Luna Musib

The Protein Kinase Cβ–Selective Inhibitor, Enzastaurin (LY317615.HCl), Suppresses Signaling through the AKT Pathway, Induces Apoptosis, and Suppresses Growth of Human Colon Cancer and Glioblastoma Xenografts

Activation of protein kinase Cbeta (PKCbeta) has been repeatedly implicated in tumor-induced angiogenesis. The PKCbeta-selective inhibitor, Enzastaurin (LY317615.HCl), suppresses angiogenesis and was advanced for clinical development based upon this antiangiogenic activity. Activation of PKCbeta has now also been implicated in tumor cell proliferation, apoptosis, and tumor invasiveness. Herein, we show that Enzastaurin has a direct effect on human tumor cells, inducing apoptosis and suppressing the proliferation of cultured tumor cells. Enzastaurin treatment also suppresses the phosphorylation of GSK3betaser9, ribosomal protein S6(S240/244), and AKT(Thr308). Oral dosing with Enzastaurin to yield plasma concentrations similar to those achieved in clinical trials significantly suppresses the growth of human glioblastoma and colon carcinoma xenografts. As in cultured tumor cells, Enzastaurin treatment suppresses the phosphorylation of GSK3beta in these xenograft tumor tissues. Enzastaurin treatment also suppresses GSK3beta phosphorylation to a similar extent in peripheral blood mononuclear cells (PBMCs) from these treated mice. These data show that Enzastaurin has a direct antitumor effect and that Enzastaurin treatment suppresses GSK3beta phosphorylation in both tumor tissue and in PBMCs, suggesting that GSK3beta phosphorylation may serve as a reliable pharmacodynamic marker for Enzastaurin activity. With previously published reports, these data support the notion that Enzastaurin suppresses tumor growth through multiple mechanisms: direct suppression of tumor cell proliferation and the induction of tumor cell death coupled to the indirect effect of suppressing tumor-induced angiogenesis.

Phase III Study of Enzastaurin Compared With Lomustine in the Treatment of Recurrent Intracranial Glioblastoma

PURPOSE This phase III open-label study compared the efficacy and safety of enzastaurin versus lomustine in patients with recurrent glioblastoma (WHO grade 4). PATIENTS AND METHODS Patients were randomly assigned 2:1 to receive 6-week cycles of enzastaurin 500 mg/d (1,125-mg loading dose, day 1) or lomustine (100 to 130 mg/m(2), day 1). Assuming a 45% improvement in progression-free survival (PFS), 397 patients were required to provide 80% power to achieve statistical significance at a one-sided level of .025. RESULTS Enrollment was terminated at 266 patients (enzastaurin, n = 174; lomustine, n = 92) after a planned interim analysis for futility. Patient characteristics were balanced between arms. Median PFS (1.5 v 1.6 months; hazard ratio [HR] = 1.28; 95% CI, 0.97 to 1.70), overall survival (6.6 v 7.1 months; HR = 1.20; 95% CI, 0.88 to 1.65), and 6-month PFS rate (P = .13) did not differ significantly between enzastaurin and lomustine, respectively. Stable disease occurred in 38.5% and 35.9% of patients and objective response occurred in 2.9% and 4.3% of patients, respectively. Time to deterioration of physical and functional well-being and symptoms did not differ between arms (HR = 1.12; P = .54). Four patients discontinued enzastaurin because of drug-related serious adverse events (AEs). Eleven patients treated with enzastaurin died on study (four because of AEs; one was drug-related). All four deaths that occurred in patients receiving lomustine were disease-related. Grade 3 to 4 hematologic toxicities were significantly higher with lomustine (46 events) than with enzastaurin (one event; P < or = .001). CONCLUSION Enzastaurin was well tolerated and had a better hematologic toxicity profile but did not have superior efficacy compared with lomustine in patients with recurrent glioblastoma.

Phase I Dose Escalation and Pharmacokinetic Study of Enzastaurin, an Oral Protein Kinase C Beta Inhibitor, in Patients With Advanced Cancer

