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Mol Cancer Ther. 2005;4:948-955
© 2005 American Association for Cancer Research

A phase I clinical trial of a ribozyme-based angiogenesis inhibitor targeting vascular endothelial growth factor receptor-1 for patients with refractory solid tumors

David E. Weng1, Paul A. Masci1, Susan F. Radka2, T. Elise Jackson2, Patricia A. Weiss1, Ram Ganapathi1, Paul J. Elson1, William B. Capra3, Vann P. Parker2, Jennifer A. Lockridge2, J. Wayne Cowens2, Nassim Usman2 and Ernest C. Borden1

1 Taussig Cancer Center, Cleveland Clinic Foundation, Cleveland, Ohio; 2 Sirna Therapeutics, Inc., Boulder, Colorado; and 3 Chiron Corp., Emeryville, California

Requests for reprints: David E. Weng, Taussig Cancer Center, Cleveland Clinic Foundation, Desk R-35, 9500 Euclid Avenue, Cleveland, OH 44195. Phone: 216-444-8375; Fax: 216-444-9464. E-mail: wengd{at}ccf.org


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Purpose: This study intended to determine the maximum tolerated dose, safety, pharmacokinetic variables, clinical response, and pharmacodynamic markers of daily s.c. administration of Angiozyme. Patients and Methods: Patients with refractory solid tumors were enrolled in a dose escalation and expanded cohort design. Dose escalation involved cohorts of patients at doses of 10, 30, 100, or 300 mg/m2/d for 29 days. A second component enrolled 15 additional patients at a daily dose of 100 mg/m2. Patients were eligible to continue on therapy until disease progression. Results: Thirty-one patients were enrolled and 28 were evaluable (range, 29–505 days; median, 89.5 days). A maximum tolerated dose was not defined by toxicity but rather by the maximal deliverable dose of 300 mg/m2/d. Grade 1 to 2 injection site reactions were the most common toxicities. One patient in the 300 mg/m2 group experienced a reversible grade 3 injection site reaction. Angiozyme showed dose-dependent plasma concentrations with good bioavailability. Surrogate markers showed Angiozyme localization in tumor biopsies and a significant increase in serum von Willebrand factor antigen, a marker for endothelial cell dysfunction. Although Angiozyme-reactive antibody production was noted for some patients, no antibody-related adverse events were noted. Seven of 28 (25%) evaluable patients had stable disease for ≥6 months, with the longest treatment duration of ≥16 months. Two patients (nasopharyngeal carcinoma and melanoma) showed minor responses. Conclusion: Angiozyme was well tolerated with satisfactory pharmacokinetic variables for daily s.c. dosing. Results have provided the basis for subsequent clinical trials of this first-of-class biologically targeted therapeutic.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Angiozyme is the first synthetic ribozyme to be tested as a therapeutic agent for human disease, targeting the conserved region of human, mouse, and rat vascular endothelial growth factor receptor (VEGFR)-1 mRNA. Ribozymes are catalytic RNA molecules that can cleave other RNA molecules in a target-specific manner, thereby down-regulating the expression of any pathogenic gene product, thus making them potentially a broad new class of therapeutic agents (1, 2). Angiozyme is an angiogenesis inhibitor targeting the VEGF angiogenic pathway and selectively down-regulating the VEGFR-1 through targeted cleavage of VEGFR-1 mRNA (3).

Prior studies showed that ribozymes targeting VEGFR-1 and VEGFR-2 can specifically cleave RNA substrates, reduce RNA levels in cell culture, and significantly inhibit VEGF-stimulated proliferation and angiogenesis (3, 4). Only disruption of VEGFR-1 by Angiozyme resulted in significant and dose-dependent antimetastatic activity for a xenograft model of human colorectal cancer, Lewis lung carcinoma model, and the mouse 4T1 tumor model (3–5). In the 4T1 animals, Angiozyme significantly decreased VEGFR-1 immunohistochemical staining in the normal kidneys of treated animals (5).

