Abstract
Clinical and laboratory observations support the view that angiogenesis is necessary for prostate cancer progression. The angiogenesis inhibitor TNP-470 has demonstrated in vivo antitumor activity in a series of clinical models. To evaluate a possible therapeutic clinical value, we conducted a Phase I dose escalation trial of alternate-day i.v. TNP-470 in 33 patients with metastatic and androgen-independent prostate cancer. The patients were evaluated during therapy for evidence of neurological toxic effects. An assay of endothelial and vascular proliferation “markers” and a sequential assay of serum prostate-specific antigen concentration were performed. The effects of TNP-470 could be evaluated in 32 of the 33 patients. The maximum tolerated dose was 70.88 mg/m2 of body surface area. The dose-limiting toxic effect was a characteristic neuropsychiatric symptom complex (anesthesia, gait disturbance, and agitation) that resolved upon cessation of therapy. The times to clinical recovery of neurological side effects were 6, 8, and 14 weeks. No definite antitumor activity of TNP-470 was observed; however, transient stimulation of the serum prostate-specific antigen concentration occurred in some of the patients treated. Additional studies of TNP-470 should be conducted using an alternate-day i.v. injection of 47.25 mg/m2 body surface area and should focus on understanding and overcoming the neurological toxic effects. In addition, valid intermediate end points that reflect the status of tumor-associated neovascularity are needed to facilitate effective development of treatment strategies.
INTRODUCTION
Inhibition of angiogenesis may be an effective strategy for the treatment of cancer (1, 2, 3, 4) . Clinically, the importance of angiogenesis in prostate cancer progression is supported by the evidence that increased microvessel density is predictive of disease recurrence after local therapy (5, 6, 7, 8) . Furthermore, IFN-α effectively inhibits angiogenesis and may exert some of its antitumor activity through this mechanism, providing further evidence that angiogenesis inhibition may be a feasible treatment strategy (9) .
The therapeutic application of this knowledge has been made possible by the development of specific angiogenesis inhibitors that have an acceptable preclinical toxicity profile. Among these agents is TNP-470, an analogue of fumagillin. Fumagillin is an antibiotic derived from the fungus Aspergillus fumigatus Fresenius. Ingber et al. (5) first reported that fumagillin inhibited endothelial cell proliferation in the presence of saturating levels of βFGF.3 They also found that s.c. administration of 100 mg/kg of fumagillin inhibited tumor-induced neovascularization in the mouse dorsal air sac. Because of the toxicity of fumagillin, synthetic analogues were developed by Takeda Chemical Industries Limited. One of these, O-(chloroacetyl carbamyl) fumagillol or TNP-470, retains the antiangiogenic effects while having reduced toxicity.
We initiated a cancer-specific Phase I trial with the primary goal of identifying the MTD of TNP-470 given as an alternate-day i.v. infusion on a 6-week schedule and of fully characterizing the toxicity profile. A secondary goal of this trial was to develop the clinical foundation for the study of angiogenesis inhibitors in prostate cancer. This latter aim was to be achieved by the incorporation of candidate markers of angiogenesis in the study. The design of this trial was based on the perceived advantages of treating a uniform group of patients when developing a new therapeutic concept. Prostate cancer was considered an ideal target for four reasons: (a) the availability of a reliable tumor marker, PSA, for this cancer; (b) the indolent clinical course of this cancer, which permits investigators to observe the therapeutic and biological effects of treatment; (c) specific clinical data supporting the predictive value of microvessel density in prostate cancer progression; and (d) preclinical in vivo data suggesting that TNP-470 may be effective against prostate cancer by inhibiting angiogenesis.
MATERIALS AND METHODS
Study Eligibility Criteria.
All of the study patients had confirmed metastatic and androgen-independent prostate cancer that was progressing after adequate androgen-ablative therapy. All of them had an estimated survival time of greater than 3 months, with no significant intercurrent medical illnesses. Objective criteria of androgen-independent progression included the following: castrate levels of serum testosterone, disease progression confirmed by two consecutive elevations in PSA concentration, radiographic evidence of cancer confirmed by two consecutive elevations in PSA concentration, and radiographic evidence of cancer (new sites consistent with metastasis and detected by bone scan or nodal enlargement detected by computed tomographic scan). Biopsy of metastatic sites was not required in patients with an elevated PSA concentration. A trial of withdrawal of the antiandrogen flutamide was not required when the study was started, but it became a requirement when the phenomenon of flutamide withdrawal was recognized. Five patients did not have ≥6-week trial of flutamide withdrawal before enrollment in the study; the initial two patients were excluded from the analysis of PSA response.