PURPOSE This phase I study was conducted to determine the recommended dose of enzastaurin, an oral protein kinase C beta (PKCbeta) inhibitor, for phase II trials. Secondary objectives were maximum-tolerated dose (MTD), pharmacokinetics (PK), toxicity, and response. PATIENTS AND METHODS Patients at least 18 years of age with advanced cancer and an Eastern Cooperative Oncology Group performance status of 0 or 1 lower received enzastaurin orally once daily at a starting dose of 20 mg. Dose escalation proceeded using a modified Simon design. RESULTS All 47 patients enrolled (mean age, 58 years) received at least one dose of enzastaurin, with a median of two cycles (range, one to 17 cycles). Prevalent malignancies were lung (n = 10) and head and neck cancers (n = 9). Although no MTD was identified up to 700 mg/d, 525 mg was chosen as the recommended dose, and 12 additional patients were accrued at that level. Three dose-limiting toxicities (QTc changes) occurred: one at the 700-mg dose (patient discontinued), and two in the expansion cohort at the 525-mg dose. Total analytes (enzastaurin and its metabolites) exposure increased with increasing doses up to 240 mg, and appeared to plateau at 525 and 700 mg. Grade 1 chromaturia, fatigue, and other GI toxicities were the most common, while no clinically significant grade 3/4 toxicities occurred. Two deaths, unrelated to enzastaurin, occurred. Twenty-one patients (45%) achieved stable disease (SD) for two to 16 cycles. CONCLUSION On the basis of plasma exposures and safety data, enzastaurin 525 mg once daily is the recommended phase II dose. Enzastaurin is well tolerated up to 700 mg/d. Evidence of early activity was seen with significant stable disease.

A phase I/II trial of enzastaurin in patients with recurrent high-grade gliomas.

Enzastaurin, a potent inhibitor of protein kinase C-beta, inhibits angiogenesis and has direct cytotoxic activity against glioma cells in preclinical studies. Patients with recurrent high-grade gliomas were stratified by histology and use of enzyme-inducing antiepileptic drugs (EIAEDs). Patients on EIAED were treated on the phase I dose-escalation portion of the trial with evaluation of serum pharmacokinetics as the primary endpoint. Patients not on EIAED were treated on the phase II portion of the trial with radiographic response and progression-free survival (PFS) as primary objectives. Patients in phase I received enzastaurin 525-900 mg/d. Phase II patients received 500 or 525 mg/d. One hundred and eighteen patients were accrued to this trial. Therapy was well tolerated with thrombosis, thrombocytopenia, hemorrhage, and elevated alanine aminotransferase as the most commonly observed drug-associated grade 3 or higher toxicities. Patients on EIAED had serum enzastaurin exposure levels approximately 80% lower than those not on EIAED. Dose escalations up to 900 mg/d did not substantially increase serum exposure levels and a maximally tolerated dose was never reached. Twenty-one of 84 evaluable patients (25%) experienced an objective radiographic response. The 6-month PFS was 7% for patients with glioblastoma and 16% for patients with anaplastic glioma. Phosphorylation of glycogen synthase kinase-3 in peripheral blood mononuclear cells was identified as a potential biomarker of drug activity. Enzastaurin has anti-glioma activity in patients with recurrent high-grade glioma, but does not appear to have enough single-agent activity to be useful as monotherapy.

Phase I Pharmacokinetic and Pharmacodynamic Study of the Oral Protein Kinase C β-Inhibitor Enzastaurin in Combination with Gemcitabine and Cisplatin in Patients with Advanced Cancer

Purpose: Enzastaurin targets the protein kinase C and phosphatidylinositol 3-kinase/AKT pathways to reduce tumor angiogenesis and cell proliferation and to induce cell death. A phase I trial was conducted to evaluate the feasibility of combining enzastaurin with gemcitabine and cisplatin. Experimental Design: Patients with advanced cancer received a 14-day lead-in treatment with oral enzastaurin followed by subsequent 21-day cycles of daily enzastaurin, gemcitabine on days 1 and 8, and cisplatin on day 1. Enzastaurin doses were escalated between 350 mg once daily to 500 mg twice daily, whereas gemcitabine doses were either 1,000 or 1,250 mg/m2 and cisplatin doses were either 60 or 75 mg/m2. Circulating endothelial cell numbers and CD146 and CD133 mRNA expression were evaluated as pharmacodynamic markers. Results: Thirty-three patients (median age, 58 years) were enrolled in seven dose levels. The maximum tolerated dose was not identified. Two dose-limiting toxicities (grade 2 QT interval corrected for heart rate prolongation and grade 3 fatigue) were reported. Other toxicities included grade 3/4 neutropenia (3 of 6 patients), thrombocytopenia (1 of 6 patients), grade 3 leukopenia (2 patients), and fatigue (5 patients). Enzastaurin twice daily (≥250 mg) resulted in more discontinuations and low-grade toxicities. In the combination, enzastaurin exposures decreased slightly but remained above the target of 1,400 nmol/L, whereas gemcitabine/cisplatin exposures were unaltered. Three patients (9.1%) had partial responses and 13 (39.4%) had stable disease. Measurement of circulating endothelial cell numbers and CD146 and CD133 mRNA expression did not contribute to decision-making on dose escalation. Conclusions: Recommended phase II dose is 500 mg enzastaurin once daily, 1,250 mg/m2 gemcitabine, and 75 mg/m2 cisplatin. This regimen is well tolerated with no significant alterations in the pharmacokinetic variables of any drug.