Based on the pharmacokinetic and toxicity profile in repetitive-dose animal studies and favorable pharmacokinetics of single-dose studies in normal volunteers, we initiated a phase I trial for this first representative of a new class of agents (6–8). Primary end points were safety, tolerability, and determination of the maximum tolerated dose. Secondary end points were plasma pharmacokinetics, clinical activity, and efforts to identify pharmacodynamic markers.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Patient Eligibility
Thirty-one adult patients with biopsy-proven solid tumors refractory to standard therapy were eligible for treatment from November 1999 to July 2000. Consenting subjects had an Eastern Cooperative Oncology Group performance status of 0 or 1, without complicating concurrent illnesses or other cancer therapies, had a life expectancy of at least 3 months, and had adequate hematologic (granulocyte count >1,500/mm3, hematocrit >28, and platelet count >125,000/mm3), renal (serum creatinine <2.0 mg/dL, unless measured creatinine clearance is >50 mL/min), hepatic (aspartate aminotransferase less than two times the upper limit of normal; direct bilirubin less than two times the upper limit of normal unless the patient has a previous diagnosis of a congenital hyperbilirubinemia), and coagulation (activated partial thromboplastin time <37 seconds, international normalized ratio <1.5) values. This study met all institutional and federal government requirements for research involving human subjects.

Treatment and Assessment Schedule
Patients received s.c. injections of Angiozyme daily for 28 consecutive days followed by a 1-week rest. A tumor biopsy was done before treatment and after 21 days of dosing for patients with easily accessible tumor. Patients underwent staging evaluations before treatment and again on day 36. Patients with progressive disease, significant toxicities (grade 3 or 4), or other complicating medical conditions were withdrawn from study and followed for 30 days to assess post-treatment toxicities. Patients without irreversible toxicities (grade 3 or 4) with stable to improved disease were offered continued treatment without dose adjustment, except for patients at the 300 mg/m2/d dose level, who had subsequent therapy at 100 mg/m2/d. Patients were restaged monthly. Angiozyme was reconstituted using USP normal saline for injection and provided to the patient in weekly kits.

Dose Escalation and Modification of Angiozyme
Angiozyme was assessed at four dose levels (10, 30, 100, and 300 mg/m2/d). Cohorts of three patients were treated at each increasing dose level until the maximum tolerated dose. The maximum tolerated dose was defined as 300 mg/m2/d (the maximal deliverable dose for s.c. injection) or the highest dose where no more than two of six evaluable patients developed a grade 3 or 4 toxicity (possibly drug related) during 29 days of treatment. If no cohort showed grade 3 or 4 toxicity (possibly drug related) in three or more of six patients, 300 mg/m2 was determined as the maximum tolerated dose because the volume for injection of the dose of 300 mg/m2/d was at the limit of patient tolerability. Following dosing of the first four cohorts, an additional cohort of 15 patients was administered 100 mg/m2/d to expand the safety and response database. The dose level of 100 mg/m2/d was chosen because of pharmacokinetic data and the tolerable volume for daily injections. Patients who developed grade 3 or 4 toxicity, possibly related to Angiozyme treatment, were started using the next lowest dose level.

Monitoring of Toxicity and Response
Treatment toxicities were assessed using the National Cancer Institute Common Toxicity Criteria (version 2.0). Patients with measurable tumor were assessed by physical or radiographic assessment within 21 days before the beginning of treatment and then monthly. Response criteria involved prospective serial assessment of the sum of the products of bidimensional perpendicular diameters of at least three index measurable lesions. Complete response was defined as disappearance of all measurable and evaluable disease, with no new lesions. Partial response was defined as at least 50% decrease in the sum of the products of bidimensional diameter lesions. Progressive disease was defined by a >25% increase in the sum of the products of bidimensional diameter measurements. Stable disease was defined as any measurement that did not qualify as a complete response, partial response, or progressive disease. Adverse events were classified according to MeDRA version 5.0.

Pharmacokinetic Methods
Pharmacokinetic Sampling. Blood was collected before and at 1, 2, 4, 6, 8, 12, 16, and 24 hours after injection on days 1 and 29 of treatment. Additional plasma pharmacokinetic samples were collected before injection on days 8, 15, and 22. Plasma was stored at –20°C until analyzed by high-performance liquid chromatography method.

Quantitation of Angiozyme in Plasma. Quantitation of Angiozyme in human plasma was carried out at PPD Development (Richmond, VA). Plasma samples were analyzed using a method involving liquid-liquid extraction with high-performance liquid chromatography analysis that was a modification of methods described previously (7).

