic and safety profile that supports continued clinical development, including in pancreatic, gastric, colorectal and potentially other cancers. Because current evidence suggests that resistance to pancreatic cancer is driven largely by inadequate drug penetration into these often poorly vascularized, stromally dense and hypoxic tumors, we sought to better understand how this relatively large (100nm) liposomal nanotherapeutic could potentially result in increased efficacy in very advanced gemcitabine-resistant pancreatic cancer and other cancer types. We developed a mechanism-based PK model of MM-398 and free irinotecan designed to predict intratumor SN-38 levels. Sensitivity analysis revealed that for MM-398 local activation of irinotecan to SN-38 was a far more important parameter than systemic activation. Through cellular uptake studies, we demonstrated in vitro that MM-398 was preferentially internalized by phagocytic macrophage/monocyte cell lines and, to a far lesser extent, by tumor cell lines. Furthermore, tumor microdistribution studies by flow cytometry and IHC showed uptake of MM-398 liposomes in both tumor cells and tumor-associated macrophages with more liposomal material being present in the macrophages. This distribution also suggests that macrophages may contribute to the postulated rate limiting process of irinotecan activation. The sensitivity analysis also suggested that tumor permeability and vascularization are important determinants of tumor-associated SN-38 levels for both free irinotecan and MM-398. To determine the effect of MM-398 on these parameters we treated mice bearing HT29 (colorectal cancer) xenografts with a single dose of MM-398 and measured hypoxic markers (CAIX) and microvessel density (CD31) by IHC. Tumors treated with MM-398 showed a greater degree of CD31 staining and lower CAIX staining, indicating that MM-398 may be able to affect tumor characteristics that traditionally have contributed to therapy resistance and limited the delivery of cancer therapeutics and resistance. In summary, encapsulation of irinotecan alters rate-limiting processes that determine tumoral SN-38 levels. Delivery of MM-398 is believed to alter tumor microvessel density and decrease hypoxia. These intriguing mechanisms of action findings support the continued clinical development of MM-398 as a differentiated therapeutic for several cancer types. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C207.

Abstract 655: Combination of MM-111, an ErbB2/ErbB3 bispecific antibody, with endocrine therapies as an effective strategy for treatment of ER+/HER2+ breast cancer

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Approximately 75% of breast cancers are estrogen receptor (ER) positive. Although endocrine therapies such as tamoxifen, fulvestrant, and letrozole have demonstrated significant efficacy in treating ER+ breast cancer patients, intrinsic or acquired resistance has limited their success. Recent studies suggest that crosstalk between ErbB receptor signaling and ER signaling may contribute to resistance to endocrine therapy. Overexpression of human epidermal growth factor receptor 2 (HER2, synonymous with ErbB2) and upregulation of the ErbB3 ligand heregulin are associated with poor prognosis and reduced overall survival. MM-111 is a novel bispecific antibody fusion protein that specifically targets the ErbB2/ErbB3 heterodimer and blocks heregulin binding to ErbB3. MM-111 inhibits ligand-induced ErbB3 phosphorylation, tumor cell cycle progression, and tumor growth when ErbB2 is overexpressed. We hypothesized that combination of endocrine therapies with MM-111 may improve anti-tumor efficacy. In an estrogen-stimulated BT474-M3 ER-positive breast cancer cell three-dimensional spheroid assay, MM-111, when used as a single agent, showed growth inhibitory effects similar to the anti-estrogen drugs tamoxifen and fulvestrant. Combination of MM-111 with anti-estrogen therapy showed superior activity to either drug alone. In the presence of heregulin, MM-111 maintained its growth inhibitory activity, whereas the inhibitory effect of tamoxifen and fulvestrant was diminished. This suggests that activation of ErbB3 confers tumor cell resistance to anti-estrogen therapies. When both estrogen and heregulin were present, the combination of MM-111 and the anti-estrogen drugs demonstrated a significantly greater inhibitory effect than either drug alone. Western blot analysis showed that treatment of BT-474-M3 cells with the combination of MM-111 and the anti-estrogen drugs significantly increased apoptosis markers such as cytochrome C and BAX. Furthermore, an in vivo BT474-M3 xenograft model showed resistance to tamoxifen treatment (5 mg/pellet, 60-day release). In this xenograft model MM-111 sensitized tumor response to tamoxifen and the combination treatment dramatically inhibited tumor growth. In conclusion, the combination of MM-111 and endocrine therapies may provide a potent regimen that overcomes acquired resistance to endocrine therapies in ER+, ErbB2-overexpressing breast cancer patients. 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 655. doi:10.1158/1538-7445.AM2011-655