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Clinical Cancer Research Vol. 10, 7244-7251, November 1, 2004
© 2004 American Association for Cancer Research

Clinical Trials

Delivery of a Liposomal c-raf-1 Antisense Oligonucleotide by Weekly Bolus Dosing in Patients with Advanced Solid Tumors

A Phase I Study

Charles M. Rudin1, John L. Marshall2, Chao Hui Huang3, Hedy L. Kindler1, Chuanbo Zhang2, Deepak Kumar2, Prafulla C. Gokhale2, Joyce Steinberg4, Steve Wanaski4, Usha N. Kasid2 and Mark J. Ratain1

1 The University of Chicago, Chicago, Illinois; 2 Georgetown University Medical Center, Washington, DC; 3 Temple-Fox Chase Cancer Center, Philadelphia, Pennsylvania; and 4 NeoPharm Inc., Lake Forest, Illinois


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Purpose: Rapid cleavage in vivo and inefficient cellular uptake limit the clinical utility of antisense oligonucleotides (AON). Liposomal formulation may promote better intratumoral AON delivery and inhibit degradation in vivo. We conducted the first clinical evaluation of this concept using a liposomal AON complementary to the c-raf-1 proto-oncogene (LErafAON).

Experimental Design: A dose escalation study was done to determine the maximum tolerated dose and to characterize the toxicities of LErafAON given as weekly intravenous infusion for 8 weeks to adults with advanced solid tumors. Pharmacokinetic analysis and evaluation of c-raf-1 target suppression in peripheral blood mononuclear cells were included.

Results: Twenty-two patients received LErafAON (median 7 infusions; range 1–27) at doses of 1, 2, 4, and 6 mg/kg/week. Across all dose cohorts patients experienced infusion-related hypersensitivity reactions including flushing, dyspnea, hypoxia, rigors, back pain, and hypotension. Prolonged infusion duration and pretreatment with acetaminophen, H1- and H2-antagonists, and corticosteroids reduced the frequency and severity of these reactions. Progressive thrombocytopenia was dose-limiting at 6 mg/kg/week. No objective responses were observed. Two patients treated at the maximum tolerated dose of 4 mg/kg/week had evidence of stable disease, with dosing extended beyond 8 weeks. Pharmacokinetic analysis revealed persistence of detectable circulating rafAON at 24 hours in 7 of 10 patients in the highest 2 dose cohorts. Suppression of c-raf-1 mRNA was noted in two of five patients analyzed.

Conclusions: Dose-independent hypersensitivity reactions and dose-dependent thrombocytopenia limited tolerance of LErafAON. Future clinical evaluation of this approach will depend on modification of the liposome composition.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The Raf-1 protein, encoded by the c-raf-1 gene, is a 75 kDa serine-threonine kinase that functions as a key regulator of cell growth, proliferation, and survival (1) . Raf-1 is a critical component of multiple signal transduction pathways, integrating signals from cell membrane-bound growth factor receptors and apoptotic regulators (2) . Activated Raf-1 in turn interfaces with a many downstream targets controlling proliferation and survival, including activation of the mitogen-activated protein kinase/extracellular signal-regulated kinase kinases MEK1 and MEK2, activation of the nuclear factor {kappa}B survival and proliferation pathway, and inhibition of the proapoptotic factor Bad (3) .

Deregulated Raf-1 activity has been implicated in oncogenic transformation (4 , 5) . Constitutive Raf-1 activation leads to morphological changes consistent with a malignant phenotype, to growth factor-independent proliferation, and to increased resistance to cytotoxic agents (6) . Raf-1 promotes full malignant transformation of c-Myc-expressing human epithelial cells, and cooperates with Akt to increase apoptotic resistance of hematopoietic progenitors (7 , 8) . Conversely, homozygous deletion of the c-raf-1 gene or expression of a dominant-negative mutant c-raf-1 allele promotes apoptotic induction and markedly inhibits proliferative potential after exposure to growth factor or serum (9 , 10) .

Recent observations suggest that Raf-1 may also be an important regulator of angiogenesis in human tumors. Constitutive expression of activated Raf-1 is associated with up-regulation of vascular endothelial growth factor production, and a dominant-negative mutant Raf-1 inhibits angiogenesis in response to either fibroblast growth factor or vascular endothelial growth factor (11 , 12) .