Pharmacokinetic Analysis. Pharmacokinetic analysis was done using WinNonlin (version 3.1, Pharsight Corp., Mountain View, CA). Methods used were based on statistical moment theory (noncompartmental approaches). Briefly, the maximum observed plasma concentration (Cmax) and the time to reach maximum concentration were observed variables. Area under the concentration-time curve was estimated using the linear trapezoidal rule up to Cmax and then a log trapezoidal rule for the remainder of the curve. Extrapolation of the plasma concentration-time curve from the last measurable point to infinity was calculated using log-linear regression. The terminal elimination half-life (T1/2) was calculated from regression analysis of the last three values that were detectable.

Localization of Angiozyme by Immunohistochemistry
Immunohistochemistry detection of Angiozyme in biopsy samples was done by Pathology Associates International (Frederick, MD). Paraffin sections of formalin-fixed material were stained with CA1-USR, a monoclonal antibody directed against 2'-C-allyl-uridine, reactive with Angiozyme (9). An isotype-matched antibody was used as staining control for adjacent sections. Pretreatment biopsies were also stained as controls. Antibody was detected by the DAKO EnVision+ System (Carpinteria, CA). Cells were identified morphologically as endothelial cells, fibroblasts, macrophages, or tumor cells.

Assessment of Patient Antibody Response to Angiozyme
An enzyme immunoassay employing biotinylated Angiozyme bound to Strep-a-Well Hi Bind streptavidin 96-well plates (Roche Molecular Systems, Indianapolis, IN) was used to detect Angiozyme-reactive antibodies in patient sera. Pre-dosing serum (1:50 dilution) from each patient was compared with post-dosing samples. Peroxidase-conjugated goat anti-human IgM, IgG, and IgA (ICN Biomedicals, Aurora, OH) were used to detect antibodies bound to biotinylated Angiozyme. Titer was determined as the dilution of a patient's serum with the same absorbance reading as the 1:50 dilution of that patient's pre-dosing serum.

Assessment of Tumor Response and Tissue Perfusion by Magnetic Resonance Imaging
Serial two-dimensional gradient echo studies during bolus administration of i.v. gadolinium-diethylenetriaminepentaacetic acid (0.1 mmol/kg; 2 cc/s Medrad Spectris Injector) were done using a T1-weighted FLASH sequence (flip angle 60°, repetition time 65 milliseconds, echo time 7 milliseconds, 20 cm field of view, 128 x 256, 5 mm slice). A series of four slices were obtained through the region of the soft tissue mass before, during, and after the contrast injection for a total of 40 acquisitions. Regions of interest were drawn with conventional software provided by the magnetic resonance manufacturer to include the necrotic center, soft tissue along the periphery of the mass, and background muscle spatially removed from the mass. Time-intensity plots were constructed for each study and compared qualitatively between the initial and follow-up visits.

Assessment of Serum von Willebrand Factor Protein Levels
Nineteen patients had paired samples evaluable for von Willebrand factor levels before and following ~4 weeks of treatment. A control cohort consisted of 21 patients with advanced malignancy, receiving standard care (14 chemotherapy treated and 7 observation only). Total serum von Willebrand factor levels were determined at baseline and at median follow-up time of 28 days (range, 15–43 days) for 20 patients (1 patient at the 30 mg/m2/d dose level and 19 patients at the 100 mg/m2/d dose level). Serum levels of von Willebrand factor antigen protein were measured by an immunoturbidometric assay, reported as a percent activity with a normal reference range of 45% to 150% based on kit standards (Liatest-vWf, Diagnostica Stago, Asnieres-Sur-Seine, France). All statistical analyses were done using the SAS software system (Cary, NC). von Willebrand factor levels and platelet counts were compared using the Kruskal-Wallis test. The difference between baseline and follow-up values were then compared within the cohorts using the Wilcoxon signed rank test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Patients
Thirty-one patients were enrolled and 28 patients were evaluable for pharmacokinetics and response (Table 1). Three patients withdrew from study before completion of 29 days of initial dosing due to patient ineligibility, voluntary withdrawal, or treatment noncompliance. Three patients voluntarily withdrew from the study before documented disease progression to pursue other treatment options. The mean age was 53.5 years (range, 24–83 years), with 45% male patients and 55% female patients. There were 16 histologies among the 31 patients, with melanoma (7 patients) and breast cancer (6 patients) as the predominant types.


