A Phase I Study of CNI-1493, an Inhibitor of Cytokine Release, in Combination with High-Dose Interleukin-2 in Patients with Renal Cancer and Melanoma¹ Free

IL-2³, produced naturally by activated T lymphocytes, is involved in immune regulation and has demonstrated antitumor properties in a variety of experimental systems (1). High-dose recombinant IL-2 produces significant regressions in 15–20% of eligible patients with metastatic renal cancer or melanoma (2, 3). However, morbidity and the difficulty and expense of toxicity management limit high-dose IL-2 to a select group of patients treated by experienced clinicians at established treatment programs (4). Very few patients can actually receive the planned total number of IL-2 doses. Lower doses, with less toxicity, alone or in combination with IFN-α, have been used and give similar overall response rates (5, 6, 7). However, the proportion of patients with complete responses does not seem to be as high nor are the responses as durable with lower-dose as with high-dose IL-2 (8).

IL-2 induces production of several proinflammatory cytokines such as IL-1, tumor necrosis factor (TNF-α), and IFN-γ. These and other substances (9, 10, 11, 12, 13), including nitric oxide (14, 15), are thought to be involved in the toxicity of IL-2.

CNI-1493 is a tetravalent guanylhydrazone, N,N′-bis[3,5-bis[1(aminoiminomethyl)hydrazonoethyl]phenyl]decanediamide tetrahydrochloride. It suppresses LPS-induced nitric oxide production as well as production of several proinflammatory cytokines, including TNF-α, IL-1, IL-6, and macrophage inflammatory proteins 1α and 1β, from human peripheral blood mononuclear cells (16). CNI-1493 has been shown to be protective in preclinical models of endotoxic shock, sepsis, pancreatitis (17), experimental allergic encephalomyelitis (18), stroke (19), and rheumatoid arthritis⁴.

A protective effect of CNI-1493 was demonstrated in rats given high doses of IL-2 (20). In Sprague Dawley rats, IL-2 given by continuous infusion at a dose ≥6 × 10⁶ IU/m² daily for up to 14 days was uniformly fatal. Rats receiving 1–10 mg/kg CNI-1493 i.p. once daily were protected against toxicity of IL-2 administered at doses up to 30 × 10⁶ IU/m² daily. At a dose of 6 × 10⁶ IU/m² daily by continuous hepatic artery infusion, IL-2 is not very effective against Novikoff hepatoma. By administering CNI-1493, the dose of IL-2 could be increased to 30 × 10⁶ IU/m² daily. At that dose, in a group of 10 animals with established tumors, 5 had a reduction of the tumor mass to <1 mm³ and 5 had complete regression of their tumors. Tumors continued to grow in all untreated control animals.

On the basis of these findings in a rodent model, we conducted a Phase I study of CNI-1493 given alone and in combination with high-dose IL-2 in patients with renal cancer and melanoma. The primary objective of the study was to determine the MTD and side effect profile of CNI-1493 alone and in combination with high-dose IL-2. Secondary objectives were to examine the effect of CNI-1493 on IL-2 toxicity, the biological parameters known to be affected by IL-2, and number of doses of IL-2 administered; determination of the pharmacokinetic profile of CNI-1493 alone and in combination with IL-2; and to assess tumor responses in patients treated with the combination of CNI-1493 and high-dose IL-2.

Patients were eligible to participate in the study if they were considered for high-dose IL-2 for treatment of renal cancer or melanoma. Before treatment, all patients underwent physical examinations, standard hematological and biochemical tests, pulmonary function evaluation, electrocardiogram, and tumor assessment. Those patients above age 40 and those with risk factors for coronary artery disease underwent cardiac stress testing. To qualify, a patient had to have an Eastern Cooperative Oncology Group performance status of 0 or 1 and be free of major organ dysfunction. All patients had to have a serum creatinine ≤1.5 mg/dl or creatinine clearance >60 ml/min. At least 4 weeks were to have elapsed from the end of previous therapy (6 weeks for nitrosoureas or mitomycin), and concurrent therapy was not permitted. No more than one prior biological response modifier treatment regimen for metastatic disease was permitted. If the patient had previously received IL-2, at least 6 months were to have elapsed since that therapy.

The study was approved by the institutional review board at each participating institution. All patients gave signed informed consent.

Patients received treatment according to the schema in Fig. 1. CNI-1493 was diluted in 100 ml 5% dextrose and administered i.v. once daily over 30 min. The drug was first administered alone daily for 5 days. After a 9-day rest period, CNI-1493 was administered again once daily in conjunction with high-dose IL-2 in two 5-day courses separated by a 9-day rest period, days 1–5 and 15–19. IL-2 was administered at the standard recommended dose and schedule, 600,000 IU/kg/dose every 8 h for up to 14 doses/course. Individual IL-2 doses could be held for toxicity, but there were no IL-2 dose modifications. CNI-1493 was discontinued after a decision that no further IL-2 would be administered during a course of therapy.