Development and Validation of a Drug Activity Biomarker that Shows Target Inhibition in Cancer Patients Receiving Enzastaurin, a Novel Protein Kinase C-β Inhibitor

Purpose: To evaluate the effects of the novel protein kinase C (PKC) inhibitor enzastaurin on intracellular phosphoprotein signaling using flow cytometry and to use this approach to measure enzastaurin effects on surrogate target cells taken from cancer patients that were orally dosed with this agent. Experimental Design: The activity of PKC was assayed in intact cells using a modification of published techniques. The U937 cell line and peripheral blood mononuclear cells were stimulated with phorbol ester, fixed, permeabilized, and reacted with an antibody specific for the phosphorylated forms of PKC substrates. The processed samples were quantitatively analyzed using flow cytometry. The assay was validated for selectivity, sensitivity, and reproducibility. Finally, blood was obtained from volunteer cancer patients before and after receiving once daily oral doses of enzastaurin. These samples were stimulated ex vivo with phorbol ester and were assayed for PKC activity using this approach. Results: Assay of U937 cells confirmed the selectivity of the antibody reagent and enzastaurin for PKC. Multiparametric analysis of peripheral blood mononuclear cells showed monocytes to be the preferred surrogate target cell. Day-to-day PKC activity in normal donors was reproducible. Initial results showed that five of six cancer patients had decreased PKC activity following enzastaurin administration. In a following study, a group of nine patients displayed a significant decrease in PKC activity after receiving once daily oral doses of enzastaurin. Conclusion: An inhibition of surrogate target cell PKC activity was observed both in vitro and ex vivo after exposure to the novel kinase inhibitor, enzastaurin.

A phase I trial of enzastaurin in patients with recurrent gliomas.

Purpose: Enzastaurin is a selective inhibitor of protein kinase C β. Prior phase I studies did not show increased drug exposures with escalating once daily administration. Limits from gastrointestinal absorption may be overcome by twice daily dosing, potentially improving antitumor effects. Experimental Design: We conducted a phase I dose escalation study in 26 patients with recurrent malignant glioma, stratified by use of enzyme-inducing antiepileptic drugs, to investigate whether divided twice daily dosing results in higher exposures compared with once daily dosing. Phosphorylated glycogen synthase 3 β was analyzed as a potential biomarker of enzastaurin activity. Results: Enzastaurin was poorly tolerated at all dose levels evaluated (500, 800, and 1,000 mg total daily), with thrombocytopenia and prolonged QTc as dose-limiting toxicities. The average drug concentration of enzastaurin under steady-state conditions was doubled by twice daily dosing compared with daily dosing [1.990; 90% confidence interval (CI), 1.450-2.730]. Additionally, geometric mean ratios doubled with 800 versus 500 mg dosing for both daily (2.687; 90% CI, 1.232-5.860) and twice daily regimens (1.852; 90% CI, 0.799-4.292). Two patients achieved long-term benefit (over 150 weeks progression free). Conclusions: Higher and more frequent dosing of enzastaurin resulted in improved drug exposure but with unacceptable toxicity at the doses tested. Phosphorylated glycogen synthase 3 β may be a useful biomarker of the biological activity of enzastaurin. Enzastaurin has activity in a subset of malignant glioma patients and warrants continued study in combination with other agents using a maximal once daily dose of 500 mg.

Enzastaurin plus temozolomide with radiation therapy in glioblastoma multiforme: A phase I study†

We conducted a phase I study to determine the safety and recommended phase II dose of enzastaurin (oral inhibitor of the protein kinase C-beta [PKCbeta] and the PI3K/AKT pathways) when given in combination with radiation therapy (RT) plus temozolomide to patients with newly diagnosed glioblastoma multiforme or gliosarcoma. Patients with Karnofsky performance status > or =60 and no enzyme-inducing anti-epileptic drugs received RT (60 Gy) over 6 weeks, concurrently with temozolomide (75 mg/m(2) daily) followed by adjuvant temozolomide (200 mg/m(2)) for 5 days/28-d cycle. Enzastaurin was given once daily during RT and adjuvantly with temozolomide; the starting dose of 250 mg/d was escalated to 500 mg/d if < or =1/6 patients had dose-limiting toxicity (DLT) during RT and the first adjuvant cycle. Patients continued treatment for 12 adjuvant cycles unless disease progression or unacceptable toxicity occurred. Twelve patients enrolled. There was no DLT in the first 6 patients treated with 250 mg enzastaurin. At 500 mg, 2 of 6 patients experienced a DLT (1 Grade 4 and 1 Grade 3 thrombocytopenia). The patient with Grade 3 DLT recovered to Grade <1 within 28 days and adjuvant temozolomide and enzastaurin was reinitiated with dose reductions. The other patient recovered to Grade <1 toxicity after 28 days and did not restart treatment. Enzastaurin 250 mg/d given concomitantly with RT and temozolomide and adjuvantly with temozolomide was well tolerated and is the recommended phase II dose. The proceeding phase II trial has finished accrual and results will be reported in 2009.