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  		Table 1. Patient demographics, cancer history, and best overall response

 
Adverse Events
The most common adverse side effect was injection site reactions (Table 2). There were no significant clinical toxicities greater than grade 3 or laboratory-based toxicities greater than grade 2 that were attributable to Angiozyme. The only grade 3 clinical toxicity occurred in one patient who experienced a reversible grade 3 injection site reaction with pain after the first injection of the highest dose (300 mg/m2/d). Because of Angiozyme solubility, 4 mL was the required volume to deliver the calculated dose. This subject subsequently received a dose reduction to 100 mg/m2/d and tolerated therapy without further significant toxicities. Subsequent patients received their dose in divided volumes of a maximum of 2 mL. There were no significant changes in patient laboratory values, performance status, or body weight.


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  		Table 2. Summary of adverse events

 
The injection site reactions were characterized by pain, erythema, pruritis, and burning, occurring within 1 to 2 days after s.c. injection, typically resolved within 3 to 7 days. Injection site reaction was not correlated to dose level, duration of therapy, anatomic site of injection, or prior history of hypersensitivity reactions and did not worsen with cumulative dosing. Topical measures, such as antihistamine, corticosteroids, or heat or cold pack treatments, did not affect injection site reactions. There were no associated side effects of systemic hypersensitivity (fevers, hypotension, bronchospasm, or edema).

Punch biopsies were obtained for three patients experiencing injection site reactions, with dermatopathologic interpretation of dermal edema with perivascular and interstitial acute and chronic inflammation composed of neutrophils, eosinophils, and lymphocytes, compatible with a nonspecific drug reaction. There were no significant toxicities or treatment limitations for patients who experienced regular injection site reactions, with one patient treated for >16 months.

Two adverse events possibly related to Angiozyme included atrial fibrillation and a gastrointestinal hemorrhage. The first patient had metastatic non–small cell lung cancer with a prior history of atrial fibrillation and was withdrawn from study after one dose. The second patient had metastatic melanoma with a prior history of gastrointestinal polyps with one episode of melanotic stool during the initial 29-day dosing interval. Subsequent upper and lower gastrointestinal endoscopy showed multiple polyps but no active bleeding site. He showed progressive disease and was withdrawn from study.

Twelve of the 31 patients underwent biopsy after a minimum of 21 days of drug administration. Biopsies included percutaneous liver biopsy, skin biopsy, tumor excision, and nodal excision. None of the biopsies were associated with delayed wound healing or abnormal bleeding.

One patient experienced a grade 3 injection site reaction in the 300 mg/m2/d dose cohort, but the additional three patients in this dose cohort did not have any dose-limiting toxicity. Therefore, the maximum tolerated dose for Angiozyme was defined as 300 mg/m2/d based on the upper limit of drug solubility and the practically deliverable dose volume and not by toxicity.

Pharmacokinetics of Angiozyme
The pharmacokinetics were determined over an extended period of 29 days (Table 3). Angiozyme at all dose levels showed acceptable bioavailability (average, >50%) with dose-dependent plasma concentration-time curves (Fig. 1). The average elimination T1/2 was ~3 to 9 hours. Comparing pharmacokinetic variables for days 1 and 29 dosing, values were similar and there was no evidence of drug accumulation in plasma with repeated dosing. Based on murine models of antitumor activity, the minimum effective plasma concentration for biological efficacy was estimated to be 0.5 µg/mL (3, 4, 6). Repetitive dosing at the 100 mg/m2/d level maintained plasma concentrations above this extrapolated value for the majority of the dosing interval with a peak concentration of 3.46 µg/mL. Dose-dependent increases in trough concentrations were observed (Fig. 2). However, there was no apparent increase in trough levels over time.


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  		Table 3. Pharmacokinetic variables obtained from noncompartmental analysis of plasma concentration-time profiles of patients administered 10, 30, 100, or 300 mg/m2/d Angiozyme for 29 days

 


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  		Figure 1. Plasma concentrations of Angiozyme after administration of 10 mg/m2 (n = 3), 30 mg/m2 (n = 3–4), 100 mg/m2 (n = 18–21), or 300 mg/m2 (n = 3). Doses were administered daily. Plasma concentrations were evaluated after the first dose (day 1) and on day 28. Points, mean; bars, SD.

