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Phase I Study of Liposome-Encapsulated c-raf Antisense Oligodeoxyribonucleotide Infusion in Combination with Radiation Therapy in Patients with Advanced Malignancies

Authors' Affiliations: ¹ Departments of Radiation Medicine and Medicine, Lombardi Comprehensive Cancer Center, Georgetown University, Washington, District of Columbia; ² Temple-Fox Chase Cancer Center, Philadelphia, Pennsylvania; ³ The University of Chicago, Chicago, Illinois; and ⁴ NeoPharm, Inc., Lake Forest, Illinois

Requests for reprints: Anatoly Dritschilo, Department of Radiation Medicine, E202, Research Building, Georgetown University, Washington, DC, 20007. E-mail: dritscha{at}georgetown.edu.

	Abstract

Top Abstract Patients and Methods Results Discussion References

 
Purpose: Raf proteins are key elements of growth-related cellular signaling pathways and are a component of cancer cell resistance to radiation therapy. Antisense oligonucleotides to c-raf-1 permit highly selective inhibition of the gene product and offer a strategy for sensitizing cancer cells to radiation therapy. In this dose escalation study, we evaluated the safety of combined liposomal formulation of raf antisense oligonucleotide (LErafAON) and radiation therapy in patients with advanced malignancies.

Experimental Design: Patients with advanced solid tumors were treated with LErafAON in a phase I dose escalation study while receiving palliative radiation therapy. Drug-related and radiation-related toxicities were monitored. Pharmacokinetics and expression of c-raf-1 mRNA and Raf-1 protein were determined in peripheral blood mononuclear cells.

Results: Seventeen patients with palliative indications for radiation therapy were entered into this study. Thirteen patients received daily infusions of LErafAON and four received twice-weekly infusions. Radiation therapy was delivered in daily 300-cGy fractions over 2 weeks. Patients tolerated radiation, and no unexpected radiation-related side effects were observed. Drug-related reactions (grade 2), such as back pain, chills, dyspnea, fatigue, fever, flushing, and hypertension, were observed in most patients and were managed by premedication with corticosteroids and antihistamines. Serious adverse events occurred in five patients, including acute infusion-related symptoms, abnormal liver function tests, hypoxia, dehydration, diarrhea, esophagitis, fever, hypokalemia, pharyngitis, and tachypnea. Twelve of 17 patients were evaluable for tumor response at completion of treatment; four showed partial response, four showed stable disease, and four experienced progressive disease. The intact rafAON was detected in plasma for 30 minutes to several hours. Six patients with partial response or stable disease were evaluable for c-raf-1 mRNA and/or Raf-1 protein expression. Inhibition of c-raf-1 mRNA was observed in three of five patients. Raf-1 protein was inhibited in four of five patients.

Conclusion: This is the first report of the combined modality treatment using antisense oligonucleotides with radiation therapy in patients with advanced cancer. A dose of 2.0 mg/kg of LErafAON administered twice weekly is tolerated with premedication and does not enhance radiation toxicity in patients. The observation of dose-dependent, infusion-related reactions has led to further modification of the liposomal composition for use in future clinical trials.

Our previous studies have identified the activated c-raf-1 gene as a factor in the radiation resistance of squamous carcinoma cells obtained from a patient with progressive cancer of head and neck origin (1, 5). These observations have been supported by further elucidation of Raf-1 involvement in signaling through the epidermal growth factor receptor/Ras/Raf/mitogen-activated protein kinase and the nuclear factor-B–mediated pathways (6–8). Taken together, these data support a central role for Raf-1 in cancer growth and identify it as an attractive target for therapeutic gain.

Raf-1 is a cytoplasmic serine/threonine kinase with roles in signal transduction pathways that are involved in cell growth and survival (9). Inhibition of Raf-1 expression using antisense strategies results in radiation sensitization of radioresistant cancer cells (5, 10, 11). A liposomally encapsulated antisense oligonucleotide preparation (LErafAON) that targets the translation initiation site of human c-raf-1 mRNA has been developed and approved for clinical testing (12). The terminal base linkages at the 5' and 3' ends of the antisense oligonucleotide sequence are modified to phosphorothioate groups (13). The liposomes are based on a formulation containing dimethyldioctadecyl ammonium bromide, egg phosphatidylcholine, and cholesterol, as previously reported (12). Liposomal delivery of antisense oligonucleotides offers potential benefits related to extended circulation time, reduced drug-related toxicity, and improved efficacy.