A phase I safety, tolerability, and pharmacokinetic study of enzastaurin combined with capecitabine in patients with advanced solid tumors

Enzastaurin, an oral inhibitor of protein kinase C&bgr;, affects signal transduction associated with angiogenesis, proliferation, and survival. Capecitabine is converted to 5-fluoruracil by thymidine phosphorylase, a putative angiogenic factor. The all-oral combination of the two drugs offers the potential for targeting angiogenesis in capecitabine-sensitive tumors with nonoverlapping toxicities. Patients with advanced cancer initially received single-agent enzastaurin to achieve steady-state concentrations (cycle 1). In subsequent 21-day cycles, enzastaurin was given orally, once daily, on days 1–21 and capecitabine orally, twice daily (b.i.d.), on days 1–14 in three dose-level cohorts. Three dose-escalation cohorts were studied: cohort 1 (n=8), 350 mg of enzastaurin +capecitabine (750 mg/m2 b.i.d.); cohort 2 (n=7), enzastaurin (350 mg)+capecitabine (1000 mg/m2 b.i.d.); cohort 3 (n=12), 525-mg capsules or 500-mg enzastaurin+capecitabine (1000 mg/m2 b.i.d.). Further dose escalation was not pursued because of emerging data that enzastaurin systemic exposure did not increase at doses above 525 mg. Although a traditional toxicity-based maximum tolerated dose was not achieved, the highest dosing cohort represented a biologically relevant dose of enzastaurin, on the basis of preclinical data and correlative pharmacodynamic biomarker assays of protein kinase C&bgr; inhibition in peripheral blood mononucleocytes, in combination with a standard dose of capecitabine. For the 500/525-mg dose, ratios of total enzastaurin analyte geometric means (i.e. enzastaurin alone versus enzastaurin with capecitabine) reflected a trend toward decreased enzastaurin exposure, but did not reach statistical significance. The pharmacokinetic parameters of capecitabine with enzastaurin were similar to those previously reported for single-agent capecitabine. The regimen was well tolerated, without any consistent pattern of drug-related grade 3 or grade 4 toxicities being observed. Although no objective tumor responses were documented, five patients maintained stable disease for ≥6 months (range: 6–9.7 months). The recommended phase II dose of this combination, based on the results of this study, is enzastaurin at a daily dose of 500 mg (tablet formulation) and capecitabine (1000 mg/m2, b.i.d.) on days 1–14 every 21 days. Further disease-directed studies are warranted, such as in malignancies in the treatment of which both capecitabine and inhibitors of angiogenesis have previously been benchmarked as being effective.

Phase I and Pharmacokinetic Study of Pemetrexed plus Cisplatin in Chemonaive Patients with Locally Advanced or Metastatic Malignant Pleural Mesothelioma or Non ^ Small Cell Lung Cancer

Purpose: Pemetrexed is approved as monotherapy and in combination with cisplatin. The established combination dose was identified before the addition of folic acid and vitamin B12 to the treatment regimen. We evaluated the toxicity and pharmacokinetics (PK) of higher pemetrexed doses with cisplatin and vitamin supplementation. Experimental Design: Patients with malignant pleural mesothelioma or non–small cell lung cancer received pemetrexed doses from 500 to 900 mg/m2 + 75 mg/m2 cisplatin once every 21 days. Folic acid and vitamin B12 were administered per label recommendations. Results: Twenty-one patients received a combined total of 84 cycles. The maximum tolerated dose was 900 mg/m2 pemetrexed + 75 mg/m2 cisplatin. Dose-limiting toxicities at this dose included grade 3 anemia, bronchopneumonia, and neutropenia, and 1 death from sepsis secondary to grade 4 febrile neutropenia, considered possibly related to study drugs. The recommended dose was 800 mg/m2 pemetrexed + 75 mg/m2 cisplatin. Pemetrexed PK were consistent across doses; pemetrexed did not seem to affect total or free platinum PK. Conclusions: Pemetrexed with vitamin supplementation was safe and well tolerated at higher doses than the currently established 500 mg/m2 + 75 mg/m2 cisplatin. Based on this study, the recommended dose would be 800 mg/m2 pemetrexed + 75 mg/m2 cisplatin. However, recent studies showed a lack of improved efficacy for 900 or 1,000 mg/m2 single-agent pemetrexed versus 500 mg/m2 and a lack of PK/pharmacodynamic exposure-response relationship for the pemetrexed/cisplatin combination across pemetrexed exposures corresponding to this dose range. Based on currently available evidence, we recommend retaining the established dose.