 


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  		Figure 2. Pre-dose (trough) plasma concentrations after administration of 10 mg/m2 (n = 3), 30 mg/m2 (n = 3–4), 100 mg/m2 (n = 18–21), or 300 mg/m2 (n = 3). The trough levels for each dose cohort were obtained on days 8, 15, and 22 of daily dosing schedule. Points, mean; bars, SD.

 
Immunohistochemistry of Tumor Samples from Angiozyme-Treated Subjects
Biopsies were collected from nine subjects before and following 28 days of treatment. Immunohistochemical studies for VEGFR-1, VEGFR-2, and CD105 were done as outlined in Materials and Methods. CD105 staining, indicative of vascular endothelial cells, was clearly present in all samples. However, VEGFR-1 and VEGFR-2 staining was heterogeneous in vascular endothelial cells. In numerous pre-dose samples, there was little or no detectable staining for VEGFR-1. Therefore, it was not possible to determine the relative effect of Angiozyme on VEGFR-1 or VEGFR-2 expression in vascular endothelial cells.

Angiozyme distribution within tumors was assessed by immunohistochemical staining of paired biopsy samples obtained before drug treatment and then following at least 21 days of drug treatment. A murine monoclonal antibody recognizing 2'-C-allyl-uridine (one of the modified synthetic oligoribonucleotides present in Angiozyme) was used to determine the localization of Angiozyme in tissue sections (9). Antibody-positive specific staining within the tumor biopsy was confirmed with accumulation in endothelial cells, fibroblasts, and macrophages (data not shown). Cells were identified by morphologic criteria.

Humoral Response to Angiozyme
Because substituted synthetic ribonucleotides might be immunogenic, patients were evaluated to determine whether they generated a humoral response to Angiozyme. Anti-Angiozyme antibodies were detected in 2 of 28 patients after 29 days dosing with 1:100 titers for patients at 100 and 300 mg/m2/d levels.

Of the five patients with serum samples available after >100 days of dosing at 100 mg/m2/d, four patients had detectable titers of anti-Angiozyme antibody. In these patients' sera, there were significant levels of free Angiozyme. Although three patients became borderline positive for ANA after chronic dosing, one remained negative and one was borderline positive at the start of the study. All patients were negative for anti-dsDNA antibodies. No patients had evidence for immune complex deposition syndromes or rheumatologic diseases.

Analysis of Clinical Outcomes
Twenty-eight of 31 enrolled patients were evaluable for clinical response. Thirteen of the 14 patients during the dose-escalation phase, and 15 of the 17 patients during the expanded cohort at 100 mg/m2/d were evaluable for response. Patients were assessed monthly for response. The median duration for time to disease progression was 120 days. Seven of the 28 patients had stable disease beyond 6 months, with tumor pathologies that included colorectal cancer, renal cell carcinoma, hemangioendothelioma, breast carcinoma, parotid adenocarcinoma, peritoneal mesothelioma, and endometrial sarcoma. The patient with endometrial sarcoma exhibited the longest duration of stable disease, with total duration of therapy of 505 days.

Two patients had evidence of a minor response. The first patient (1003) with nasopharyngeal squamous cell carcinoma, who was treated at the lowest dose level of 10 mg/m2/d, had a minor response with decreased size of his measurable lesion. Dynamic contrast magnetic resonance imaging has been investigated as means to evaluate vascular perfusion and angiogenesis inhibitor effect (10). Evaluation revealed decreased perfusion in the tumor lesion with central necrosis. Direct visualization of the posterior pharynx of this patient confirmed tumor necrosis. The second patient (1015), treated at the 100 mg/m2/d dose level, with a minor response had metastatic melanoma. He showed a mixed response with a decrease of one index lesion but an increase of a second lesion.

Serum von Willebrand Factor Antigen Levels Increase with Angiozyme Treatment
Several surrogate serum markers, including VEGF, basic fibroblast growth factor, soluble vascular cell adhesion molecule-1 soluble VEGFR-1, and von Willebrand factor antigen, were assessed, comparing pretreatment and post-treatment levels and correlating to clinical course. Although detectable levels could be measured, there were no statistically significant correlations of increase or decrease between pretreatment and post-treatment levels. A statistically significant increase in both VEGF and soluble vascular cell adhesion molecule-1 for patients who had progressive disease was noted (11).