The dose of CNI-1493 was escalated by cohorts from 2–32 mg/m²; there were no intrapatient dose escalations. The dose was doubled for each successive cohort. Dose was escalated initially in cohorts of three patients until DLTs attributable to CNI-1493 occurred. Once at least one patient in a cohort experienced a DLT attributable to CNI-1493, up to six patients were entered at that dose level. When the MTD was reached, a decision was made to add three additional patients at the 4-mg/m² dose, so a total of seven patients were treated at that level.

During high-dose IL-2 treatment, patients received standard prophylaxis for IL-2-induced toxicity, including acetaminophen, indomethacin, and either cimetidine or ranitidine. Patients with indwelling catheters received oral ciprofloxacin or cephalexin. Patients received other supportive care for IL-2 toxicity as appropriate (21).

The primary objective of the study was determination of the MTD of CNI-1493 either alone or in conjunction with high-dose IL-2. The MTD was defined as the dose below that at which at least two patients experienced a DLT. DLT was defined as any adverse event falling into a category in Table 1 and which was: (a) felt to be possibly or probably related to CNI-1493; and (b) not ameliorated by symptomatic measures.

Adverse events were defined and graded by National Cancer Institute Common Toxicity Criteria, Version 1. Investigators assessed the causality of adverse events. In the case of adverse events occurring during combined therapy with CNI-1493 and high-dose IL-2, the investigator could attribute the event to either or both medications or to other conditions, such as tumor progression or intercurrent illness. Adverse events felt by an investigator to be possibly or probably related at least in part to CNI-1493 were considered as DLTs if they fit the criteria in Table 1.

Secondary end points were evidence of amelioration of IL-2 toxicity by CNI-1493 and an increase in the number of IL-2 doses administered relative to historical controls.

Patients underwent physical examination and hematological and biochemical testing before and periodically during treatment. No statistical comparisons of clinical laboratory parameters were planned for this study. However, on review of the adverse events observed during combined therapy with CNI-1493 and IL-2, it was noted that there was less liver toxicity than anticipated for high-dose IL-2 alone. Therefore, post hoc analyses were performed to examine changes in bilirubin and liver enzymes as a function of dose. Regression analyses (22) were performed using results of each dose-group independently to look at the significance of these changes from day 1, just before the start of IL-2, through day 6, the day after IL-2 treatment was scheduled to stop. Included in these analyses as a reference group were 10 patients fulfilling the same entry criteria as study patients, but who were receiving high-dose IL-2 alone at one of the investigative sites.

For most patients treated with ≥4 mg/m² CNI-1493, TNF levels were measured at baseline and periodically through 24 h after the initial dose of combined therapy. TNF levels through 8 h after IL-2 administration in a reference group (three patients) treated with high-dose IL-2 only at one of the participating institutions were used for comparison. Serum specimens for TNF levels and pharmacological determinations were frozen at −20°C until analysis. TNF levels were measured using a commercially available ELISA kit (Endogen, Woburn, MA).

Pharmacokinetic sampling for CNI-1493 and IL-2 levels was performed on selected patients. A high pressure liquid chromatography assay was developed for CNI-1493 in biological fluids and performed by Microconstants, Inc., San Diego, CA⁵. Plasma samples containing EDTA as the anticoagulant were extracted by protein precipitation with acetonitrile. The extract was analyzed by direct injection. The mobile phase was nebulized using heated nitrogen in a Z-spray source/interface, and the ionized compounds were detected using a triple quadrupole mass spectrometer. Assays for IL-2 were performed on selected patients using a commercial ELISA method (Quantikine Human IL-2 immunoassay kit, R&D Systems, Minneapolis, MN) by Performance Bioanalytical, Inc., San Diego, CA. Descriptive pharmacokinetic parameters were determined by standard model independent methods (23) based on each of the individual sets of plasma concentration-time data.

Repeat tumor assessment was conducted at least once after treatment was completed. Patients with stable disease or better were eligible for retreatment with combined CNI-1493 and IL-2 at 12–14 week intervals.

A total of 24 patients were treated in this study; 22 received at least one course of combined therapy with CNI-1493 and IL-2.

Patients were typical of those receiving high dose IL-2 at the participating institutions. Patient characteristics are shown in Table 2 by low (2 or 4 mg/m²; n = 10), intermediate (8 or 16 mg/m²; n = 7), and high (25 or 32 mg/m²; n = 7) dose groups, as well as all patients together. Patient characteristics were similar among dose groups.