Together the above data suggest that suppression of Raf-1 activity could have substantial anticancer therapeutic potential. Strategies for Raf-1 suppression that we and others have explored include the use of small molecule inhibitors of the Raf-1 protein and the use of antisense oligonucleotides (AON) directed against the c-raf-1 mRNA (13, 14, 15) . Preclinical models have shown that AON can inhibit translation and accelerate degradation of c-raf-1 mRNA, leading to a decrease in Raf-1 intracellular concentration (16) . Cancer cells transfected with an antisense c-raf-1 cDNA show specific suppression of Raf-1 level, growth inhibition, and increased sensitivity to both chemotherapy and {gamma}-radiation (17 , 18) .

Unmodified short oligonucleotides are subject to rapid hydrolysis by extracellular nucleases present in plasma. One strategy to inhibit nuclease degradation is to modify the nucleotide backbone, with phosphorothioate rather than phosphodiester linkages between bases. Nearly all published clinical trials of AON have used phosphorothioate oligonucleotides (19 , 20) . A number of characteristic sequence-independent toxicities have been associated with the use of phosphorothioate oligonucleotides, including fever, malaise, hypotension, and in some cases prolongation of the activated partial thromboplastin time. Animal studies of phosphorothioate oligonucleotides have reported complement activation as a sequence-independent toxicity, and we have reported previously dose-dependent serum complement activation in human patients treated with a phosphorothioate oligonucleotide directed against c-raf-1 (13) .

An alternative to the use of phosphorothioate oligonucleotides is liposomal formulation of minimally modified oligonucleotides. Liposomal formulation may prolong AON stability by limiting nuclease access and secondarily may reduce nonspecific toxicity by minimizing the need for backbone modifications such as phosphorothioate linkages. In addition, liposomal formulation may increase intracellular AON delivery by facilitating endocytosis. Using the phosphorothioate c-raf-1 AON ISIS 5132, liposomal formulation has been reported to increase the area under the plasma concentration curve over 5-fold after intravenous administration in mice and to improve Raf-1 suppression in human tumor cells in vitro relative to use of an identical AON alone (21) .

LErafAON incorporates a c-raf-1 AON within a cationic liposome approximately 400 nm in diameter (22) . The negatively charged c-raf-1 AON in LErafAON is entrapped within the cationic lipid bilayer with an encapsulation efficiency of over 85% (22 , 23) . In contrast to the cationic lipid formulation used here, neutral and anionic liposomes exhibit relatively poor oligonucleotide entrapment efficiencies. All internal linkages in this AON are nuclease-sensitive phosphodiester linkages; phosphorothioate modifications are limited to the 5' and 3' termini. Preclinical analysis of LErafAON in nude mice bearing PC-3 human prostate cancer xenografts showed intratumoral Raf-1 suppression, single agent inhibition of tumor growth, and enhancement of radiation sensitivity and chemosensitivity leading to tumor regression (22 , 24) .

Here we report the first clinical evaluation of single agent LErafAON. The primary goals of this study were to evaluate the toxicity and determine the maximally tolerated dose of LErafAON when given by weekly intravenous infusion in patients with advanced malignancies.


   PATIENTS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patients.
The trial was limited to adults with histologically confirmed recurrent or progressive solid tumor for which no curative therapy was available. All patients had Eastern Cooperative Oncology Group performance status ≤2. Any number of prior chemotherapy regimens was allowed. Adequate hematologic (absolute neutrophil count ≥1500/µL, and platelets ≥100,000/µL), hepatic (bilirubin within normal institutional limits, and aspartate aminotransferase and alanine aminotransferase ≤2.5 x institutional upper limit of normal), renal (creatinine within normal institutional limits), and coagulation (prothrombin time and activated thromboplastin time function within normal institutional limits) status was required. All patients provided written informed consent before study enrollment or performance of study-related procedures, in accordance with institutional and federal guidelines.

Study Drug Preparation.
Lyophilized c-raf-1 AON (NeoPharm, Lake Forest, IL) was resuspended in sterile saline and added to vials containing a desiccated liposomal preparation containing the cationic lipid dimethyldioctadecyl ammonium bromide (NeoPharm). This reconstituted mixture was vortexed for 2 minutes, allowed to hydrate at room temperature over 0.5 to 2.5 hours, and then sonicated at maximum intensity in a bath-type sonicator for 10 minutes before infusion.