Because VEGFR-1 is thought to be essential for endothelial cell viability, serum von Willebrand factor antigen levels can increase when endothelial cell viability is compromised (12). Post-treatment levels of serum von Willebrand factor antigen showed a significant increase compared with pretreatment levels in relation to Angiozyme treatment. Two cohorts of subjects were analyzed for von Willebrand factor levels: Angiozyme-treated subjects and subjects with advanced malignancies undergoing standard care treatment for their disease. Patients in the control cohort were similar to Angiozyme-treated subjects.

Changes in von Willebrand factor levels were assessed using two approaches. To assess the intrapatient effect of treatment, serum von Willebrand factor levels after 1 month of daily treatment were compared with baseline levels. This approach assessed the direct individual effect of Angiozyme treatment by measuring percent change from baseline. The significance of percent change from baseline was determined for both controls and treated subjects using the Wilcoxon signed rank test. The second approach involved comparison of serum von Willebrand factor levels between cohorts in relation to baseline and follow-up levels (Fig. 3). This approach assessed the aggregate cohort effect of prolonged Angiozyme treatment.



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  		Figure 3. Changes in von Willebrand factor (VWf) levels for cohorts of treated and control patients. A, average value of von Willebrand factor percent activity before and after treatment for each cohort. B, platelet concentration was stable within and between cohorts.

 
Increased serum von Willebrand factor levels showed a significant correlation with Angiozyme treatment for both intrapatient and group effect. Of the 31 patients on study, 19 patients (1 patient at dose level 30 mg/m2/d and 18 patients at dose level 100 mg/m2/d) had complete data for von Willebrand factor analysis. Baseline von Willebrand factor levels were not significantly different between the two cohorts (P = 0.58). Serum levels of total von Willebrand factor protein increased for all of the Angiozyme-treated patients. Median time from baseline to follow-up level was 29 days (range, 29–36 days). The mean baseline level was 174.1 ± 13.6 (range, 100–351) and mean follow-up level was 256.7 ± 19.2 (range, 119–458; P < 0.001; Fig. 3A).

Total serum von Willebrand factor protein was measured at baseline and median follow-up time was 28 days (range, 15–43 days) in control subjects. Total serum von Willebrand factor protein increased over the study period in 9 of the 21 (43%) control cohort patients. No significant difference was observed in the mean baseline value compared with the follow-up value (215.7 versus 207.6; P = 0.54; Fig. 3A). Because all subjects in the control cohort showed stable or progressive disease over the sampling interval but had no significant increase in von Willebrand factor levels during the sampling interval, it is unlikely that von Willebrand factor levels were a marker for progressive disease only. Therefore, it was more likely that change in von Willebrand factor levels was specific for Angiozyme effect.

Because platelets are the other source of von Willebrand factor, a possible cause for increased von Willebrand factor levels in Angiozyme-treated subjects could be increased platelet counts. To control for a platelet source of von Willebrand factor, total platelet counts were analyzed for all three cohorts, with no significant differences in baseline (P = 0.82) or changes from baseline for treated or control subjects (P > 0.21 for each cohort; Fig. 3B).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This phase I trial for patients with refractory solid tumors showed that Angiozyme could be administered daily on a repetitive dosing schedule via a s.c. route without significant side effects with some subjects receiving >6 months of treatment. There were no dose-limiting toxicities for the dose range tested. The top dose of 300 mg/m2/d was deemed to be the maximum deliverable dose because of the significant discomfort associated with the volume of injection required for drug delivery at that dose level. We concluded that the dose level of 100 mg/m2/d was the appropriate dose for future clinical development based on the favorable pharmacokinetics and the tolerability of long-term injection at this dose level.

Clinical studies of other VEGF antiangiogenesis agents showed side effects of thromboembolic complications, tumor hemorrhage, hypertension, proteinuria, headache, nausea, and vomiting (13–16). The lack of similar toxicities may be due to inherent differences in ribozyme tolerability, the possibility that the optimal Angiozyme dose for biological activity was not reached or maintained with the administered dose, the s.c. route of drug delivery for Angiozyme, the additional factor of concurrent chemotherapy in other trials, or the relatively small sample size of trials subjects. Studies of Angiozyme as a single agent or in combination with chemotherapy are ongoing and will expand the safety and efficacy profile of this agent.

