Antisense strategies targeting protein kinase C: preclinical and clinical development

Antisense Strategies Targeting Protein Kinase C: Preclinical and Clinical Development Giampaolo Tortora and Fortunato Ciardiello Altered protein kinase C-␣ (PKC-␣) expression has been implicated in tumor promotion and carcinogenesis. One potentially attractive therapeutic intervention may be the use of selective antisense oligonucleotides to inhibit production of PKC-␣. In preclinical studies, the antisense oligonucleotide LY900003 (ISIS 3521;Affinitak; Isis Pharmaceuticals, Carlsbad, CA) has shown selective inhibition of PKC-␣ mRNA and protein expression and has shown antitumor activity. In clinical studies, LY900003 has shown activity as a single agent, but the most promising data have been obtained in combination with chemotherapy, particularly in patients with non–small cell lung cancer. Data from phase I and II studies have led to ongoing randomized phase III trials in combination with either cisplatin and gemcitabine or carboplatin and paclitaxel. Studies in other tumor types will also investigate the benefit of combining LY900003 with conventional chemotherapy. Semin Oncol 30 (suppl 10):26-31. © 2003 Elsevier Inc. All rights reserved.

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ROTEIN KINASE C (PKC) exists in mammalian cells as a family of at least 12 closely related isozymes with serine-threonine kinase activity. Protein kinase C is involved in the intracellular transduction of a variety of signals, including proliferation, differentiation, migration, adhesion, transformation, and protection from apoptosis, that are triggered by G-protein coupled receptors, tyrosine kinase receptors and other membrane signaling molecules.1-4 Altered PKC expression has been mechanistically implicated in tumor promotion and carcinogenesis.2-4 Among the different isoforms, the ubiquitously expressed PKC-␣ is considered the most consistently altered in human tumor cells. Abnormal PKC-␣ expression has been found in

From the Division of Medical Oncology, Department of Molecular and Clinical Endocrinology and Oncology, Universita˘ di Napoli “Federico II,” Napoli, Italy; and the Department of Experimental and Clinical Medicine “F. Magrassi,” Seconda Universita˘ di Napoli, Napoli, Italy. Address reprint requests to Giampaolo Tortora, MD, PhD, Cattedra di Oncologia Medica, Dipart. Endocrinologia e Oncologia Molecolare e Clinica, Universita˘ di Napoli Federico II, Via S. Pansini 5, 80131, Napoli, Italy. © 2003 Elsevier Inc. All rights reserved. 0093-7754/03/3004-1005$30.00/0 doi:10.1016/S0093-7754(03)00282-3 26

several human tumor types, including prostate, breast, colon, pancreatic, and lung, and in many transformed cell lines.3-10 Overexpression of PKC-␣ in MCF-7 breast cancer cells confers a more aggressive growth in vitro and tumorigenicity in nude mice.11 PKC activity has been shown to correlate with the growth rate of several glioblastoma multiforme cell lines and primary tumors.12 In pancreatic adenocarcinoma cell lines, a positive correlation exists between PKC-␣ activity and more biologically aggressive phenotypes; when a moderately differentiated pancreatic cell line (HPAC) was stably transfected to overexpress PKC-␣, the cells showed a marked decrease in epithelioid cell appearance, and an increase in proliferation rate and anchorage-independent growth in vitro. Animals implanted with these transfected cells died 6 days earlier than animals implanted with control HPAC cells; after 58 days, all the animals implanted with transfected cells had died while 40% of the animals implanted with control cells survived.10 PKC-␣ inhibitors appear to block proliferation, affect growth and survival of tumors, promote apoptosis, and sensitize tumor cells to chemotherapeutic agents; inhibition of PKC-␣ has been shown to reduce neoplastic properties in human lung carcinoma cells.3,12,13 In addition, overexpression of PKC-␣ has been associated with increased expression of the multidrug resistance phenotype that may in part be explained by the promoter region of the multidrug resistance genes possessing a recognition site for PKC-␣.3,14-19 For instance, ovarian cancer cells have shown an increased uptake of gemcitabine following down-regulation of PKC-␣.20 Moreover, 12-O-tetradecanoylphorbol-13-acetate-mediated depletion of PKC-␣ isoform, but not of PKC-␨, selectively increased cisplatin and carboplatin sensitivity in wild type and platinum-resistant ovarian cancer cells, suggesting a central role for PKC-␣ in modulating platinum sensitivity.21 For the above reasons, PKC has been considered an attractive target for therapeutic intervention. However, the similarity in the structural characteristics of PKC isozymes prevented the development of selective inhibitors targeting PKC-␣ proSeminars in Oncology, Vol 30, No 4, Suppl 10 (August), 2003: pp 26-31