Study Design.
LErafAON was administered weekly by intravenous infusion, with the initial plan being for infusion over 30 minutes. As described in detail below, infusion duration was increased in the course of the trial to a minimum of 60 minutes. Cohorts of at least three patients were entered at escalating dose levels, starting at 1 mg/kg/week, with subsequent cohorts receiving 2, 4, and 6 mg/kg/week, respectively (Table 1)Citation . Intrapatient dose escalation was not allowed. Each cohort was observed for at least 10 days after the first dose of LErafAON before initiation of treatment of a subsequent cohort. Dose escalation proceeded until an maximum tolerated dose was defined. Maximum tolerated dose was defined as the dose level below that associated with dose-limiting toxicity (DLT, defined below) in two or more of up to six patients. If tolerating therapy, patients were allowed to continue on LErafAON weekly until progression.


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  		Table 1 Dose cohorts

 
Toxicity and Response Evaluation.
We assessed toxicity was assessed using NCI Common Toxicity Criteria (version 2.0) on all patients enrolled on study. DLT was defined as any grade 4 toxicity considered probably or definitely related to LErafAON or any grade 3 toxicity considered probably or definitely related to LErafAON excluding alopecia, malaise, fever, and hepatic transaminase elevation on a postdose sample. Emesis was considered dose-limiting only if continuing despite maximal antiemetic therapy. Allergic and/or hypersensitivity reactions in general were not considered dose-limiting, because these reactions are typically not dose-dependent: such reactions were considered dose-limiting if the reaction met the following criteria: (a) pretreatment regimen and specified infusion rate were followed, (b) >50% of the planned dose was administered, and (c) at least one of the symptoms of the reaction was a grade 3 or worse, or one of the symptoms of the reaction was a grade 2 bronchospasm considered to be probably or definitely related to study medication.

Response was evaluated by comparison of baseline computed tomography scan done 0 to 4 weeks before the first dose of LErafAON with re-evaluation computed tomography scans done at least every 8 weeks while on study drug, using the Response Evaluation Criteria in Solid Tumors (RECIST).

Pharmacokinetic Evaluation.
Blood for pharmacokinetic evaluation was collected on week 1 of therapy 30 minutes before initiation of LErafAON, 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. Trough levels were drawn before infusion in weeks 2 and 8. Plasma concentrations of rafAON were determined as described previously (22) . 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 a portion of the predose plasma. A negative control consisted of predose (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, 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 were quantified with ImageQuant software (Amersham Biosciences, Uppsala, Sweden). In clinical samples, 0.01 µg/mL or a lesser concentration was designated unevaluable.

Determination of c-raf-1 mRNA Expression and Raf-1 Protein Levels.
Blood for exploratory analysis of c-raf-1 mRNA and Raf-1 protein was collected in heparinized tubes before and after LErafAON infusion on days 1 and 8 in a subset of patients in the 4 or 6 mg/kg/week dose cohorts. Sample sets were adequate for RNA analysis in a total of five patients and for protein analysis in six patients. Heparinized blood was stored overnight at 4°C in tubes containing protease inhibitors (20 µg/mL aprotinin; 20 µg/mL leupeptin; Roche, Basel, Switzerland). For total RNA, the lymphocytes were isolated from the heparinized blood with RNA aqueous blood module (Ambion, Austin, TX). Total RNA was isolated from lymphocytes with RNA aqueous-4PCR kit following the manufacturer’s instructions (Ambion, Austin, TX). Radiolabeled reverse transcription (RT)-PCR was done with Titan one tube RT-PCR kit according to the manufacturer’s instructions (Roche Molecular Biochemicals, Mannheim, Germany), using c-raf-1-specific primers (forward primer, 5'-TCAGAGAAGCTCTGCTAAG-3'; reverse primer, 5'-CAATGCACTGGACACCTTA-3'; Invitrogen). For RT-PCR, the RT was done at 50°C for 30 minutes followed by denaturation at 94°C, 2 minutes and 25 cycles of 94°C for 30 seconds, 55°C for 30 seconds, 68°C for 1 minute, final extension at 68°C for 7 minutes, and incubation at 4°C. In parallel, radiolabeled RT-PCR procedure was done with 18S rRNA-specific primers (QuantumRNA 18S Internal Standards, Ambion, Austin, TX) to detect the expression of internal standard, 18S rRNA. The amplified c-raf-1 (494 bp) and 18S (324 bp) fragments were resolved by 5% polyacrylamide gel electrophoresis and autoradiography. The relative amount of c-raf-1 mRNA was 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).