Although clinical efficacy could not be established in this dose-finding and pharmacokinetic study, seven patients had prolonged stable disease for >6 months while on Angiozyme. Five of these seven patients had progressive disease within 6 months before enrollment. Additionally, dynamic contrast magnetic resonance imaging of one patient (1003), who was treated at the lowest dose level of 10 mg/m2/d, showed tumor hypoperfusion and necrosis, suggesting antitumor activity after ~2 months of treatment. A second patient (1015), treated at the 100 mg/m2/d dose level, showed a mixed response with partial response in some lesions and progressive disease in others. These may be clinical indicators suggesting that the dose levels tested in this study were within the appropriate range for biological and clinical activity.

Multiple surrogate markers have been investigated during antiangiogenesis trials (14–17). We noted a striking correlation of increased serum von Willebrand factor in relation to Angiozyme treatment. Serum von Willebrand factor is produced exclusively in megakaryocytes and endothelial cells (18). A portion of endothelial-derived von Willebrand factor is stored in intracellular vesicles termed Weibel-Palade bodies where the protein aggregates into higher molecular weight multimers. Although several well-described vasoactive substances and clinical conditions can cause the acute and chronic release of von Willebrand factor from platelets and endothelial cells as a normal physiologic response, inhibition of endothelial cell viability can also cause increased serum von Willebrand factor protein (12). As described previously, inhibition of VEGFR-1 can lead to disruption of endothelial cell biology (19). Therefore, it is possible that serum von Willebrand factor will be useful as a marker of endothelial cell damage, induced by disruption of the VEGF signaling system.

Serum von Willebrand factor levels significantly increased after Angiozyme treatment for both intrapatient and interpatient studies, suggesting biological activity of Angiozyme against endothelial cells. We did not have sufficient patient data to correlate von Willebrand factor changes to Angiozyme dose levels and therefore are not able to hypothesize whether von Willebrand factor levels could be used as a surrogate marker to determine the optimal dose level for biological or clinical activity. Although platelets were unchanged during Angiozyme treatment, it is still possible that Angiozyme effect on megakaryocytes could account for the von Willebrand factor change. Similarly, SU5416, a small-molecule inhibitor of VEGFR-1 and VEGFR-2, showed elevated serum von Willebrand factor levels in patients treated with SU5416, suggesting that disruption of VEGF signal pathways leads to endothelial activation/dysfunction (20).

In summary, Angiozyme was well tolerated as a potential novel antiangiogenesis agent targeting the VEGFR-1 receptor. This multidose trial showed Angiozyme to have minimal toxicities with good bioavailability. Additionally, increased von Willebrand factor levels suggest biological activity of Angiozyme to perturb endothelial cell homeostasis. Phase II trials for specific tumor types are ongoing to further assess the biological and clinical activity of Angiozyme.


   Acknowledgments
 
We thank Dr. Paul Ruggieri, Judy Smith, Dr. Kayvon Jalali, Dr. Tom Budd, Dr. Maurice Wolin, Deni Robson, Shelly Robinson, and Dr. Ronald Bukowski for their assistance in the design and conduct of this work and Drs. Patrice Lee, Pamela Pavco, and Roger Aitchison for reviewing the article.


   Footnotes
 
Grant support: Sirna Therapeutics, Inc. (Boulder, CO).

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.