ANTISENSE STRATEGIES TARGETING PROTEIN KINASE C

tein. Therefore, the antisense oligonucleotide (ASO) strategy has been pursued as an alternative approach to block selectively PKC-␣.22 ANTISENSE OLIGONUCLEOTIDES

Antisense oligonucleotides are devised to selectively inhibit the expression of specific genes by targeting mRNA. Antisense technology has developed relatively recently, as the main translation of biochemical work to preclinical models dates back only to the 1990s.23,24 Antisense oligonucleotides bind to the target single-strand mRNA or premRNA by Watson-Crick base pairing, and modulate pre-mRNA processing or inhibit mRNA translation through several mechanisms.25 The most important one is the recruitment of endogenous RNase H, a ubiquitous endonuclease that cleaves the RNA strand of a DNA-RNA heteroduplex. Other mechanisms include inhibition of translation by disrupting ribosome assembly on the target mRNA (“translational arrest”). Inhibition of translation through translational arrest mechanisms typically involves the targeting of 5⬘-untranslated region or the translational initiation AUG site of a targeted mRNA.25 Chemical modifications of ASOs have been made to avoid the natural degradation of the phosphodiester backbone by cellular nucleases and to improve ASO efficacy. The most common modification is the replacement of a nonbridging oxygen atom of the phosphodiester moiety by sulfur (phosphorothioates). Phosphorothioate segments of ASOs are very efficient in inducing RNase H cleavage, and have also high affinity/specificity for RNA binding. The majority of ASOs belong to this class. Other chemical modifications of the phosphodiester backbone include the substitution of a methyl group (methylhosphonates) or amino group (phosphoramidates), or substitutions at the 2⬘-sugar position with methyl (2⬘-O-methyl ASO) or methoxy-ethyl (2⬘-O-methoxy-ethyl) groups (“second-generation” compounds). PRECLINICAL STUDIES WITH ANTISENSE OLIGONUCLEOTIDES

Early studies were conducted with a 20-mer phosphorothioates oligodeoxynucleotide targeting the initiation codon of the mRNA encoding the murine PKC-␣. This compound, mixed with cationic liposomes, selectively inhibited, in a doseand time-dependent fashion, the expression of

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PKC-␣ in cell culture.26 Intraperitoneal administration of this ASO to normal mice showed dose- and oligonucleotide sequence-dependent inhibition of PKC-␣ expression in liver that was selective for the PKC-␣ isoform.26 Several 20-mer phosphorothioates antisense oligodeoxynucleotides were screened for their ability to inhibit expression of human PKC-␣ in A549 human lung carcinoma cells in vitro.26 This exercise resulted in the identification of ISIS 3521, a highly potent and selective inhibitor of PKC-␣ expression that targets the 3⬘-untranslated region of human PKC-␣ mRNA. Inhibition of PKC-␣ expression by ISIS 3521 was accompanied by inhibition of phorbol myristate acetate-stimulated intercellular adhesion molecule (ICAM-1) expression.27 In this study, a chemically modified version of ISIS 3521 that does not support RNase H activity was used to show that cleavage by RNAse H is the mechanism primarily responsible for mediating the activity of ISIS 3521 in cells. ISIS 3521, which was later named LY900003 and (Affinitak; Isis Pharmaceuticals, Carlsbad, CA), was selected for further evaluation. When glioblastoma multiforme cells (A172 GBM) were cultured in the presence of 200 nmol/L LY900003, PKC-␣ levels were selectively reduced within 24 to 72 hours without affecting the expression of other PKC isoforms. A scrambled control oligonucleotide did not have an effect on PKC-␣ levels. Apoptosis was evident within 48 hours after initiating LY900003 treatment and the pro-apoptotic proteins p53 and IGFBP3 were markedly increased after 24 hours of treatment with LY900003; IGFBP3 levels increased to eight times higher than control cells after 72 hours. Cells treated with the scrambled control appeared similar to untreated cells.12 Highly metastatic human melanoma cells (C8161) cultured with LY900003 showed 70% inhibition of PKC-␣ m-RNA when compared with cells treated with a control oligonucleotide. Notably, when the treated cells were injected into athymic mice, metastasis to the lung was suppressed by 75%. Cells treated with the control oligonucleotide produced lung metastases at a rate similar to untreated cells.28 Additionally, it has been shown that a phosphorothioate oligonucleotide with the same antisense sequence as LY900003 down-regulated PKC-␣ mRNA and protein in T24 bladder and PC3 prostate carci-

