1 Section of Hematology and Oncology, University of Chicago, Chicago, USA; 2 Committee on Clinical Pharmacology and Pharmacogenomics; 3 University of Chicago Cancer Research Center, Chicago, USA and 4 Lorus Therapeutics, Toronto, Canada
*Correspondence to: Dr R. L. Schilsky, MD, Biological Sciences Division, University of Chicago, 5841 S. Maryland Ave, MC 1000, Chicago, IL 60 637, USA. Tel: +1-773-834-3914; Fax: +1-773-834-3915; Email: rschilsk{at}medicine.bsd.uchicago.edu
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Abstract |
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Patients and methods:: GTI-2040 was administered as a continuous intravenous infusion for 21 days every 4 weeks. Dose escalation was performed using an accelerated, dose-doubling schedule until any drug related toxicitygrade 2 was observed; subsequent dose escalation followed a more conservative dose escalation scheme with three patients/cohort.
Results:: A total of 49 cycles of therapy were administered to 36 patients at GTI-2040 doses ranging from 18.5 mg/m2/day to 222 mg/m2/day. GTI-2040 was generally well tolerated. At the highest dose level examined, two patients experienced dose limiting reversible hepatic toxicity. Constitutional toxicities consisting of fatigue and anorexia were the most common toxicities.
Conclusions:: The recommended dose of GTI-2040 given on this infusion schedule is 185 mg/m2/day. GTI-2040 appears to have a manageable toxicity profile and is generally well tolerated as a single agent.
Key words: antisense therapies, GTI-2040, pharmacokinetics, phase I trial, phosphorothioate oligonucleotides, ribonucleotide reductase inhibition
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Introduction |
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Ribonucleotide reductase (RNR) catalyzes the synthesis of 2'-deoxyribonucleotides from the corresponding ribonucleoside 5'-diphosphates. This step is the rate-limiting reaction in the production of 2'-deoxyribonucleoside 5'-triphosphates required for DNA synthesis. RNR consists of two subunits: R1 and R2. Different genes on separate chromosomes encode the two subunits, and the mRNAs for the two proteins are differentially expressed throughout the cell cycle [79
]. The mRNA levels for the R2 subunit are highest during late G1/early S phase. GTI-2040 is a 20-mer phosphorothioate oligonucleotide that is complementary to the mRNA of the R2 subunit of RNR [10
].
Various cytotoxic agents in current use inhibit RNR as part of their mechanism of action. However, their lack of specificity potentially limits their effectiveness [1113
]. An antisense oligonucleotide such as GTI-2040, designed to specifically bind and inhibit RNR mRNA, might be more effective in inhibiting the target, and the increased specificity might lead to a more favorable therapeutic index. Non-clinical data also suggest that the R2 protein plays a role in determining the malignant potential of cells via synergism with activated oncogenes, and overexpression of the R2 protein has been associated with increases in membrane-associated Raf-1 protein and mitogen-activating protein kinase-2 activity [14
, 15
]. Thus, a specific inhibitor of the R2 mRNA might exert antineoplastic effects through multiple pathways.
GTI-2040 inhibits the growth of human lung, prostate, colon, bladder, liver, ovary, brain, skin, breast and pancreas tumor cells in colony forming assays [10]. GTI-2040 also significantly inhibits growth of human colon, pancreas, liver, lung, breast, renal, ovarian, melanoma, brain, prostate and cervical tumors in xenograft models [10
, 16
]. GLP toxicology and toxicokinetic studies were conducted in Sprague-Dawley rats and rhesus monkeys. In rats, consistent, dose-related changes in hematology and clinical chemistry parameters, with significant differences relative to controls in mid- and high-dose animals were found. Some common hematologic abnormalities observed were: a decrease in hemoglobin concentration, platelet counts and hematocrit. At the end of the recovery period, the changes on the hematological parameters were less marked amongst the two groups of animals. Chemistry parameters such as total bilirubin, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) also increased with escalating doses. At the end of the recovery period for the high-dose group, AST and ALT were still elevated. In general, most of the changes were small in magnitude.
In rhesus monkeys administered single escalating doses of GTI-2040 by continuous intravenous infusion for 24 h, an increase in aPTT at 80 mg/kg and a dose-related increase in concentrations of complement split product Bb were observed. No treatment related clinical signs or changes in other toxicity parameter evaluations such as body weight, blood pressure, heart rate, serum chemistry and hematology were observed in the treated animals. Similar to the acute regimen, when GTI-2040 was administered on a 21-day continuous intravenous infusion to monkeys, an increase in aPTT (at 50 mg/kg dose) and a dose-related increase in concentrations of complement split product Bb were observed. The 21-day continuous intravenous infusion study performed in the rhesus monkey demonstrated that most toxicities of GTI-2040 were minor and reversible upon discontinuation of GTI-2040 [10].