The activity of LErafAON alone or in combination with radiation therapy has been evaluated in human tumor xenografts, including squamous cell carcinomas, prostate, breast, and pancreatic cancers (12, 14, 15). Temporary delays in tumor growth have been observed in animals given LErafAON as monotherapy, and tumor regression or eradication has been observed in animals given LErafAON in combination with radiation therapy (12–15). LErafAON has been evaluated in toxicity studies in mice and rabbits, and maximum tolerated doses (MTD) of LErafAON by i.v. infusion in combination with radiation therapy have been determined. More recently, a dose escalation study evaluated the MTD and toxicities of LErafAON given as weekly i.v. infusion in patients with advanced solid tumors (16). The present study reports the first clinical use of LErafAON in combination with radiation therapy in patients with advanced malignancies.

	Patients and Methods

Top Abstract Patients and Methods Results Discussion References

 
Eligibility and patient evaluation. Patients with histologically confirmed malignancy that recurred or progressed after initial definitive treatment and for which no effective standard therapy was available were candidates for this study. Palliative radiation therapy indications for relief of pain or tumor mass effects were required. Other eligibility criteria included (a) age 18 to 80 years; (b) Eastern Cooperative Oncology Group performance status 0 to 2 (ambulatory and able to care for self); (c) >4 weeks since any prior therapy and recovery from any side effects; (d) measurable or evaluable tumor documentation 2 weeks before study entry; (e) adequate hematopoietic, hepatic, and renal functions; (f) no history of excessive toxicity from prior radiation therapy; (g) no infections requiring i.v. antibiotics, no HIV infection, and no hepatitis B or hepatitis C; (h) no central nervous system metastasis; and (i) no previous course of radiation therapy to the targeted tumor site. Written informed consent was obtained in accordance with federal and institutional guidelines on a protocol approved by the Institutional Review Boards at Georgetown University, Temple University, and University of Chicago.

Drug preparation and administration. LErafAON was supplied by NeoPharm, Inc. (Lake Forest, IL) in a two-vial system. Lyophilized c-raf-1 antisense oligodeoxynucleotide (rafAON; 50 mg) and blank liposomes (750 mg) were provided in separate glass vials. To reconstitute, 25 mL of normal saline was added to the rafAON and swirled to dissolve. The rafAON solution was then transferred to the vial containing blank liposomes, vortexed vigorously for 2 minutes, allowed to hydrate at room temperature for 2 hours, and then sonicated at maximum intensity in a bath-type sonicator for 10 minutes at room temperature. Drug administration was done i.v. 2 hours before radiation therapy. Planned infusion times ranged from 30 minutes to 3 hours.

Radiation therapy. Radiation therapy was delivered by linear accelerators with energies of 4 MeV. The treatment schedule was designed according to conventional daily radiation therapy scheduling; 5 days of treatment (Monday to Friday), a 2-day break followed by an additional 5 days of treatment (Monday to Friday). The gross tumor volume was documented by physical examination and diagnostic imaging studies using a margin of 1 to 2 cm. Conventional clinical considerations were used for shielding critical normal tissue structures with adherence to dose and volume limitations of lung, liver, kidney, heart, and spinal cord.

All fields were simulated before the start of treatment. The prescribed dose encompassed the tumor volume and the maximum dose did not exceed 115% of the prescribed dose. The daily fraction size was 300 cGy.