For protein analysis, lymphocytes were isolated from heparinized blood containing protease inhibitors with Ficoll-Paque PLUS (Amersham Biosciences) and stored at –80°C until use. Cells were lysed in lysis buffer [0.15 mol/L sodium chloride, 0.05 mol/L Tris-HCl (pH 7.4), 1 mmol/L EDTA, 1% Triton X-100, 0.1% SDS, 1% sodium deoxycholate, 1 mmol/L phenylmethylsulfonyl fluoride, 100 µmol/L sodium orthovanadate, 20 µg/mL aprotinin, and 20 µg/mL leupeptin]. 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) and detection of Raf-1 band (74 kDa) with the enhanced chemiluminescence method as described previously (22, 23, 24) . The blots were reprobed with glyceraldehyde-3-phosphate dehydrogenase polyclonal antibody (Trevigen, Gaithersburg, MD) to detect the expression of internal standard, glyceraldehyde-3-phosphate dehydrogenase protein (30 kDa) in the same sample. The relative amount of Raf-1 protein was calculated by densitometric scanning of the Raf-1 and glyceraldehyde-3-phosphate dehydrogenase bands (ImageQuant Software, Amersham Biosciences), followed by normalization of the Raf-1 band density (arbitrary value) against glyceraldehyde-3-phosphate dehydrogenase band density (arbitrary value).


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Patient Characteristics.
A total number of 22 patients was enrolled on this study. Patient characteristics are summarized in Table 2Citation . The majority of patients were extensively pretreated, with 18 of 22 patients having received at least three prior regimens. All but two patients had performance status 0 or 1. The most common disease type was colorectal cancer.


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  		Table 2 Patient demographics

 
Dose Cohorts and Toxicity Analysis.
Patients were treated in 4 dose cohorts. Treatment-related adverse events common toxicity criteria grade ≥2 are summarized by dose cohort in Table 3Citation . A total of four patients were treated at dose level 1, 1 mg/kg/week LErafAON, initially without pretreatment. Two of these four patients experienced grade 3 or 4 hypersensitivity reactions, manifest by flushing, diaphoresis, low-grade fever, back pain, and dyspnea. These reactions occurred within minutes of initiation of therapy, on day 1 or day 15 of cycle 1. Because of the association of these reactions with initiation of infusion, these hypersensitivity reactions were judged to be clearly study drug-related but not evidently dose-related. The protocol was modified to include oral acetaminophen (650 mg) before all subsequent infusions, and infusion duration was increased from 30 minutes to at least 60 minutes in all subsequent patients. Neither of these patients was rechallenged with LErafAON. Although the other two patients in this dose cohort did not have acute systemic hypersensitivity reactions, both experienced rigors during LErafAON infusion, and one of these patients electively withdrew from study on day 15. The final patient in this cohort discontinued therapy after 8 weeks because of progressive disease.


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  		Table 3 Related adverse events ≥grade 2

 
Cohort 2 consisted of three patients treated at a dose of 2 mg/kg/week. One substantial (grade 3) hypersensitivity reaction was observed in week 2, characterized by flushing, back pain, chills, low-grade fever, and dyspnea. The pretreatment regimen was further modified to include oral diphenhydramine (50 mg), oral famotidine (20 mg), and intravenous methylprednisolone (100 mg) before each LErafAON infusion. With these premedications, this patient received two additional LErafAON doses without complication but discontinued therapy because of progressive disease. No dose-related and dose-limiting toxicity was observed in cohort 2.

A total of seven patients were treated in cohort 3, at a dose of 4 mg/kg/week. One patient in this cohort experienced a hypersensitivity reaction in the first 20 minutes of infusion in week 2, with rigors and grade 3 hypoxia. The patient was unable to complete the infusion, either on this day or 2 days later after repeat premedication, and was taken off study. The other six patients in this cohort were treated successfully for at least 7 weeks. One of these patients developed grade 3 thrombocytopenia (dose-limiting toxicity) in week 6 but went off study in week 7 because of progressive disease.