Required hematological criteria were a granulocyte count of ≥1500/ml, a platelet count of ≥100,000/ml, hemoglobin ≥10 g/dl, a normal coagulation profile (thrombin time, partial thromboplastin time), and no recent history of bleeding. A Zubrod performance status of ≥2 was required. Written informed consent approved by the Institutional Review Board of The University of Texas M. D. Anderson Cancer Center was obtained from all of the patients before entry into the trial. Specific exclusion criteria were a greater than 4-fold increase in serum transaminase level, a serum creatinine level >2 mg/dl, clinical congestive heart failure, and uncontrolled angina.
Pretreatment Evaluation.
A medical history and a physical examination (including an ophthalmological evaluation) were required in the initial trial design. After recognition of the neuropsychiatric effects of TNP-470, routine neuropsychiatric evaluation was also performed on all of the study patients. Laboratory studies included the following: complete blood count, differential and platelet count; SMA-12, triglyceride, cholesterol, PSA, and transferrin levels; screening for stool guaiac; hepatitis and the human immunodeficiency virus screening; bone marrow aspiration and biopsy; and electrocardiogram and urinalysis.
Measurement of Plasma-soluble E-Selectin and Thrombomodulin.
Plasma-soluble E-selectin levels were quantified using an ELISA kit (obtained from British Biotechnology Products Limited, Abingdon, United Kingdom; Refs. 10, 11, 12, 13 ), according to the manufacturer’s procedure. In brief, to each well of a 96-well plate coated with murine monoclonal antibody against human E-selectin, 100 μl of anti-E-selectin were added. The contents were decanted, and the wells were washed six times; 100 μl of HRP substrate (tetramethylbenzidine) were added to each well. The reaction proceeded for 30 min and was then stopped by the addition of Stop solution. The absorbance of each well sample was determined in an ELISA reader. Quantities of soluble E-selectin in the test samples were determined by relating the absorbance to a calibration curve. Positive and negative controls were included on each assay plate.
Plasma-soluble thrombomodulin levels were measured using an enzyme immunoassay kit (Diagnostic Stage, Asnières-Sur-Seine, France), as described in the manufacturer’s manual. In brief, 200 μl of standard, control, or test sample was added to each well of a 16-well microtiter plate. The plate was kept at room temperature for 2 h, and each well was then repeatedly washed. As soon as the last washing cycle was finished, 200 μl of anti-thrombomodulin-HRP conjugate was added to each well. After 2 h, each well was washed five times with washing buffer. Then 200 μl of HRP substrate (o-phenylenediamine) was added to each well, and the mixture was incubated at room temperature for 8 min. The reaction was stopped, and the absorbance was determined in an ELISA reader. The quantity of soluble thrombomodulin in the test sample was determined from the calibration curve.
Treatment.
Patients received a 60-min i.v. infusion of TNP-470 through a central venous catheter every other day for 28 days (14 treatment days) followed by a 2-week treatment-free interval. Dose level zero (starting dose) was 9.3 mg/m2 BSA. Dose escalation was at 50% increments until either a biological effect or a toxic effect occurred. Initially, the study design called for three patients to be treated at each dose level. If a biological effect but no toxic effect occurred at a given level, the number of patients at that dose level was increased from three to six. The trial design was based on preclinical evidence suggesting that antitumor activity would be encountered before evidence of any toxicity.
A biological effect was considered to have occurred when a given level of TNP-470 suppressed the PSA concentration to less than half the baseline serum concentration.
Patients were examined at weekly intervals for specific evidence of toxic reactions.
RESULTS
Study Population.
Thirty-three patients were placed on study: 29 were evaluable for analysis of PSA kinetics and toxicity; two patients did not have flutamide withdrawal before initiation of TNP-470 (one patient in whom toxicity only could be evaluated), and one patient was inevaluable. The patient evaluable only for toxicity had withdrawal of flutamide simultaneously with initiation of TNP-470; therefore, his PSA response could be assessed. One patient had a declining platelet count that dropped below 100,000/ml before the first infusion of TNP-470, although the patient was registered on the clinical trial. Therapy was stopped for individual patients when the dose-limiting toxicity was observed. Six patients each (rather than the prescribed three) received TNP-470 at dose levels 1, 2, and 3 to help clarify the influence of TNP-470 on PSA kinetics.
Treatment.