Toxicity and dose modifications. Toxicities were graded in accordance with the National Cancer Institute Common Toxicity Criteria version 2. Toxicities were elicited from patients at the end of each course of treatment. Safety was assessed through the performance of periodic physical examination, vital sign measurements, clinical chemistry and hematology assessments, and adverse event surveillance. The MTD was defined on the daily schedule (five doses per week) as the dose level below the dose that caused unacceptable toxicity in two or more of up to six patients. Dose-limiting toxicity was defined as grade 3 or 4 hematologic or radiation-related adverse events. Allergic and/or hypersensitivity reactions were not considered dose limiting, because they are not generally dose dependent, and were managed with a premedication regimen consisting of diphenhydramine (50 mg), famotidine (20 mg), and i.v. methylprednisolone (100 mg) or oral dexamethasone (4 mg, bid).

Clinical monitoring. Patient history, physical examination, diagnostic studies, and laboratory data were obtained before study entry. Physical examination, performance status, and laboratory studies were recorded on days 5, 12, 19, and 50. Pre- and post-infusion platelet counts, partial thromboplastin time, alanine aminotransferase, C3, C4, and CH₅₀ were obtained on days 1, 3, and 10. Pharmacokinetic assays were obtained on days 1 and 11, and c-raf-1 mRNA and Raf-1 protein determinations were done on days 1, 4, 11, and 30.

Tumor measurements by diagnostic studies (when appropriate) were done at the start of treatment and end of treatment (days 29-57). Follow-up for delayed toxicities was done at 2-month intervals for 6 months after completion of therapy.

Quantification of rafAON in human plasma: bioanalytic method validation. Pre-study validation experiments were done to quantify rafAON in human plasma using a gel electrophoresis method (12). The effects of dilution, freeze-thaw cycles, and storage at –80°C and 4°C on the precision and accuracy of the limits of quantification of rafAON were determined. Other variables included the integrity of rafAON during sample processing, specificity of rafAON sequence, and interference of lipids in the assays. The acceptance criteria were <25% coefficient of variation (CV) for precision and 100 ± 25% analytic recovery (AR). The precision and accuracy of the four standard curve pools of rafAON (0.01-0.5 µg/mL, designated as S1-S4) ranged from 8.80% to 17.2% CV and 93.9% to 104% AR, respectively (n = 6). The precision and accuracy of three control concentration pools (0.015, 0.075, and 0.45 µg/mL, designated as C1-C3) ranged from 11.9% to 19.5% CV and 90.0% to 99.3% AR, respectively (n = 6). The precision and accuracy of C1-C3 tested in one experiment with each concentration in triplicate ranged from 4.75% to 9.28% CV and 108% to 120% AR, respectively. Based on these data, four plasma standard concentration pools (0.01, 0.05, 0.1, and 0.5 µg/mL) were selected to be used per run in clinical studies.

Three dilutions (10-, 100-, and 500-fold) were made using blank plasma from a total of two stocks of the rafAON solution (1 and 5 µg/mL) followed by a within-run experiment. The accuracy of these three dilutions was in the 110% to 120% AR range. These data suggest that if necessary, a maximum dilution of 500-fold from a 5 µg/mL rafAON specimen was acceptable in clinical studies. The rafAON preparations (0.02 and 0.4 µg/mL) were stored at –80°C for up to 14 days, before analysis. The accuracies were 85% and 93% AR for 0.02 and 0.4 µg/mL rafAON, respectively, suggesting that rafAON samples could be stored at –80°C for up to 14 days. The rafAON solution was freeze-thawed consecutively thrice, and the rafAON concentration was measured. The accuracy of 0.03 or 0.06 µg/mL rafAON was 93% AR, implying that three cycles of freeze-thaw did not impair the quality of the rafAON specimens.

The integrity of 15-mer rafAON was determined by comparing the size of the 15-mer rafAON with a size marker representing a mixture of 15-mer, 14-mer, and 13-mer rafAON. In addition, a 15-mer random oligonucleotide was used to test the specificity in two independent experiments. The sample processing procedure did not compromise the integrity and specificity of 15-mer rafAON. Furthermore, precision and accuracy of quantification of rafAON using LErafAON were within the acceptable ranges (0.01-0.5 µg/mL: <22% CV; 78.0-120.0% AR; n = 3). These observations suggest that lipids did not interfere in the quantification of rafAON in human plasma.