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noma cells. This agent also appeared to improve the activity of traditional chemotherapeutics.15 When mice bearing both subcutaneous and intracranial U-87 human glioblastoma tumors (U-87 cells overexpress PKC-␣) were treated with LY900003, tumor growth was inhibited and mortality was decreased, whereas treatment with a control oligonucleotide was ineffective. Mice bearing intracranial tumors experienced a doubling of median survival time with 40% long-term survivors. No systemic toxicity was observed following daily intraperitoneal injection for 21 or 80 days in mice bearing subcutaneous or intracranial tumor, respectively. In mice bearing subcutaneous implanted U-87 tumors, the oligonucleotide achieved a tumor concentration of 2 ␮mol/L when administered at a dose of 20 mg/kg for 21 days. The antitumor effect was accompanied by selective reduction of PKC-␣, but not of PKC-⑀ and PKC-␨ levels in subcutaneous tumors.29 When a moderately differentiated pancreatic cell line (HPAC) was treated with LY900003 in vitro, PKC-␣ mRNA was shown to be downregulated. When mice that had been orthotopically implanted with HPAC cells were treated with LY900003, the LY900003-treated animals survived statistically significantly longer than those treated with vehicle alone. Interestingly, animals treated with a scrambled ASO also showed a survival benefit in this study, albeit less robustly than that seen in the LY900003-treated mice.10 LY900003 has also shown antitumor activity against a series of tumor types that were established subcutaneous in nude mice. Between 2 and 3 weeks following introduction of the tumor cells, LY900003 was administered daily intravenously (IV) for 2 to 6 weeks. Antitumor activity was studied in MDA-MB.231 breast carcinoma, Calu-1 lung carcinoma, and MIA PACA 2 pancreatic carcinoma xenograft models. Significant antitumor activity has been observed against all three tumor types, and the observed antitumor effect was highly oligonucleotide sequence-specific (B. Monia, unpublished results, Isis Pharmaceuticals). Finally, studies in prostate cancer xenograft models are ongoing (M. Chedid, Eli Lilly and Company, personal communication, January 2003). These results show that LY900003 displays good antitumor and toxicologic profile in preclinical

TORTORA AND CIARDIELLO

cancer models at relatively low doses, with effective intratumor concentrations of intact oligonucleotide that result in the selective lowering of PKC-␣ protein levels in tumor models. Moreover, in cell culture studies, LY900003 showed the ability to reduce selectively production of PKC-␣ while leaving other PKC isoforms unaffected, thus potentially reducing cumulative toxicity when used with conventional cytotoxic agents. Collectively, these preclinical results justify the progression of LY900003 from the laboratory into a clinical setting. CLINICAL STUDIES WITH LY900003

Based on the broad spectrum of antitumor activity of LY900003, phase I dose-escalation studies in patients with solid tumors resistant to conventional treatments were initiated. In one study (CS02),30 LY900003 was administered to 21 patients with advanced malignancies for 3 weeks by continuous IV infusion followed by a 1-week treatment rest. The maximum tolerated dose was 2.0 mg/kg/day, while dose-limiting thrombocytopenia and fatigue were observed at the dose of 3 mg/kg/ day. Antitumor activity was observed in three patients with ovarian cancer. In particular, one patient obtained a reduction of 60% of measurable disease with a time to progression of 11 months, and two patients experienced a marked reduction in the levels of the serum CA125 tumor marker. This study showed a good correlation between the target doses achieved in humans with those showing antitumor activity in mouse models.30 In a trial conducted with the same schedule in patients with recurrent high-grade astrocytomas (CS10), LY900003 was well tolerated with mild and reversible toxicity, but no clinical benefit was observed.31 In another phase I study (CS01), 36 patients with advanced disease received 99 cycles of LY900003 administered by a 2-hour IV infusion three times a week, for 3 consecutive weeks followed by 1 week of rest. Moderate nausea, fever, fatigue, and thrombocytopenia were observed, although a dose-limiting toxicity level was not achieved. Two patients with heavily pretreated non-Hodgkin’s lymphoma had complete responses.32 A phase II study (CS06) in which patients were randomized to receive either LY900003 or an ASO targeting raf-1 was conducted in chemother-