The treatment level and number of courses administered/patient are outlined in Table 1⇓ . A course was defined as 6 weeks of alternate-day TNP-470 infusion (4 weeks of alternate-day infusion followed by a 2-week treatment-free interval).
Number of courses of TNP-470 delivered, by dose level
Toxic Effects.
The toxic effects of TNP-470 were modest and reversible (Table 2)⇓ . Because most of the patients had high-volume prostate cancer, it was sometimes difficult to distinguish with certainty the origin of all of the symptoms, because they may have been caused in part by the advanced cancer. There appeared to be no evidence of excessive bleeding in this population, although one patient developed upper gastrointestinal tract bleeding that was attributed to gastritis and that resolved with conservative management. Excessive bleeding was not detected at venipuncture sites with minor injuries or at areas of cancer invasion into the bladder.
Toxic effects of TNP-470
The major dose-limiting side effect was a reversible neurological toxic reaction. Fatigue, possibly multifactorial in origin, was present. It was not clear to the treating physicians whether the fatigue resulted from the TNP-470 treatment. Although the fatigue appeared related to TNP-470 administration, it may have been attributable in part to the progressive prostate cancer. Some features of the fatigue were similar to those experienced by patients treated with IFN-α. In some instances, the fatigue was associated with other, more definite symptoms of neurological toxicity.
Neuropsychiatric Toxicity.
Asthenia was common and occurred in a dose-dependent but reversible fashion during treatment with TNP-470. Asthenia increased in severity and frequency parallel to dose escalation (Table 3)⇓ . Anxiety and dysphoria also occurred with increasing doses of TNP-470. The anxiety was characterized by a sense of “reduced mental acuity” that resolved upon cessation of therapy. After recognition of the phenomenon, three patients at the final dose level 6 had a baseline EEG when symptoms of central nervous system toxicity first appeared. Results revealed a diffuse mental slowing that was completely resolved on the follow-up EEG.
Neuropsychiatric toxicity
The case histories of three patients with clinical neuropsychiatric effects after the TNP-470 treatment are described below.
Patient 1 (Dose Level 6).
The patient was a 60-year-old, well-educated white man who had a history of alcohol abuse but had not used alcohol for more than 8 years. Three weeks after initiation of therapy (TNP-470 dose, 106 mg/m2), the patient reported decreased concentration ability, fear, anxiety, and tightness in his throat. The objective clinical findings were a score of 17 on the Beck Depression Inventory and impaired visual scanning speed, verbal memory, and concentration. Extrapyramidal signs detected were reduced blinking, a wide gaze, and a slow intention tremor. Within 6 weeks of stopping therapy, the patient showed a marked improvement, and within 8 weeks, no objective or subjective symptoms of neurotoxicity could be detected. An EEG performed upon recognition of the clinical findings revealed diffuse mental slowing that completely resolved with cessation of therapy. A computed tomographic scan of the brain revealed no abnormalities to account for the toxic effect.
Patient 2 (Dose Level 5).
Patient 2, a 67-year-old white farmer, reported feeling “like a zombie” and like being “in a shell” 2 weeks after therapy with TNP-470 (dose, 70.88 mg/m2). He felt detached as if he had the flu. Neurological examination revealed that he had a positive palmomental reflex and was bradykinetic, but he did not have a glabelar or suck reflex. In addition, his ankle reflexes were absent, but both toes were down going. Results of neuropsychological testing were consistent with subfrontal cortical decline. Repeat neuropsychiatric testing revealed a total recovery within 6 weeks of experiencing mild language difficulties. The initial EEG revealed diffuse slowing of speech; follow-up findings were improved. Further testing was not performed because the patient returned home to continue care of his advancing prostate cancer.
Patient 3 (Dose Level 5).
Patient 3, a 65-year-old white man, had been receiving methylphenidate therapy for 15 years. After his first 4 weeks of receiving TNP-470 (dose, 70.88 mg/m2), his wife reported that the patient was “not thinking clearly.” The patient reported fatigue and balance and coordination problems. Neuropsychological testing revealed mild frontal lobe dysfunction with mild extrapyramidal features. The patient’s Beck Depression Inventory score was 9. The findings were interpreted as being most consistent with subcortical and frontal dysfunction. Eight weeks after cessation of therapy, the neurological findings resolved.
Hematological and Hepatic Toxicity.
Serial blood counts and measurement of liver function (transaminase, bilirubin, and alkaline phosphatase concentrations) showed no evidence of hepatic or hematological toxic effects.
Gastrointestinal Toxicity.
