Phase I Trial of the Angiogenesis Inhibitor TNP-470 for Progressive Androgen-independent Prostate Cancer

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.

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).

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.

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.

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.

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Fig. 1.

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)⇓ .

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)⇓ .

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).