ANTISENSE STRATEGIES TARGETING PROTEIN KINASE C

apy-naive hormone-refractory prostate cancer patients. Fifteen patients received 43 courses of LY900003 given as a 21-day continuous IV infusion repeated every 4 weeks. Mild to moderate toxicities were observed in 21% of patients. No prostate-specific antigen objective responses were observed, but three patients experienced ⱖ 5 months of stable disease.33 Because single-agent activity with minimal toxicity was observed in phase I trials, a phase I/II trial of combination therapy was initiated. As mentioned above, cell culture data showed that PKC-␣ levels decrease 24 to 72 hours after initiation of LY900003 treatment, with signs of apoptosis beginning after 48 hours; in one study, a proapoptotic protein, IGFBP3, reached its highest levels 72 hours after LY900003 initiation.12,26 Assuming target inhibition should occur before combination therapy, some combination studies have been designed to initiate their cytotoxic chemotherapy 3 days after initiating the LY900003 infusion; however, in the absence of conclusive clinical data linking mechanism of action, pretreatment dosing, and antitumor efficacy, some combination studies have been designed to initiate cytotoxic chemotherapy on the same day that the LY900003 infusion is initiated. In the first phase I/II combination therapy trial (CS3/3N), LY900032 mg/kg/day, was administered by continuous IV infusion on days 0 through 14, while carboplatin (area under the concentration time curve ⫽ 6) and paclitaxel 175 mg/m2 were administered on day 4, in patients with stage IIIB or IV NSCLC. In 53 patients, 83% in stage IV, 258 cycles of therapy were administered, with a median of six cycles and a range of one to nine cycles. Thirteen patients had received one previous therapeutic regimen, while 12 patients had already received at least two former regimens. Toxicity consisted mostly of neutropenia and thrombocytopenia, which were responsible for the delay of treatment in 12 cycles (6 patients) in a total of 19 cycles delayed of more than 1 week. In the 48 evaluable patients, the response rate was 48%, with 2% (1 patient) obtaining a complete response and 46% (22 patients) obtaining a partial responses, while stable disease was observed in 35% (17 patients). Median time to progression and median survival were 6.3 months and 15.9 months, respectively. Thus, the combination of LY900003, carboplatin, and pacli-

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taxel was well tolerated and showed promising activity in NSCLC.34 In a second phase I/II trial (CS15), LY900003 at 2 mg/kg/day was administered by continuous IV infusion days 0 through 14, while cisplatin 80 mg/m2and gemcitabine 1,000 mg/m2 were administered at the same time (day 0), with gemcitabine administered again on day 7. This schedule was evaluated in a cohort of advanced NSCLC patients, with 93% of patients with stage IV disease. The combination was well tolerated, with manageable neutropenia and thrombocytopenia as the main toxicities. No pharmacokinetic interactions were observed. In the phase II portion of the study, 44 evaluable, chemotherapy-naive, advanced NSCLC patients received a median of three cycles of treatment. Toxicity was moderate with thrombocytopenia, neutropenia, anemia, fatigue, dehydration, sepsis, and neutropenic fever. In the updated analysis of the trial, the response rate was 37%, including one complete remission and 11 partial remissions, while 50% of patients obtained a stable disease. LY90003 combined with cisplatin and gemcitabine is well tolerated. Survival data are still pending.35 A third phase II trial was conducted in patients with advanced NSCLC to evaluate the safety and efficacy of LY900003 administered by continuous IV infusion for 14 days plus docetaxel 75 mg/m2 on day 3 of each cycle. In the 53 patients treated, toxicity was moderate and consisted of thrombocytopenia, neutropenia, neutropenic fever, and fatigue. In the 36 evaluable patients, five partial responses (14%) and 15 with stable disease (42%) were observed.36 Collectively, the results of the above studies conducted in NSCLC with LY90003 in combination with different chemotherapy regimens, especially CS3/3N and CS15, were encouraging. In fact, in three studies with comparable NSCLC patient cohorts receiving chemotherapy alone, the median time to disease progression and median survival ranged between 4.2 to 6.9 and 8.1 to 9.1 months, respectively.37-39 On the basis of the above results, two large randomized phase III trials have started as first-line treatment in NSCLC patients: A 600 patient study of LY90003 in combination with carboplatin and paclitaxel and a 1,000-patient study of LY90003 in combination with cisplatin and gemcitabine (the ALERT study). The second phase III