Expression analysis of biomarkers (c-raf-1 mRNA and Raf-1 protein) in human peripheral blood lymphocytes: method certification. Pre-study validation experiments were done to quantify the relative levels of c-raf-1 mRNA and Raf-1 protein in human lymphocytes, using a reverse transcription-PCR (RT-PCR) method for RNA and a Western blotting method for protein as described below. The acceptance criteria were <25% CV for precision and 100 ± 25% AR. Leukocytes for mRNA extraction were isolated from heparinized human blood obtained from healthy donors. The precision and accuracy of the relative level of c-raf-1 mRNA expression were calculated using two to three replicates of 40, 80, and 160 ng total RNA. The precision and accuracy values ranged from 6.49% to 25.0% CV and 76.4% to 121.9% AR, respectively (n = 3). Accordingly, the recommended lowest and highest amounts of total RNA to be used in clinical studies were 40 and 160 ng, respectively. Three cycles of consecutive freeze-thaw of the RNA preparation resulted in accuracy values outside the acceptable range (59.27-122.36% AR, n = 3). The total RNA preparation was stored at –80°C for 3 weeks, and the relative c-raf-1 expression was compared with that of the freshly prepared samples in three independent runs. The accuracy values for 120 ng sample ranged from 75% to 100.7% AR; however, the % AR for 60 ng total RNA did not meet the acceptance criteria (20.6-149.3% AR). Based on these data, total RNA samples were unacceptable for expression analysis following three cycles of freeze-thaw and/or storage at –80°C for 3 weeks before analysis. The mRNA data presented used the conditions found to give acceptable results.

The Raf-1 protein validation experiments were done using whole-cell lysates from donor human lymphocytes. The precision and accuracy of the relative level of Raf-1 expression were calculated using four different amounts of total protein (100-300 µg). The precision and accuracy values ranged from 5.7% to 17.3% CV and 80.9% to 115.0% AR, respectively (n = 3). Therefore, the lowest and highest amounts of protein to be used in clinical studies were 100 and 300 µg, respectively. Lymphocytes subjected to three cycles of freeze-thaw were unacceptable for Raf-1 expression determinations in clinical studies (160 µg, 65.2% AR). However, lymphocytes stored frozen at –80°C for 2 months before assay were found to be acceptable for Raf-1 expression in clinical studies (140 µg, 82.8% AR).

Pharmacokinetic evaluation. Blood samples for pharmacokinetic evaluation were collected in a subset of patients on days 1 and 11 at the following times: 30 minutes before initiation of LErafAON infusion; at the end of infusion; 5, 15, and 30 minutes after infusion; and 1, 2, 3, 4, 6, and 24 hours after the end of infusion. Plasma concentrations of rafAON were determined using a previously described (12) and validated gel electrophoresis method. In brief, rafAON was extracted from plasma by phenol-chloroform extraction. The rafAON concentration standards (0.01, 0.05, 0.1, and 0.5 µg/mL) were prepared by adding known amounts of rafAON to an aliquot of pre-dose plasma. A negative control consisted of pre-dose (blank) plasma from the same patient. The extracts were loaded onto 20% polyacrylamide/8 mol/L urea gels and electrophoresed in Tris-borate EDTA buffer (Invitrogen/Life Technologies, Carlsbad, CA). The gels were electroblotted and the blots were probed with a ³²P-5'-end-labeled c-raf-1 sense oligonucleotide probe. The autoradiographs were scanned and signals quantified using ImageQuant software (Amersham Biosciences, Uppsala, Sweden). In clinical samples, concentrations of 0.01 µg/mL were designated as unevaluable.