Nausea and vomiting were not reported; however, one patient developed gastric bleeding and a second had constipation.
Bone Pain.
No convincing increase in bone pain beyond that attributed to the prostate cancer was detected. Assessment of bone pain was difficult in these patients because of the inability to distinguish between pain from the progressive cancer metastasis and that potentially related to TNP-470. Those patients with a rising PSA concentration and bone metastasis experienced pain that resolved on cessation of therapy and was coincident with the decline in PSA. In each instance of a reported increase in the characteristic osseous-type pain, this pain occurred at a site of known preexistent bone metastasis. We detected no arthralgias or bony pain that was not at sites of previous existent disease.
Infectious Complications.
Patients were specifically evaluated for infectious complications of therapy. No clinical infection was found despite the fact the patients were at increased risk because of the long central catheter line used for blood sampling and administration of therapy.
Glucose Concentration.
Serum glucose concentrations were measured before each course of TNP-470 therapy. During therapy, one patient not known to be diabetic was found to have a substantially elevated glucose concentration (215 mg/dl). The patient never required therapy, and his glucose concentration returned to normal after cessation of TNP-470 therapy.
PSA Kinetics during TNP-470 Treatment.
PSA kinetics was measured only in those patients who were withdrawn from flutamide before initiation of TNP-470. A clear, reproducible pattern of increased serum PSA concentrations was seen in some patients treated and rechallenged with TNP-470 (Fig. 1)⇓ . A TNP-470-induced increase in serum PSA concentration was defined as a rise in serum PSA that upon cessation of therapy resolved on sequential measurements of PSA concentration to below 50% of the peak level on two consecutive measurements. A decline below the baseline level was considered as possible evidence of antitumor activity.
Serial serum concentrated prostate-specific antigen in relationship to TNP-470 therapy.
Two patterns of PSA kinetics were seen. In some patients, PSA concentration increased when therapy began, although the concentration in the other patients was not influenced by TNP-470. This biological effect did not appear to be influenced by the TNP-470 dose level used (Table 4)⇓ .
Transient increase in serum PSA concentration
Serum Adhesion Molecule Concentration.
Thrombomodulin concentrations were assayed in the serum, and levels were tabulated in relation to the PSA concentrations. The rise in thrombomodulin concentration seemed to parallel the rise in serum PSA concentration.
There was no clear trend for E-selectin serum concentrations during sequential measurements while patients received TNP-470. These concentrations did not parallel the rise in serum PSA and appeared to randomly change (Table 5)⇓ .
Comparison of pre-TNP-470 and 5-week values for 26 cases
Urine βFGF Concentration.
βFGF concentration was measured sequentially in the urine of 12 patients. No pattern for βFGF concentration was seen, and no relationship to baseline serum concentration was found (data not shown).
DISCUSSION
In this Phase I clinical trial, we evaluated the toxicity of the specific angiogenesis inhibitor TNP-470 and studied candidate markers of angiogenesis that might prove useful in the clinical study of cancer-associated neovascularity. The antitumor activity of agents that inhibit angiogenesis may not be detected with the clinical methods used to assess the efficacy of cytotoxic agents. Therefore, we attempted to establish the MTD of TNP-470 and develop intermediate end points useful for the clinical study of such inhibitors in prostate cancer. The dose escalation schema we adopted permitted us to safely reach the predicted biologically active dose and treat sufficient numbers of patients at each level to assess them for unanticipated side effects by the first of a new class of agents. The MTD was identified as 70.88 mg/m2 BSA. Therefore, the starting dose of TNP-470 on the alternate-day schedule is 47.25 mg/m2 BSA.
The dose-limiting toxicity we encountered was neuropsychiatric in nature. After receiving 70.88 mg/m2 BSA of TNP-470, two patients experienced neurological symptoms (agitation, gait disturbance, and fatigue). Although the exact mechanism of the neuropsychiatric toxicity is unknown, it correlated closely with the TNP-470 infusion and resolved after cessation of the TNP-470. The symptom complex was not clinically attributed to other causes and suggested frontal subcortical toxicity. In the three patients on whom an EEG was performed, diffuse cortical abnormalities were detected. These abnormalities were considered not specific for any disease and resolved after the cessation of therapy and upon clinical improvement of the symptoms. Although we believe that the patients fully recovered from the neurological complication, follow-up of these patients has been short. Thus, the ultimate severity of the neurological toxicity of TNP-470 and this dose and schedule will be known only with further follow-up of additional patients. In our experience, the neurological findings and symptoms resolved within 2 months of cessation of therapy. It has also been reported that the neurological toxic effects of drugs increase in frequency and severity with advancing patient age. Thus, the prevalence of this side effect may be partially attributed to our patient population, whose median age was 65 years (range, 38–81 years). No other major toxic effects were attributed to the therapy. Excess bleeding at venipuncture sites was not reported, and those patients with active cancers invading the bladder did not have increased bleeding. The increased bone pain correlated with the rise in serum PSA concentration was most severe at the site of known cancer involvement and was not attributed to the TNP-470.