Thus, based on the non-clinical evidence of antitumor activity, we undertook this phase I study of continuous infusion GTI-2040 with the following primary objectives: to determine the dose-limiting toxicity (DLT) and maximum-tolerated dose (MTD) of GTI-2040 administered as a continuous intravenous infusion in patients with advanced solid tumors or lymphoma; to describe the safety profile of this agent; and to describe the pharmacokinetics and pharmacodynamics of the drug.
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Patients and methods |
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Study design
This was an open-label, single-institution, phase I study. All laboratory tests that assessed eligibility had to be completed within 3 weeks before start of treatment. The starting dose was based on the non-clinical toxicology studies, which demonstrated that minimal and reversible toxicity was observed at the 59 mg/m2/day and 123 mg/m2/day dose levels in the rat and monkey, respectively. The no-effect dose levels in the rat and monkey were 11.8 mg/m2/day and 24.6 mg/m2/day, respectively. Hence, a starting dose of 18.5 mg/m2/day (approximately one-sixth of 123 mg/m2/day) was chosen for this phase I trial. GTI-2040 was administered as a continuous intravenous infusion (via an implanted venous access device) for 21 days followed by 7 days of rest. Each 28-day period was defined as one treatment cycle.
Dose escalation was performed in two parts. During part I, the GTI-2040 dose was escalated based on an accelerated, dose-doubling schedule. A minimum of one and a maximum of three patients could be treated per dose level. Escalation to the next dose level was allowed if the first patient at a dose level completed cycle 1 of therapy without any toxicity greater than grade 1. Observation of any toxicitygrade 2 required the enrollment of three patients at that dose level, and also required subsequent dose escalation to occur according to the part II dose escalation plan. Once dose level 4 (148 mg/m2/day) was reached, dose escalation was to proceed according to the part II escalation scheme, even if no toxicity had been observed at lower doses. In part II, it was required that three patients complete at least one cycle of therapy prior to entry of patients at the next higher dose level. Dose increments of 20% were employed for each dose escalation. Dose escalation was permitted if none of the initial three patients developed DLT. If one of the initial three patients experienced DLT in cycle 1, the dose cohort was expanded to six evaluable patients. The MTD was defined as one dose level below the level at which two (or more) of six patients experienced a DLT.
For the purposes of determining the MTD, only DLT occurring during the first cycle of therapy was considered. Toxicity was assessed using National Cancer Institute Common Toxicity Criteria (version 2.0). Patients not completing cycle 1 for reasons other than DLT were replaced. DLT was defined as: (a) grade 4 neutropenia associated with fever or lasting 3 days or longer; (b) any grade 4 thromobocytopenia or grade 3 thrombocytopenia associated withgrade 1 hemorrhage; (c) any
grade 3 coagulation abnormality associated with
grade 1 hemorrhage; (d) nausea/vomiting
grade 3 despite maximal antiemetic therapy; (e) diarrhea
grade 3 despite maximal anti-diarrheal therapy; (f) any other non-hematological toxicity
grade 3 with the exception of alopecia.
Pretreatment and follow-up studies
Before start of therapy, all patients were assessed with a complete history and physical examination. Assessment of Karnofsky performance status, radiographic evaluation of sites of disease, a chemistry panel, complete blood count, and serum tumor markers (when applicable) were also performed. Hematologic parameters were assessed weekly and complete serum chemistries were assessed every 2 weeks while on therapy. Physical examination and performance status evaluations were performed every 2 weeks during treatment. Coagulation parameters, total hemolytic complement activity (CH50) and complement split product levels (such as Bb levels) were obtained before the start of therapy and monitored every week thereafter while patients were on study. All of the above parameters were monitored more frequently if deemed necessary by the treating physicians. Tumor response assessments were performed after the first two cycles of therapy and every two cycles thereafter. Two-dimensional measurements were used to classify response using World Health Organization response criteria. Patients with partial and complete response or stable disease were allowed to continue therapy if they did not experience dose-limiting toxicity.