c-raf-1 mRNA and Raf-1 protein in peripheral blood lymphocytes. Blood samples were collected for exploratory analyses of c-raf-1 mRNA and Raf-1 protein in heparinized tubes before LErafAON infusion on days 1, 4, 11, and on day 30 in a subset of patients in the 0.35, 0.70, 1.40, or 2 mg/kg dose cohorts. Heparinized blood samples were stored overnight at 4°C in tubes containing protease inhibitors (aprotinin, 20 µg/mL; leupeptin, 20 µg/mL; Roche, Basel, Switzerland). Expression of c-raf-1 mRNA was measured using RT-PCR method. In brief, the lymphocytes were isolated from the heparinized blood using the RNA aqueous blood module (Ambion, Austin, TX). Total RNA was isolated from lymphocytes using the RNA aqueous-4PCR kit following manufacturer's instructions (Ambion). Radiolabeled RT-PCR was done using a Titan one tube RT-PCR kit according to the manufacturer's instructions (Roche Molecular Biochemicals, Mannheim, Germany) and c-raf-1–specific primers (forward primer, 5'-TCAGAGAAGCTCTGCTAAG-3'; reverse primer, 5'-CAATGCACTGGACACCTTA-3'; Invitrogen/Life Technologies). In parallel, radiolabeled RT-PCR was done using 18S rRNA–specific primers (QuantumRNA 18S Internal Standards, Ambion) to detect the expression of an internal standard, 18S rRNA. The amplified c-raf-1 (494 bp) and 18S (324 bp) fragments were resolved by 5% PAGE and autoradiography. The relative amounts of c-raf-1 mRNA were calculated by densitometric scanning of the c-raf-1 and 18S bands followed by normalization of the c-raf-1 band density (arbitrary value) against 18S band density (arbitrary value).

Expression of Raf-1 protein was measured using Western blotting method. Lymphocytes were isolated from heparinized blood containing protease inhibitors using Ficoll-Paque PLUS (Amersham Biosciences) and stored at –80°C until use. Proteins in the freshly prepared whole-cell lysates were resolved by 7.5% SDS-PAGE followed by immunoblotting with monoclonal anti-Raf-1 antibody (BD Biosciences, San Jose, CA) detecting Raf-1 band (74 kDa) using the enhanced chemiluminescence method (12). The blots were reprobed with G3PDH polyclonal antibody (Trevigen, Gaithersburg, MD) to detect the expression of the internal standard G3PDH protein (30 kDa) in the same samples. The relative amounts of Raf-1 protein were calculated by densitometric scanning of the Raf-1 and G3PDH bands (ImageQuant Software, Amersham Biosciences) followed by normalization of the Raf-1 band density (arbitrary value) against G3PDH band density (arbitrary value).

	Results

Top Abstract Patients and Methods Results Discussion References

 
Patient characteristics. Seventeen patients (10 male and 7 female) were enrolled between March 2001 and February 2003, as summarized in Table 1. The median age was 66 years (range, 23-78 years). Fourteen patients had completed one or more prior chemotherapy regimens. The histologic diagnosis distribution included four patients with colon cancers and five with lung cancers. All patients had an indication for palliative radiation therapy (e.g., pain, mass effect, obstruction, and bleeding).

Pharmacokinetic analysis. A total of 92 samples were available for day 1 pharmacokinetic evaluation from 10 patients. End of infusion rafAON levels in plasma showed high interpatient variability but were highest in patients in the 1.4 mg/kg cohort (1.061 ± 0.289 µg/mL; n = 4). The highest concentrations achieved in the 0.70 and 0.35 mg/kg cohorts were 0.526 µg/mL (n = 3) and 0.415 ± 0.132 µg/mL (n = 3), respectively (Fig. 1A and B). The rafAON was detectable in plasma for several hours following completion of infusion in patients in the 1.4 and 0.70 mg/kg dose cohorts and for 30 minutes in patients in the 0.35 mg/kg dose cohort. The rafAON concentrations were below the limit of quantification by 24 hours after infusion.