Median age of all patients was 54 years (range, 36–73 years). Thirty-eight percent of patients had melanoma; 62% had renal carcinoma. Twelve patients (50%) had received prior therapy with biological-response modifiers alone or in addition to chemotherapy; 6 (25% of all patients) had received prior IL-2. All but one patient had undergone surgery, radiation therapy, or some form of systemic therapy.

Table 3 shows CNI-1493 and IL-2 dosing by cohort. All but one patient received all five doses of CNI-1493 administered alone. That one patient, enrolled in the 4-mg/m² cohort, opted to discontinue study participation after receiving four doses of CNI-1493.

The number of patients/cohort varied. Once the MTD was reached, it was decided to add three more patients at the 4-mg/m² dose. One patient treated at 16 mg/m² developed problems unrelated to study medication that necessitated temporary cessation of study therapy, and a fourth patient was added at that dose. Subsequently, the patient was able to resume therapy, so four patients are evaluable at that dose level. After DLT occurred in two patients at 32 mg/m², one patient was treated at an intermediate dose of 25 mg/m². Only one patient was treated at this dose, however, as the patient developed moderate nephrotoxicity similar to that which was dose-limiting.

Most patients received the full five doses of CNI-1493 when administered with each course of IL-2, as shown in Table 3. One patient, who received 32 mg/m², received five doses of CNI-1493 alone but was taken off study before receiving IL-2, as two patients had already experienced DLT with the combination of IL-2 and CNI-1493 at that dose level.

Forty courses of IL-2 were administered. One patient, who received 16 mg/m² of CNI-1493, received the full 28 doses of IL-2. One patient each treated at 2, 8, and 16 mg/m² received 14 doses of IL-2 during one of the two courses of that drug. In all cases, the reasons for discontinuing or withholding IL-2 were established toxicities of that drug. In 22 of 35 courses (63%) in which IL-2 was discontinued at <14 doses, the reason for discontinuation was refractory hypotension, either alone or along with other toxicities.

As can be seen from Table 3, CNI-1493 did not seem to increase the number of doses of IL-2 that could be administered. At doses between 2 and 16 mg/m², there is no suggestion that the number of IL-2 doses administered increased with escalation of CNI-1493, although the number of patients in each dose group is too small to demonstrate anything but a very marked effect. At 25 and 32 mg/m² CNI-1493, toxicity of the combination supervened, and the number of IL-2 doses administered per course decreased.

There was no trend toward a decreasing incidence or severity of cardiovascular toxicity (mainly hypotension) as the dose of CNI-1493 was increased, nor was the frequency of discontinuation of therapy attributable to cardiovascular toxicity decreased as the dose of CNI-1493 was increased.

CNI-1493 was well tolerated when given as a single agent daily for five days. Injection site reactions in two patients were the only clinical adverse events attributed to study medication. One patient treated at 4 mg/m² developed moderate and one treated at 32 mg/m² developed mild treatment-related phlebitis at the injection site, the former on the fifth day of CNI-1493 treatment and the latter on the second day of treatment. In both patients, the phlebitis resolved without sequelae.

Five patients had by National Cancer Institute Common Toxicity Criteria grade 1 increases in serum creatinine while on CNI-1493 alone: one of seven treated at 4 mg/m², one of one treated at 25 mg/m², and three of six treated at 32 mg/m². The increases in three of these patients were only 0.1–0.3 mg/dl. The patient treated with 25 mg/m² had an increase from 0.9 mg/dl at baseline to 1.6 mg/dl just before starting combination therapy; one patient treated with 32 mg/m² had an increase from 1.2 mg/dl at baseline to 1.7 mg/dl just before starting IL-2. While on CNI-1493 alone, patients experienced a variety of other adverse events that were felt by the investigators to be related to the patients’ underlying malignancies or to intercurrent illnesses.

During treatment with the combination of CNI-1493 and IL-2, patients experienced the spectrum of side effects typically attributed to high-dose IL-2 (24). Except as noted below with regard to MTD and pharmacological effects, there was no correlation between incidence or severity of toxicities and dose of CNI-1493.When 32 mg/m² CNI-1493 was given along with IL-2, two patients developed markedly increased creatinine levels, to 8.0 and 5.7 mg/dl after only eight and five doses of IL-2, respectively. Both of these patients had metastatic melanoma, and neither had evidence of preexisting renal disease nor had they received cisplatin. One patient required transient hemodialysis. In both instances, the creatinine returned to normal (≤1.3 mg/dl) within 6 weeks of discontinuation of CNI-1493 and IL-2. One patient was treated at an intermediate dose level, 25 mg/m², but that patient’s creatinine increased to 3.0 mg/dl after five doses of IL-2 during the first treatment course, and therapy was discontinued. No additional patients were treated at 25 mg/m². The MTD of CNI-1493 given in combination with high dose IL-2 was, therefore, determined to be 16 mg/m². The MTD for CNI-1493 alone is >32 mg/m².