Pharmacokinetics
Blood samples for pharmacokinetic analysis were collected on day 1 pretreatment and at 1, 2, 3, 4, and 6 h after starting treatment. Samples were also collected on day 22 just prior to the end of infusion, and at 0.25, 0.5, 1, 2, 4 and 6 h after the end of infusion. A single pharmacokinetic sample was also collected on days 8 and 15 of cycle 1. Finally, a single pharmacokinetic sample was collected on day 1 of cycle 2 just prior to the start of the cycle 2 infusion. PPD Development (Richmond, VA) performed pharmacokinetic (pK) analysis of GTI-2040 in plasma. Samples were stored at 80 °C until analysis. Plasma concentration of GTI-2040 was determined by capillary electrophoresis (CE) with ultraviolet absorbance detection, and metabolite concentrations for N1 to N4 were calculated using extinction coefficients, molecular weight and internal standard concentration. The method was validated to a lower limit of quantitation (LLOQ) for GTI-2040 of 0.125 µg/ml. The intra-assay coefficients of variation (CV) were 1.9% at LLOQ and 4.9%10.4% at levels above LLOQ; and inter-assay CV was 15% at LLOQ and 5.6%11.7% at levels above LLOQ. There were no endogenous electrophoretic peaks that interfered with quantitation of GTI-2040. Validation and analysis was performed using the Beckman P/ACE system MDQ 980709-15* 44 Acquisition Version 1.5, and Oracle Relational Database version 7.3.4.3. Pharmacokinetic parameters were estimated from the plasma concentration profile for each subject using non-compartmental analysis. The area under the concentration versus time curve [AUC (0t)] was calculated using the linear trapezoidal rule. AUC calculations are reported only for individuals who received the entire 21-day drug infusion and for whom plasma concentrations were available at the end of the cycle. Analysis of the peak plasma concentration (Cmax) was determined by inspection. Clearance was calculated for each patient using the following formula: dose/AUC. Steady-state drug concentration was determined as an arithmetic mean of the concentrations obtained at least five half-lives after the start of the infusion (concentrations collected and calculated after the infusions were discontinued were not included).
Pharmacodynamics
Ten ml of peripheral blood was collected from patients into an EDTA tube at baseline and day 22 of the first cycle to analyze expression of R2 before and after GTI-2040 treatment. Peripheral blood leukocytes were obtained by mixing the whole blood immediately with erythrocyte lysis buffer. The mixture was then incubated on ice for 15 min (the mixture was vortexed briefly twice during the incubation). Subsequently, the mixture was centrifuged at 500 g for 10 min at 4 °C; the supernatant was discarded and the leukocytes were frozen with liquid nitrogen and stored in a freezer at 80 °C until future use. To examine R2 gene expression, total cellular RNA was extracted from patient leukocytes using TRIzol Reagent (Invitrogen, Burlington, Ontario) according to manufacturer's instructions. The extracted RNA was treated with DNase I using a DNase-free kit using the manufacturer's instructions (Ambion Inc., Austin, TX). The RNA concentration was determined by spectrophotometric analysis of absorbance of the sample at 260 nm. RNA was reverse transcribed using a hexanucleotide primer (Amersham Pharmacia, Piscataway, NJ) and 800 units of Moloney murine leukemia virus reverse transcriptase (Invitrogen, Burlington, Ontario) using the manufacturer's instructions. Following reverse transcription all samples were treated with RNase H (Amersham Pharmacia, Piscataway, NJ) at 37 °C for 20 min. R2 mRNA analysis was performed using semi-quantitative RT-PCR using ß-actin gene as a control, and R2 message levels were expressed as a ratio of R2/actin. Murine studies to examine R2 downregulation in PBMCs after GTI-2040 treatment were considered, but could not be performed since the assay lacked the sensitivity to assess R2 changes in the volume of blood recovered from mice.
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Results |
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One patient developed arm pain and cellulitis due to GTI-2040 extravasation into the subcutaneous tissue from a ruptured catheter towards the end of the first cycle of therapy. Upon resolution of the symptoms, the patient was retreated with GTI-2040 and the toxicity data from the second cycle were included. The dose levels evaluated in the 26 fully assessable patients are listed in Table 2. A total of 49 cycles of therapy were administered at GTI-2040 doses ranging from 18.5 mg/m2/day to 222 mg/m2/day.
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The other serious non-dose-limiting adverse events observed on the trial included two patients who developed grade 3 infections (non-neutropenic) with Gram-negative pathogens during treatment cycles 2 and 4, respectively. Both patients were noted to have extensive abdominal organ metastases that might have served as a source for the infections. One patient with extensive retroperitoneal disease developed a bowel obstruction during the second cycle of treatment that was eventually confirmed to have resulted from disease progression.
Tumor response
No complete or partial objective tumor responses were observed. Four patients experienced disease stabilization: one patient with pancreatic cancer had stable disease for 2 months (therapy was discontinued in cycle 3 due to central line infection), two patients with colorectal cancer experienced disease stabilization of 4 and 6 months duration, respectively, and one patient with renal cell carcinoma experienced disease stabilization for 4 months.