View larger version (59K): [in this window] [in a new window]   Fig. 1. Plasma concentration-time profile of (A and B) LErafAON, (C) inhibition of c-raf-1 mRNA in peripheral blood mononuclear cells, and (D) inhibition of Raf-1 protein in peripheral blood mononuclear cells. A, representative autoradiograph showing intact rafAON in plasma at various time points posttreatment on day 1 of a patient in the 1.4 mg/kg/d dose level. Sample aliquots were diluted as follows: 0', 4x; 5', 4x; 15', 2x; 30', 2x; and 6 h, 0.5x; and 24 h, 0.5x; or left undiluted before electrophoresis. S1, S3, and S4 represent 0.01, 0.1, and 0.5 µg/mL of rafAON, respectively. Standard sample (0.5 µg/mL) corresponds to 7.5 ng of rafAON. –30', pre-dose sample. B, mean plasma concentrations of rafAON (±SD) are depicted by dose cohort. Time 0 is the end of infusion on day 1. Quantification data were calculated based on comparison with known concentrations of the standard samples and then normalization against sample dilution factors used for loading. C, the c-raf-1 mRNA expression was determined by RT-PCR using raf-1 and 18S-specific primers. Top, representative autoradiographs showing inhibition of the c-raf-1 mRNA relative to the pre-dose (PD) on day 4 (D4). Bottom, quantification data showing inhibition of c-raf-1 mRNA expression (versus pre-dose, day 1 sample) observed in various cohorts. Expression in patient 10 (1.4 mg/kg dose) was below the level of detection/quantification on day 4. D, Raf-1 protein expression was analyzed in peripheral blood mononuclear cells by Western blotting using anti-Raf-1 antibody followed by reprobing of the same blot with anti-GAPDH antibody. Top, representative immunoblots showing inhibition of Raf-1 expression on day 4 (D4) relative to the pre-dose (PD). Bottom, quantification data showing inhibition of Raf-1 protein expression (versus pre-dose, day 1 sample) observed in various cohorts.

c-raf-1 mRNA and Raf-1 protein expression in peripheral blood lymphocytes. Peripheral blood mononuclear cells were used for exploratory analysis of c-raf-1 expression. Full sample sets (pre-dose, day 3 or 4 and day 10 or 11) for both mRNA and protein analysis were available for five of 10 evaluable patients (patients 6, 8, 10, 11, and 14); partial sample sets were available for the remaining patients (patents 1, 5, 9, 13, and 15). A wide range of inhibition of mRNA and/or protein was observed in seven patients (Fig. 1C and D); levels for the remaining three patients (5, 6, 13) were generally increased from pre-dose (data not shown).

The observed level of inhibition of raf observed did not correlate well with the drug dose level. Six patients with partial response or stable disease were evaluable for c-raf-1 mRNA and/or Raf-1 protein expression. Although the patient numbers are insufficient to form a conclusion, three of five evaluated patients (partial response/stable disease) had inhibition of mRNA expression, one showed increased expression, and one had no change in expression. Likewise, four of five patients (partial response/stable disease) showed inhibition of protein expression, and the remaining one had increased expression of Raf-1. One patient with progressive disease showed increased expression of both c-raf-1 mRNA and Raf-1 protein in a full set of samples (data not shown).

	Discussion

Top Abstract Patients and Methods Results Discussion References

 
Sensitization of cancer cells to radiation through the inhibition of gene products involved in cell growth and survival-related signaling pathways may improve the efficacy of radiation therapy for patients with cancer. Preclinical studies suggest that the epidermal growth factor receptor, Ras, and Raf are involved in a disruptable pathway that leads to enhanced sensitization of cancer cells to radiation (9). In addition, these molecules have been shown by various laboratories to mediate cellular responses to ionizing radiation and play a role in radiation resistance in some tumors.

We enrolled patients with indications for radiation therapy in a dose escalation trial of LErafAON to determine the tolerability and recommended dose for use of LErafAON in combination with radiation therapy. The liposomal preparation of antisense oligonucleotides directed at c-raf-1 is clinically relevant. However, cationic liposomes can lead to infusion-related reactions. A basis for reactions may be in the ability of cationic lipids to induce cytokine release (18). Premedication with steroids and antihistamines was instituted early in the study and was required for the completion of this trial. Nevertheless, Raf-1–inhibiting doses of LErafAON were delivered with such premedication.