Received 8/17/04; revised 3/ 1/05; accepted 4/13/05.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Usman N, Beigelman L, McSwiggen JA. Hammerhead ribozyme engineering. Curr Opin Struct Biol 1996 Aug;6:527–33.[Medline]
  2. Usman N, Blatt LM. Nuclease-resistant synthetic ribozymes: developing a new class of therapeutics. J Clin Invest 2000 Nov;106:1197–202.[Medline]
  3. Pavco PA, Bouhana KS, Gallegos AM, et al. Antitumor and antimetastatic activity of ribozymes targeting the messenger RNA of vascular endothelial growth factor receptors. Clin Cancer Res 2000 May;6:2094–103.[Abstract/Free Full Text]
  4. Parry TJ, Cushman C, Gallegos AM, et al. Bioactivity of anti-angiogenic ribozymes targeting flt-1 and KDR mRNA. Nucleic Acids Res 1999 Jul 1;27:2569–77.
  5. Silverman D, Berstrom R, Rothchild K, Chambers K, Weng DE, Kim J. Reduction in spontaneous metastases from 4T1 murine carcinoma using a ribozyme which targets flt-1 (VEGF receptor subtype 1) mRNA. 12th NCI-EORTC-AACR Symposium on New Drugs in Cancer Therapy; 2001.
  6. Sandberg JA, Bouhana KS, Gallegos AM, et al. Pharmacokinetics of an antiangiogenic ribozyme (Angiozyme) in the mouse. Antisense Nucleic Acid Drug Dev 1999 Jun;9:271–7.[Medline]
  7. Sandberg JA, Parker VP, Blanchard KS, et al. Pharmacokinetics and tolerability of an antiangiogenic ribozyme (Angiozyme) in healthy volunteers. J Clin Pharmacol 2000 Dec;40:1462–9.[Abstract]
  8. Sandberg JA, Sproul CD, Blanchard KS, et al. Acute toxicology and pharmacokinetic assessment of a ribozyme (Angiozyme) targeting vascular endothelial growth factor receptor mRNA in the cynomolgus monkey. Antisense Nucleic Acid Drug Dev 2000 Jun;10:153–62.[Medline]
  9. Radka SF, Pasko C, Haeberli P, Beigelman L. The development of a monoclonal antibody specific for a 2(')-C-allyl modification of uridine, and its use in the localization of ribozymes in vivo. Anal Biochem 2002 Aug 1;307:40–6.[CrossRef]
  10. Padhani AR, Husband JE. Dynamic contrast-enhanced MRI studies in oncology with an emphasis on quantification, validation and human studies. Clin Radiol 2001 Aug;56:607–20.[CrossRef][Medline]
  11. Radka SF, Pasko C, Moyer CF, et al. Biomarker assessment of patient samples from a phase I/II study of Angiozyme in metastatic cancer patients. Third International Symposium on Anti-Angiogenic Agents; 2001.
  12. Mannucci PM. Von Willebrand factor: a marker of endothelial damage? Arterioscler Thromb Vasc Biol 1998 Sep;18:1359–62.[Free Full Text]
  13. Gordon MS, Margolin K, Talpaz M, et al. Phase I safety and pharmacokinetic study of recombinant human anti-vascular endothelial growth factor in patients with advanced cancer. J Clin Oncol 2001 Feb 1;19:843–50.
  14. Margolin K, Gordon MS, Holmgren E, et al. Phase Ib trial of intravenous recombinant humanized monoclonal antibody to vascular endothelial growth factor in combination with chemotherapy in patients with advanced cancer: pharmacologic and long-term safety data. J Clin Oncol 2001 Feb 1;19:851–6.
  15. Stopeck A, Sheldon M, Vahedian M, Cropp G, Gosalia R, Hannah A. Results of a phase I dose-escalating study of the antiangiogenic agent, SU5416, in patients with advanced malignancies. Clin Cancer Res 2002 Sep;8:2798–805.[Abstract/Free Full Text]
  16. Kabbinavar F, Hurwitz HI, Fehrenbacher L, et al. Phase II, randomized trial comparing bevacizumab plus fluorouracil (FU)/leucovorin (LV) with FU/LV alone in patients with metastatic colorectal cancer. J Clin Oncol 2003 Jan 1;21:60–5.
  17. Herbst RS, Mullani NA, Davis DW, et al. Development of biologic markers of response and assessment of antiangiogenic activity in a clinical trial of human recombinant endostatin. J Clin Oncol 2002 Sep 15;20:3804–14.
  18. Greenberg CS, Orthner CL. Procoagulant cofactor proteins. In: Lee GR, Foerster J, editors. Wintrobe's clinical hematology. 10th ed. Philadelphia: Lippincott; 1999. p. 702–9.
  19. Ferrara N, Gerber HP, LeCouter J. The biology of VEGF and its receptors. Nat Med 2003 Jun;9:669–76.[CrossRef][Medline]
  20. Kuenen BC, Levi M, Meijers JC, et al. Analysis of coagulation cascade and endothelial cell activation during inhibition of vascular endothelial growth factor/vascular endothelial growth factor receptor pathway in cancer patients. Arterioscler Thromb Vasc Biol 2002 Sep 1;22:1500–5.



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