A total of eight patients were treated in cohort 4, at a dose of 6 mg/kg/week. One patient experienced grade 3 allergic reaction despite premedication, in the first 30 minutes of infusion in week 2, with facial flushing, dyspnea, hypotension, and grade 3 hypoxia. Two other patients also experienced grade 3 hypoxia, in one case associated with dyspnea and rigors in the first 20 minutes of week 1 and in the other during infusion in week 3. The latter patient completed the week 3 dose but went off study the same week because of development of grade 3 thrombocytopenia. A total of three patients in cohort 4 developed grade 3 thrombocytopenia during the first 8 weeks of therapy. The dose of 4 mg/kg/week was established as the maximum tolerated dose.

Thrombocytopenia.
A decrease in platelet count was observed during cycle 1 in all dose cohorts and was most notable in patients treated at the 4 and 6 mg/kg/week dose levels (Fig. 1)Citation . Thrombocytopenia was progressive over the 8-week course of drug administration and was followed by variable platelet recovery during the subsequent 4 weeks off therapy (weeks 9–12). Only one patient on study experienced bleeding: a pancreatic cancer patient treated at 4 mg/kg/week developed a grade 4 gastrointestinal hemorrhage 1 week after completing week 8 of LErafAON, associated with dislodgement of a biliary stent. This patient had had a relatively minor change in platelet count while on study (from baseline of 121,000/µl to 80,000/µl over the course of 8 weeks; grade 1 thrombocytopenia throughout). This episode was believed to be unrelated to study drug.



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  		Fig. 1. Change in platelet count over time. The average platelet counts, normalized to pretreatment baseline, are shown by week on therapy and by dose cohort. LErafAON was administered weekly over 8 weeks. Week 12 values are given for patients who did not continue on therapy beyond 8 weeks (all but four patients). Note that values for cohort 1 (1 mg/kg/week) represent data on a single patient after week 3. Error bars represent SD.

 
Complement Activation.
A previous analysis of an anti-c-raf-1 phosphorothioate oligonucleotide was notable for dose-dependent induction of serum complement C3a and Bb (13) . Evaluation of these markers of complement activation was done on serum samples obtained immediately before and after LErafAON infusion on weeks 1, 2, 4, 6, and 8 of cycle 1. In all dose cohorts, comparison of pre- and post-therapy levels revealed a substantial increase in both C3a and Bb following LErafAON infusion (Fig. 2A and B)Citation . There was no significant change in pretreatment complement levels over consecutive weeks of therapy (not shown).



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  		Fig. 2. Complement activation. Bars represent average ratio of post infusion to preinfusion serum levels of C3a (panel A) and of Bb (panel B). Data from all patients studied within each of the four dose cohorts are included. Because pretreatment complement levels did not change significantly over the course of therapy, data from multiple weeks are included. Error bars represent SD.

 
Response.
There were no objective responses. Six patients were taken off therapy because of toxicity or patient decision within the first 8 weeks of therapy and were considered inevaluable for response. Two patients were taken off therapy because of evident disease progression before the first planned response evaluation (after 4 and 7 weeks, at dose level 2 and 6 mg/kg/week, respectively). Of the remaining 14 patients, five had stable disease on first evaluation, and nine had progressive disease. All five patients with stable disease were in cohorts treated with 4 or 6 mg/kg/week LErafAON. The median number of infusions given was ≤8 in all dose cohorts. Two patients with stable disease in the 4 mg/kg/week dose cohort received treatment over the course of 17 weeks (16 infusions) and 32 weeks (27 infusions), respectively. The latter patient was a 29-year-old woman with sarcoma.

Pharmacokinetic Analysis.
A total of 156 samples for pharmacokinetic analysis were available from 20 patients enrolled on protocol. End of infusion rafAON levels in plasma showed high interpatient variability but were highest in dose level 4, 6 mg/kg/week (1.8 ± 1.2 µg/mL). Initial plasma half-life (t1/2{alpha}) of rafAON was <15 minutes in all dose cohorts (Fig. 3)Citation . Plasma rafAON was undetectable 24 hours after infusion in five of five patients evaluated in the first 2 dose cohorts (level of detection, 10 ng/mL), but was detectable at 24 hours in seven of ten patients in the last 2 cohorts (average 25 ± 18 ng/mL, range 11–60 ng/mL).