Validated assays for levels of rafAON, c-raf-1 mRNA expression, and Raf-1 expression have been presented. Dose-dependent increase in the maximum plasma concentration of rafAON was observed. Circulating intact rafAON was seen for 30 minutes to several hours after infusion, and the levels dropped below the limits of quantification by 24 hours. A wide range of inhibition of c-raf-1 mRNA expression or Raf-1 protein expression was observed, whereas some patients exhibited increased expression. The change in expression was independent of the LErafAON dose level. The present exploratory studies offer a suggestion of correlation of partial response/stable disease status to inhibition of c-raf-1 mRNA and/or protein expression in lymphocytes based on a limited number of evaluable specimens. Six patients with partial response or stable disease were evaluable for c-raf-1 mRNA and/or Raf-1 protein expression. Inhibition of c-raf-1 mRNA was observed in three of five patients. Raf-1 protein was inhibited in four of five patients. The increase in measured Raf-1 expression in one patient with partial response, one patient with progressive disease, and one patient not evaluable for disease status requires further consideration. The changes observed in biomarker expression (mRNA or protein) were based on a single pre-dose value per patient. Multiple blood draws before infusion of LErafAON may provide more accurate baseline levels of expression of c-raf-1 mRNA or Raf-1 protein, particularly in such cases. Recently, an increase in Bcl-2 mRNA or protein was observed in bone marrow samples from some acute myeloid leukemia patients receiving a combination of phosphorothioate antisense oligo to Bcl-2 (G3139) and intensive chemotherapy (19). The study suggests an association of decreased Bcl-2 expression with complete response to chemotherapy and an association of increased Bcl-2 with nonresponders. Future clinical studies are necessary to establish a correlation between inhibition of Raf-1 in tumor tissues and therapy response in a statistically significant number of patients.

In summary, this is the first human study of LErafAON in combination with radiation therapy. The MTD is 2 mg/kg, twice weekly for 2 weeks for combined modality treatment with premedication to manage infusion-related reactions. To improve the tolerability of LErafAON, a modified liposomal formulation has been developed and preclinical therapeutic efficacy has been described (20). A phase I dose-finding study using this formulation (LErafAON-ETU) is currently in progress (21). The presence of intact circulating rafAON and inhibition of both c-raf-1 mRNA and Raf-1 protein in peripheral blood lymphocytes in a subset of patients provide the basis for future studies of this drug using modified liposomal preparations.

	Footnotes

 
Grant support: NIH grants CA74175 and MO1RR13297 and NeoPharm, Inc.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Conflict of interest statement: A. Dritschilo, J. Marshall, P.C. Gokhale, and U.N. Kasid received research support from NeoPharm, Inc. J.L. Dul, A. Ahmad, I. Ahmad, and J.W. Sherman are employees of NeoPharm.

Received 6/13/05; revised 11/18/05; accepted 12/ 9/05.

	References

Top Abstract Patients and Methods Results Discussion References

 