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TORTORA AND CIARDIELLO

trial is a three-arm study, which will examine, in a controlled randomized fashion, the optimal timing of LY900003 dosing in relation to chemotherapy. In one arm, patients are treated with a standard regimen of gemcitabine and cisplatin. In a second arm, patients receive a 14-day infusion of LY900003 at 2 mg/kg/day beginning on day 1, followed by cisplatin 80 mg/m2 on day 4 and gemcitabine 1,250mg/m2 on days 4 and 11. In the third arm, patients initiate all therapy on day 1 and receive their second gemcitabine dose on day 8. Whether pre-dosing with LY900003 maximizes efficacy is important to patients and physicians because same-day dosing will be more convenient if the two regimens show similar efficacy. This question is also important to the academic community, as is the issue of pre-dosing effects on antisense compounds in general. Other studies with LY90003 in combination with chemotherapy have been undertaken in patients affected by other types of cancer. A phase I study with LY900003 in combination with 5-fluorouracil and leucovorin was conducted in patients with refractory solid tumors, the majority affected by colorectal cancer. Fifteen patients received LY900003 at either 1.0, 1.5, or 2 mg/kg/day by 21-day continuous IV infusion, and simultaneously received 5-fluorouracil and leucovorin for 5 consecutive days; treatment was repeated every 4 to 5 weeks. Moderate toxicity was observed except for one grade 3 mucositis and five grade 4 neutropenia. There were no effects on prothrombin time and activated partial thromboplastin time. A clinically defined maximum tolerated dose or classical dose-limiting toxicity were not reached. LY900003 and 5-fluorouracil pharmacokinetics did not affect each other and were similar to other studies. About 20% tumor responses were observed, ranging from minor reduction in tumor size (4 patients) to partial response (2 patients).40 CONCLUSIONS

The ASO LY90003 (ISIS 3521) represents a novel and selective therapeutic agent that has been successfully translated from the bench to clinical trials. Toxicologic studies have shown that LY900003 is well tolerated and does not increase the toxicity of chemotherapeutic agents when used in combination. LY900003 has shown activity as a single agent, but the most promising results are those obtained in combination with chemo-

therapy, particularly in patients affected by NSCLC. The ongoing large randomized phase III trials in combination with either cisplatin and gemcitabine or carboplatin and paclitaxel will provide important information on whether this antisense PKC-␣ ASO should be included in the conventional treatment of NSCLC. Further studies are planned to evaluate whether the treatment of other tumor types will benefit from the combination of LY900003 with conventional treatments. The encouraging results obtained with this “firstin-its class” agent will surely provide a major contribution to open new avenues in the field of therapeutic .ASOs, favoring the development of this novel therapeutic strategy in the treatment of cancer. REFERENCES 1. Nishizuka Y: The molecular heterogeneity of protein kinase C and its implications for cellular regulation. Nature 334:661-665, 1988 2. Newton AC: Regulation of protein kinase C. Curr Opin Cell Biol 9:161-167, 1997 3. Basu A: The potential of protein kinase C as a target for anticancer treatment. Pharmacol Ther 59:257-280, 1993 4. O’Brian CA, Kuo JF: Protein kinase C inhibitors, in Kuo JF (ed): Protein Kinase C. New York, NY, Oxford University Press, 1994, pp 96-120 5. O’Brian C, Vogel VG, Singletary SE, et al: Elevated protein kinase C expression in human breast tumor biopsies relative to normal breast tissue. Cancer Res 49:3215-3217, 1989 6. Kopp R, Noelke B, Sauter G, et al: Altered protein kinase C activity in biopsies of human colonic adenomas and carcinomas. Cancer Res 51:205-210, 1991 7. Gescher A: Towards selective pharmacological modulation of protein kinase C – Opportunities for the development of novel antineoplastic agents. Br J Cancer 66:10-19, 1992 8. La Porta CA, Tessitore L, Comolli R: Changes in protein kinase C alpha, delta and in nuclear beta isoform expression in tumour and lung metastatic nodules induced by diethylnitrosamine in the rat. Carcinogenesis 18:715-719, 1997 9. Cornford P, Evans J, Dodson A, et al: Protein kinase C isoenzyme patterns characteristically modulated in early prostate cancer. Am J Pathol 154:137-144, 1999 10. Denham DW, Franz MG, Denham W, et al: Directed antisense therapy confirms the role of protein kinase C-alpha in the tumorigenicity of pancreatic cancer. Surgery 124:218-223, 1998 11. Ways DK, Kukoly CA, deVente J, et al: MCF-7 breast cancer cells transfected with PKC␣ exhibit altered expression of other protein kinase isoforms and display a more aggressive neoplastic phenotype. J Clin Invest 95:1906-1915, 1995 12. Shen L, Dean NM, Glazer RI: Induction of p53-dependent, insulin-like growth factor-binding protein-3-mediated apoptosis in glioblastoma multiforme cells by a protein kinase