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  		Fig. 3. Plasma concentration-time curves of LErafAON. Average plasma concentrations of rafAON are depicted by dose cohort. Time 0 is end of infusion on day 1. Error bars represent SD.

 
c-raf-1 mRNA Suppression.
Peripheral blood mononuclear cells were isolated from a subset of patients treated with 4 or 6 mg/kg/week LErafAON immediately before and after therapy on days 1 and 8 for exploratory analyses of relative c-raf-1 mRNA and Raf-1 protein concentration. Quantitative RT-PCR for mRNA evaluation showed high interindividual variation in c-raf-1 expression at baseline and suggested a suppression of c-raf-1 mRNA concentration with therapy in two of five patients analyzed (Fig. 4)Citation . Raf-1 evaluation by Western blotting showed very poor reproducibility, with apparent relative concentration varying by as much as 8-fold in blood draws from the same day (data not shown).



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  		Fig. 4. c-raf-1 mRNA level. Relative c-raf-1 mRNA level in peripheral blood mononuclear cells was determined by quantitative RT-PCR in samples collected before and after therapy on days 1 and 8 on a total of five patients. Data from each patient is depicted separately. Error bars represent SD.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This report describes the first clinical trial to date of a liposomal antisense oligonucleotide. This trial used an oligonucleotide directed against the c-raf-1 mRNA, which encodes a factor known to play a critical function in regulating cancer cell proliferation, survival, and differentiation. Antisense oligonucleotide therapies have generally been administered by prolonged continuous intervenous infusion and are associated with a spectrum of sequence-independent toxicities. Liposomal formulation was tested as a means of increasing drug stability to permit intermittent dosing and of decreasing toxicity by "shielding" the oligonucleotide and permitting use of a minimally modified DNA molecule.

Successful administration of this agent was limited by hypersensitivity reactions. These reactions occurred in patients in all dose cohorts, were typically manifest shortly after initiation of infusion, and could be only partially suppressed with the use of a combination of an antipyretic, H1 and H2 inhibitors, and a corticosteroid. Other trials involving antisense oligonucleotides, including oligonucleotides directed against c-raf-1, have relatively rarely reported substantial hypersensitivity reactions (19) . We believe that the hypersensitivity reactions frequently observed in this trial of LErafAON may be because of the liposomal preparation used. Definition of predictors or correlates of hypersensitivity would be of significant use in further development of this novel class of agents.

Liposomal formulation of a number of other agents has been used successfully to facilitate drug delivery (25 , 26) . Most (although not all) lipid preparations in clinical use are anionic, as are the majority of lipids on the cell surface. Because rafAON, like all oligonucleotides, is negatively charged, micellar formation with rafAON was promoted in this instance by the use of a mixture of cationic lipids. It is unclear whether and to what extent other factors in addition to the specific lipid formulation used may have contributed to the hypersensitivity reactions observed. Hypersensitivity with complement activation has also been noted in association with other lipid formulations, notably liposomal doxorubicin (27) .

The plasma pharmacokinetics of LErafAON in patients are consistent with preclinical data. The plasma clearance of LErafAON in cynomolgus monkeys showed a biexponential pattern with rafAON t1/2{alpha} of 9 minutes, and terminal half-life (t1/2ß) of over 30 hours (22) . It is unclear whether the initial tissue distribution of rafAON and the low level of rafAON persisting in circulation are sufficient to provide prolonged suppression of c-raf-1 levels. Pilot data on mRNA levels in peripheral blood mononuclear cells suggest that c-raf-1 expression may be inhibited in at least some patients, but Raf-1 protein levels could not be consistently measured.