Pharmacokinetics
A summary of pharmacokinetic parameters is provided in Table 5. Plasma concentrations versus time at different dose levels of GTI-2040 are shown in Figure 1. Detectable levels of GTI-2040 were observed in plasma at doses of 37 mg/m2/day and above, but detectable plasma concentrations for all four sampling days were only observed at the three highest dose levels. The pharmacokinetic parameters exhibited dose independence and high inter-individual variability. The terminal half-life of GTI-2040 was approximately 3 h at the recommended phase II dose. Plasma concentrations sufficient to suppress RNR expression in model systems were attainable in patients at the recommended phase II dose [16]. Analysis of data for association between body surface area (BSA) and clearance did not demonstrate any correlation between the two parameters (data not shown).
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Discussion |
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Consistent with findings of prior trials that used a similar drug infusion schedule, we did not observe substantial alterations in the complement pathway except in two patients. One patient was noted to have findings suggestive of complement activation associated with acute neutropenia. Similar findings have been described previously [2224
]. The patient did not experience any serious clinical complications. GTI-2040 infusion was discontinued in the patient 1 day after the abnormalities in the complement pathway were observed due to another clinical indication (extravasation of GTI-2040 in the subcutaneous tissue), leading to normalization of all the parameters. The complement pathway abnormalities observed in the second patient coincided with grade 4 hepatic toxicity. Both patients were treated at the highest dose level. Complement activation has typically been associated with high peak plasma concentrations and continuous infusion schedules often eliminate this toxicity [18
, 19
, 22
24
]. The two patients with complement activation in this trial were noted to have two of the highest peak concentrations of GTI-2040. Thus, it is possible that the high GTI-2040 plasma concentrations were responsible for the toxicity; however, the clinical situations of these patients (one patient with GTI-2040 tissue extravasation and the second patient with grade 4 hepatic toxicity) might also have contributed to activating the complement pathway.
The dose independence of the pharmacokinetics at the higher dose levels observed on this trial is in contrast to the pharmacokinetic findings described in several reports of other phosphorothioate oligonucleotides administered by continuous infusion [1721
]. The high coefficient of variation (CV) that we observed in the pharmacokinetic parameters also differs from previous reports [17
21
], and needs to be considered when interpreting the pharmacokinetic data. Missing pharmacokinetic samples for some subjects, along with several samples having drug concentrations below the lower level of quantification (BLQ), are potential explanations for the high CV of the pharmacokinetic parameters. Some patients also reported that their drug infusion had ended a few hours prior to arrival in the clinic for the early morning PK sampling on days 8, 15 or 22. This may have contributed to some of the BLQ values, given the relatively short half-life of the drug. However, other assay or sample handling errors cannot be ruled out and will need to be clarified in future trials of GTI-2040. The relatively small dosing increments at the three highest dose levels along with the high pharmacokinetic variability might well have masked the dose dependence of the pharmacokinetic parameters. The lack of association of BSA and clearance of the agent was not surprising, and also suggests that future trials of GTI-2040 could be performed using fixed dosing schedules rather than BSA-based dosing.
Although attempts were made to measure R2 mRNA levels in peripheral blood leukocytes before and after treatment with GTI-2040, the variability associated with these measurements was too large to allow adequate interpretation. Assessing changes in target gene expression in peripheral blood leukocytes has been difficult in other trials that attempted to perform such correlative studies [17, 19
]. We also considered performing western blots to assess changes in R2 levels; however, it was determined that R2 levels were too low to perform western blots from PBMCs recovered from 10 ml blood. There are significant limitations to measuring plasma concentrations of such agents, as well as performing pharmacodynamic assays in surrogate tissue, since neither are a true measure of the drug effect in the target tissue. Hence, the studies of GTI-2040 being conducted or planned as part of the NCI sponsored trials are being designed to address the limitations of this trial by expanding the correlative studies to include both surrogate and target tissues. Attempts will also be made to measure the tissue drug concentrations in some of these trials and attempts will be made to clarify the methodological issues pertaining to such biomarkers and their clinical relevance in future NCI sponsored studies of this drug.
The experience gained from other trials with antisense oligonucleotides suggests that this class of agents might be best utilized in combination with cytotoxic agents. The ideal combination should be guided by non-clinical studies, such as the high activity observed in combination with capecitabine against renal cell carcinoma [10]. Previous phase I and II trials performed with gemcitabine (an agent that also inhibits RNR) in combination with 5-fluorouracil have demonstrated good activity against renal cell carcinoma [25
, 26
]. Thus, a phase I/II trial using GTI-2040 in combination with capecitabine is now in progress for patients with refractory renal cell carcinoma.
Received for publication July 16, 2004. Revision received January 4, 2005. Accepted for publication January 10, 2005.
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