ANTISENSE STRATEGIES TARGETING PROTEIN KINASE C

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28. Dennis JU, Dean NM, Bennett CF: Human melanoma metastasis is inhibited following ex vivo treatment with an antisense oligonucleotide to protein kinase C alpha. Cancer Lett 128:65-70, 1998 29. Yazaki T, Ahmad S, Chahlavi A, et al: Treatment of glioblastoma U-87 by systemic administration of an antisense protein kinase C-␣ phosphorothioate oligodeoxynucleotide. Mol Pharmacol 50:236-242, 1996 30. Yuen AR, Halsey J, Fisher GA, et al: Phase I study of an antisense oligonucleotide to protein kinase C-alpha (ISIS 3521/CGP 64128A) in patients with cancer. Clin Cancer Res 5:3357-3363, 1999 31. Alavi J, Grossman S, Supko J, et al: Efficacy, toxicity, and pharmacology of an antisense oligonucleotide directed against protein kinase C-alpha (ISIS 3521) delivered as a 21-day continuous intravenous infusion in patients with recurrent high grade astrocytomas (HGA). Proc Am Soc Clin Oncol 19:167, 2000 (abstr 647) 32. Nemunaitis J, Holmlund JT, Kraynak M, et al: Phase I evaluation of ISIS 3521, an antisense oligodeoxynucleotide to protein kinase C-alpha, in patients with advanced cancer. J Clin Oncol 17:3586-3595, 1999 33. Tolcher AW, Reyno L, Venner PM, et al: A randomized phase II and pharmacokinetic study of the antisense oligonucleotides ISIS 3521 and ISIS 5132 in patients with hormonerefractory prostate cancer. Clin Cancer Res 8:2530-2535, 2002 34. Yuen AR, Halsey J, Fisher GA, et al: A phase I/II trial of ISIS 3521, an antisense inhibitor of protein kinase C-alpha, with carboplatin and paclitaxel in non–small cell lung cancer. Proc Am Soc Clin Oncol 20:309a, 2001 (abstr 1234) 35. Ritch PS, Belt R, George S, et al: Phase I/II trial of ISIS 3521/LY900003, an antisense inhibitor of PKC-alpha with cisplatin and gemcitabine in advanced non-small cell lung cancer (NSCLC). Proc Am Soc Clin Oncol 21:309a, 2002 (abstr 1233) 36. Moore MR, Saleh M, Jones CM, et al: Phase II trial of ISIS 3521/LY900003, an antisense inhibitor of PKC-alpha, with docetaxel in non–small cell lung cancer (NSCLC). Proc Am Soc Clin Oncol 21:297a, 2002 (abstr 1186) 37. Cardenal F, Lopez-Cabrerizo MP, Anton A, et al: Randomized phase III study of gemcitabine-cisplatin versus etoposide-cisplatin in the treatment of locally advanced or metastatic non–small-cell lung cancer. J Clin Oncol 17:12-18, 1999 38. Sandler AB, Nemunaitis J, Denham C, et al: Phase III trial of gemcitabine plus cisplatin versus cisplatin alone in patients with locally advanced or metastatic non–small-cell lung cancer. J Clin Oncol 18:122-130, 2000 39. Schiller JH, Harrington D, Belani CP, et al, and the Eastern Cooperative Oncology Group: Comparison of four chemotherapy regimens for advanced non-small-cell lung cancer. N Engl J Med 346:92-98, 2002 40. Mani S, Rudin CM, Kunkel K, et al: Phase I clinical and pharmacokinetic study of protein kinase C-alpha antisense oligonucleotide ISIS 3521 administered in combination with 5-fluorouracil and leucovorin in patients with advanced cancer. Clin Cancer Res 8:1042-1048, 2002