The etiology of the progressive and dose-limiting thrombocytopenia observed is unclear, but it could be associated with Raf-1 inhibition. Several studies have implicated Raf-1 activity and the Raf-1/MEK/ERK signaling cascade, in human megakaryocytopoiesis. The megakaryocyte growth factor thrombopoietin activates ERK, and this activity is required for maturation of human megakaryocytic precursors (28 , 29) . ERK activation by thrombopoietin has been shown to be dependent on both MEK and Raf-1 (30) . Inhibition of this signaling cascade with a MEK inhibitor blocks megakaryocyte maturation of human CD34+ cells (31) . Conversely, a constitutively active MEK construct stimulates megakaryocyte differentiation of precursor cells (32) . Megakaryocytopoiesis in mice does not require Raf-1; in fact, c-raf-1-deficient mice have slightly higher platelet counts than normal littermates (33) . Notably, thrombocytopenia was not anticipated by preclinical models of LErafAON: e.g, no evidence of decreased platelets was seen in mice treated with LErafAON at doses up to 420 mg/kg (22) . This may reflect a difference between human and murine megakaryocytopoiesis. However, thrombocytopenia has not been a prominent toxicity of the small molecule Raf-1 inhibitor sorafenib (15) .

A new formulation of lipid to be used in conjunction with rafAON has been produced and is currently in preclinical testing. This formulation facilitates drug preparation and, because of a significantly smaller and more uniform micelle size and different lipid component, may be associated with a lower incidence of acute hypersensitivity reactions. Future clinical testing of this alternative formulation will include a more comprehensive analysis of target suppression. Limited bone marrow examination to further characterize the mechanism(s) responsible for progressive dose-related thrombocytopenia also may be of interest. Given that the mechanism of thrombocytopenia is uncertain, it is plausible that this toxicity may be ameliorated through a change in lipid formulation.


   ACKNOWLEDGMENTS
 
We thank Evie Sprague RN for assistance with coordination of patient care.


   FOOTNOTES
 
Grant support: This study was sponsored by 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.

Requests for reprints: Charles M. Rudin, The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Bunting-Blaustein Cancer Research Building I, Rm. 344, 1650 Orleans Street, Baltimore, MD 21231. Phone: 410-955-8904; Fax: 410-502-0677; E-mail: rudin{at}jhmi.edu