1. Weichselbaum RR, Dahlberg W, Beckett M, et al. Radiation-resistant and repair-proficient human tumor cells may be associated with radiotherapy failure in head- and neck-cancer patients. Proc Natl Acad Sci U S A 1986;83:2684–8.[Abstract/Free Full Text]
2. Lammering G, Hewit TH, Valerie K, et al. EGFRvIII-mediated radioresistance through a strong cytoprotective response. Oncogene 2003;22:5545–53.[CrossRef][Medline]
3. Jones HA, Hahn SM, Bernhard E, et al. Ras inhibitors and radiation therapy. Semin Radiat Oncol 2001;11:328–37.[CrossRef][Medline]
4. Kasid U, Pfeifer A, Weichselbaum RR, et al. The raf oncogene is associated with a radiation-resistant human laryngeal cancer. Science 1987;237:1039–41.[Abstract/Free Full Text]
5. Kasid U, Pfeifer A, Brennan T, et al. Effect of antisense c-raf-1 on tumorigenicity and radiation sensitivity of a human squamous carcinoma. Science 1989;243:1354–6.[Abstract/Free Full Text]
6. Kasid U, Suy S, Dent P, et al. Activation of Raf by ionizing radiation. Nature 1996;382:813–6.[CrossRef][Medline]
7. Schulze A, Lehmann K, Jefferies HB, et al. Analysis of the transcriptional program induced by Raf in epithelial cells. Genes Dev 2001;15:981–94.[Abstract/Free Full Text]
8. Mayo MW, Baldwin AS. The transcription factor NF-kappaB: control of oncogenesis and cancer therapy resistance. Biochim Biophys Acta 2000;1470:M55–62.[Medline]
9. Kasid U, Dritschilo A. RAF antisense oligonucleotide as a tumor radiosensitizer. Oncogene 2003;22:5876–84.[CrossRef][Medline]
10. Gokhale PC, Soldatenkov V, Wang FH, et al. Antisense raf oligodeoxyribonucleotide is protected by liposomal encapsulation and inhibits Raf-1 protein expression in vitro and in vivo: implication for gene therapy of radioresistant cancer. Gene Ther 1997;4:1289–99.[CrossRef][Medline]
11. Gokhale PC, McRae D, Monia BP, et al. Antisense raf oligodeoxyribonucleotide is a radiosensitizer in vivo. Antisense Nucleic Acid Drug Dev 1999;9:191–201.[Medline]
12. Gokhale PC, Zhang C, Newsome JT, et al. Pharmacokinetics, toxicity, and efficacy of ends-modified raf antisense oligodeoxyribonucleotide encapsulated in a novel cationic liposome. Clin Cancer Res 2002;8:3611–21.[Abstract/Free Full Text]
13. Soldatenkov VA, Dritschilo A, Wang FH, et al. Inhibition of Raf-1 protein kinase by antisense phosphorothioate oligodeoxyribonucleotide is associated with sensitization of human laryngeal squamous carcinoma cells to gamma radiation. Cancer J Sci Am 1997;3:13–20.[Medline]
14. Mewani RR, Tang W, Rahman A, et al. Enhanced therapeutic effects of doxorubicin and paclitaxel in combination with liposome-entrapped ends-modified raf antisense oligonucleotide against human prostate, lung and breast tumor models. Int J Oncol 2004;24:1181–8.[Medline]
15. Pei J, Zhang C, Gokhale PC, et al. Combination with liposome-entrapped, ends-modified raf antisense oligonucleotide (LErafAON) improves the anti-tumor efficacies of cisplatin, epirubicin, mitoxantrone, docetaxel and gemcitabine. Anticancer Drugs 2004;15:243–53.[CrossRef][Medline]
16. Rudin CM, Marshall J, Huang CH, et al. Delivery of a liposomal c-raf-1 antisense oligonucleotide by weekly bolus dosing in patients with advanced solid tumors: a phase I study. Clin. Cancer Res 2004;10:7244–51.
17. Rudin CM, Holmlund J, Fleming GF, et al. Phase I trial of ISIS 5132, an antisense oligonucleotide inhibitor of c-raf-1, administered by 24-hour weekly infusion to patients with advanced cancer. Clin Cancer Res 2001;7:1214–20.[Abstract/Free Full Text]
18. Tan Y, Zhang JS, Huang L. Codelivery of NF-kappaB decoy-related oligodeoxynucleotide improves LPD-mediated systemic gene transfer. Mol Ther 2002;6:804–12.[CrossRef][Medline]
19. Marcucci G, Stock W, Dai G, et al. Phase I study of oblimersen sodium, an antisense to Bcl-2, in untreated older patients with acute myeloid leukemia: pharmacokinetics, pharmacodynamics, and clinical activity. J Clin Oncol 2005;23:3404–11.[Abstract/Free Full Text]
20. Lei Y, Ahmad A, Sheikh S, Zhang A, Ahmad I. Enhanced therapeutic efficacy of a novel liposome-based formulation of c-raf AON in combination with Taxol against human ovarian tumor model in SCID mice [Abstract 638]. Proc Am Assoc Cancer Res 2004;45:147.
21. Steinberg JL, Mendelson DS, Block H, et al. Phase I study of LErafAON-ETU, an easy-to-use formulation of liposome entrapped c-raf antisense oligonucleotide, in advanced cancer patients [Abstract 3214]. J Clin Oncol 2005;23:244s.

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