Traditional clinical study end points (e.g., regression of tumor) will not be valid for angiogenesis inhibitor studies. Thus, we elected to minimize the number of variables that might influence the results in this Phase I clinical trial by focusing on a specific disease. Androgen-independent prostate cancer was selected as a candidate cancer for initial study for a number of reasons: (a) clinical evidence that angiogenesis is predictive of prostate cancer progression; (b) preclinical data suggesting that angiogenesis inhibition suppresses prostate cancer growth in vivo; (c) the availability of serum markers of prostate cancer progression that may detect effects on the cancer that could not be detected by tumor regression; and (d) the fact that advanced prostate cancer is a major health problem with no effective therapy. Therefore, we designed a cancer-specific Phase I trial that reduced the heterogeneity in the clinical study. Minimizing the influence of clinical heterogeneity was thought to be essential for the establishment of valid and therapeutically relevant intermediate end points.
The endothelial markers and markers of angiogenesis that we elected to explore were detectable within serum and urine with reproducible assays and may reflect the activity of cancer-associated angiogenesis. These candidate markers did not show a pattern of urine or serum concentration that was associated with cancer progression or related to therapy. Soluble E-selectin and thrombomodulin are considered markers of endothelial damage or activation. These markers are shed in concentrations detectable in serum and are preferentially expressed in proliferating endothelial cells. Concentrations of E-selectin and thrombomodulin seemed to vary randomly from day 1 to completion of the first 28-day course of therapy. In addition, serum PSA concentration was unrelated to baseline endothelial marker concentration. Similarly, urinary βFGF concentration did not correlate with serum PSA concentration or with the introduction of therapy. Each of the candidate vascular markers seemed to fluctuate randomly throughout the treatment. Future studies will need to consider the concentration of these markers in untreated patients to obtain more clinical information concerning the baseline concentration of the markers and their fluctuation. Although further study is required, our data suggest that these markers may not be suitable as intermediate end points in the clinical study of angiogenesis inhibition in prostate cancer.
TNP-470 was biologically active in patients with advanced androgen-independent prostate cancer at the dose and schedule we used. Treatment resulted in a rise and subsequent decline in PSA coincident with the delivery of the TNP-470. This biological activity is worthy of further investigation. Although the PSA concentration rose coincident with the initiation of the treatment, its prompt decline after cessation of therapy indicates that the effect is temporary. It was noted recently that PSA production was influenced by exposure to TNP-470, further clouding the ability to interpret the significance of the PSA modulation coincident with TNP-470 therapy. There is no evidence of sustained cancer progression as a result of TNP-470. We have no explanation for the observed transient increase of bone pain coincident with the TNP-470 treatment.
We conclude from this Phase I trial that the dose-limiting toxicity of TNP-470 is neuropsychiatric and apparently reversible. An understanding of the mechanism of the neurotoxicity and its reversibility will be required before TNP-470 can be widely used in a clinical setting. The therapeutic implications of TNP-470 modulation of the PSA concentration remain to be determined and will also require further study.
Future studies of TNP-470 will need to focus on overcoming the neurological toxic effects reported here. In view of the biological effect of TNP-470 on serum PSA concentrations in some of the patients we treated, prostate cancer is a promising future target for angiogenesis inhibitors.
Footnotes
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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.
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↵1 Supported by TAP Holdings, Inc. and CaP CURE Foundation.
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↵2 To whom requests for reprints should be addressed, at the Department of Genitourinary Medical Oncology, The University of Texas M. D. Anderson Cancer Center, Box 13, 1515 Holcombe Boulevard, Houston, TX 77030. Phone: (713) 792-2830; Fax: (713) 745-1625.
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↵3 The abbreviations used are: βFGF, β-fibroblast growth factor; MTD, maximum tolerated dose; PSA, prostate-specific antigen; HRP, horseradish peroxidase; BSA, body surface area; EEG, electroencephalogram.
- Received September 17, 1999.
- Revision received January 26, 2001.
- Accepted February 6, 2001.