Received 4/ 2/04; revised 7/ 6/04; accepted 7/26/04.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Troppmair J, Rapp UR. Raf and the road to cell survival: a tale of bad spells, ring bearers and detours. Biochem Pharmacol 2003;66:1341-5.[CrossRef][Medline]
  2. Chong H, Vikis HG, Guan KL. Mechanisms of regulating the Raf kinase family. Cell Signal 2003;15:463-9.[CrossRef][Medline]
  3. Chang F, Steelman LS, Shelton JG, et al Regulation of cell cycle progression and apoptosis by the Ras/Raf/MEK/ERK pathway (Review). Int J Oncol 2003;22:469-80.[Medline]
  4. Kerkhoff E, Rapp UR. High-intensity Raf signals convert mitotic cell cycling into cellular growth. Cancer Res 1998;58:1636-40.[Abstract/Free Full Text]
  5. Troppmair J, Bruder JT, Munoz H, et al Mitogen-activated protein kinase/extracellular signal-regulated protein kinase activation by oncogenes, serum, and 12-O-tetradecanoylphorbol-13-acetate requires Raf and is necessary for transformation. J Biol Chem 1994;269:7030-5.[Abstract/Free Full Text]
  6. Nottage M, Siu LL. Rationale for Ras and raf-kinase as a target for cancer therapeutics. Curr Pharm Des 2002;8:2231-42.[CrossRef][Medline]
  7. Pfeifer AM, Jones RT, Bowden PE, et al Human bronchial epithelial cells transformed by the c-raf-1 and c-myc protooncogenes induce multidifferentiated carcinomas in nude mice: a model for lung carcinogenesis. Cancer Res 1991;51:3793-801.[Abstract/Free Full Text]
  8. McCubrey JA, Lee JT, Steelman LS, et al Interactions between the PI3K and Raf signaling pathways can result in the transformation of hematopoietic cells. Cancer Detect Prev 2001;25:375-93.[Medline]
  9. Chen J, Fujii K, Zhang L, et al Raf-1 promotes cell survival by antagonizing apoptosis signal-regulating kinase 1 through a MEK-ERK independent mechanism. Proc Natl Acad Sci USA 2001;98:7783-8.[Abstract/Free Full Text]
  10. Mikula M, Schreiber M, Husak Z, et al Embryonic lethality and fetal liver apoptosis in mice lacking the c-raf-1 gene. EMBO J 2001;20:1952-62.[CrossRef][Medline]
  11. Grugel S, Finkenzeller G, Weindel K, et al Both v-Ha-Ras and v-Raf stimulate expression of the vascular endothelial growth factor in NIH 3T3 cells. J Biol Chem 1995;270:25915-9.[Abstract/Free Full Text]
  12. Hood JD, Bednarski M, Frausto R, et al Tumor regression by targeted gene delivery to the neovasculature. Science (Wash DC) 2002;296:2404-7.[Abstract/Free Full Text]
  13. 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]
  14. Strumberg D, Voliotis D, Moeller JG, et al Results of phase I pharmacokinetic and pharmacodynamic studies of the Raf kinase inhibitor BAY 43–9006 in patients with solid tumors. Int J Clin Pharmacol Ther 2002;40:580-1.[Medline]
  15. Ratain MJ, Stadler W, Smith M, et al A phase II study of BAY 43–9006 using the randomized discontinuing design in patients with advanced refractory cancer. Clin Cancer Res 2003;9:6265s-6s.
  16. Monia BP, Johnston JF, Geiger T, et al Antitumor activity of a phosphorothioate antisense oligodeoxynucleotide targeted against C-raf kinase. Nat Med 1996;2:668-75.[CrossRef][Medline]
  17. 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 (Wash D C) 1989;243:1354-6.
  18. Rasouli-Nia A, Liu D, Perdue S, et al High Raf-1 kinase activity protects human tumor cells against paclitaxel-induced cytotoxicity. Clin Cancer Res 1998;4:1111-6.[Abstract]
  19. Stephens AC, Rivers RP. Antisense oligonucleotide therapy in cancer. Curr Opin Mol Ther 2003;5:118-22.[Medline]
  20. Jansen B, Zangemeister-Wittke U. Antisense therapy for cancer–the time of truth. Lancet Oncol 2002;3:672-83.[CrossRef][Medline]
  21. Kasid U, Dritschilo A. RAF antisense oligonucleotide as a tumor radiosensitizer. Oncogene 2003;22:5876-84.[CrossRef][Medline]
  22. 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]
  23. 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]
  24. 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]
  25. Wolff AC. Liposomal anthracyclines and new treatment approaches for breast cancer. Oncologist 2003;8(Suppl2):25-30.
  26. Herbrecht R, Natarajan-Ame S, Nivoix Y, et al The lipid formulations of amphotericin B. Expert Opin Pharmacother 2003;4:1277-87.[CrossRef][Medline]
  27. Chanan-Khan A, Szebeni J, Savay S, et al Complement activation following first exposure to pegylated liposomal doxorubicin (Doxil): possible role in hypersensitivity reactions. Ann Oncol 2003;14:1430-7.[Abstract/Free Full Text]
  28. Gaur M, Murphy GJ, de Sauvage FJ, et al Characterization of Mpl mutants using primary megakaryocyte-lineage cells from mpl(–/–) mice: a new system for Mpl structure-function studies. Blood 2001;97:1653-61.[Abstract/Free Full Text]
  29. Rouyez MC, Boucheron C, Gisselbrecht S, et al Control of thrombopoietin-induced megakaryocytic differentiation by the mitogen-activated protein kinase pathway. Mol Cell Biol 1997;17:4991-5000.[Abstract]
  30. Garcia J, de Gunzburg J, Eychene A, et al Thrombopoietin-mediated sustained activation of extracellular signal-regulated kinase in UT7-Mpl cells requires both Ras-Raf-1- and Rap1-B-Raf-dependent pathways. Mol Cell Biol 2001;21:2659-70.[Abstract/Free Full Text]
  31. Fichelson S, Freyssinier JM, Picard F, et al Megakaryocyte growth and development factor-induced proliferation and differentiation are regulated by the mitogen-activated protein kinase pathway in primitive cord blood hematopoietic progenitors. Blood 1999;94:1601-13.[Abstract/Free Full Text]
  32. Melemed AS, Ryder JW, Vik TA. Activation of the mitogen-activated protein kinase pathway is involved in and sufficient for megakaryocytic differentiation of CMK cells. Blood 1997;90:3462-70.[Abstract/Free Full Text]
  33. Kamata T, Pritchard CA, Leavitt AD. Raf-1 is not required for megakaryocytopoiesis or TPO-induced ERK phosphorylation. Blood 2003;103:2568-70.



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