The increase in serum TNF levels associated with IL-2 administration was attenuated in patients receiving CNI-1493, as shown in Fig. 2. For clarity purposes, data are displayed by low (4 mg/m²), intermediate (8 or 16 mg/m²), and high (32 mg/m²; TNF data were not obtained on the patient treated with 25 mg/m²) doses of CNI-1493. As can be seen in the figure, by 4 h after IL-2 dosing, the TNF increase was blocked in a dose-dependent manner. At 8 h after dosing, mean ± SD values were 5.75 ± 5.44 pg/ml for the 4-mg/m² group, 0.83 ± 1.33 pg/ml for the 8- or 16-mg/m² group, and 0.20 ± 0.40 for the 32-mg/m² group; the mean value was 26.67 ± 4.16 pg/ml in the reference group. Median values were similar. TNF levels remained low throughout IL-2 dosing in patients receiving CNI-1493.

IL-2 induces hepatocellular dysfunction, as manifest by increases in serum total bilirubin and transaminases. Maximum changes are generally noted at the first measurement after completion of treatment, i.e., on or about day 6. As noted in “Parameters Evaluated,” above, fewer patients in the higher- than in the lower-dose CNI-1493 or reference groups developed significant increases in bilirubin or ALT. Eleven of 19 (58%) of patients in the reference, 2-, and 4-mg/m² dose groups developed grade 4 increases in bilirubin; only 2 of 14 (14%) of patients in higher dose groups developed grade 4 bilirubin increases. No patient on the study developed grade 4 increases in ALT; however 9 of 19 (47%) of patients in the reference, 2-, and 4-mg/m² dose groups developed grade 2 or 3 increases in ALT versus none of 14 in the higher-dose groups. We therefore analyzed changes in bilirubin and ALT from the start of CNI-1493 alone to the end of the first course of combined CNI-1493 and IL-2; results are shown as scatter plots by dose group for bilirubin (Fig. 3) and ALT (Fig. 4).

In patients receiving CNI-1493, there was a dose-dependent attenuation of the increase in total bilirubin. The median bilirubin increase at day 6 in the reference group was 5.1 mg/dl. The increases in patients receiving the low (2 or 4 mg/m²) intermediate (8 or 16 mg/m²) or high (25 or 32 mg/m²) doses of CNI-1493 were 82%, 31%, and 14%, respectively, of that in the reference group.

As shown in Fig. 4, peak ALT levels were lower in patients receiving CNI-1493 than in the reference group; there seemed to be a plateau at 8 or 16 mg/m². At day 6, the median increase in ALT in the reference group was 67 units/liter. Median changes in the low-, medium-, and high-dose groups were 93%, 46%, and 74% of that for the control group. Changes in AST paralleled those in ALT (data not shown).

By regression analysis, the P for the relationship between CNI-1493 dose and the change in total bilirubin was 0.013, that for the relationship between CNI-1493 dose and ALT change was 0.068, and for AST change, 0.063. Ps for these comparisons, excluding the reference group, were similar, inasmuch as 2 mg/m² CNI-1493 was essentially a no-effect dose. Taken together, these biochemical data suggest that CNI-1493 may ameliorate hepatocellular toxicity induced by high-dose IL-2.

Serial tumor response assessments were obtained on all but four patients in the study. One patient with renal cancer (4 mg/m²) had a confirmed complete response that was ongoing at the 12 month follow-up. None of the 11 other patients with renal cell carcinoma or the 8 patients with melanoma had a major response to therapy. Four renal cancer and three melanoma patients had stable disease for at least 12 weeks.

Pharmacokinetic data on CNI-1493 were obtained from nine patients (three at 4-, two at 16-, one at 25-, and three at 32-mg/m² doses of CNI-1493) and on IL-2 from six patients (one each at 2 and 4-, and two each at 8- and 16-mg/m² doses of CNI-1493).

The half-life for IL-2 was 1.17 ± 0.33 h after the first dose of IL-2 and 1.20 ± 0.45 h after dosing on day 4. There was no change in t_1/2 as a function of CNI-1493 dose.

The half-life of CNI-1493 on days 1 and 5 of treatment without IL-2 was 1.42 ± 0.67 and 1.02 ± 0.35 h, respectively. On the first and third or fourth days of combined therapy with IL-2, t_1/2 of CNI-1493 was 1.43 ± 0.40 and 0.83 ± 0.17 h, respectively. There was no indication of variation in t_1/2 with increasing dose of CNI-1493.

C_max and area under the curve increased with increasing dose of CNI-1493. On day 1 of treatment, at 32 mg/m², C_max was 6.2 ± 0.8 μg/ml and area under the curve was 432 ± 14 μg min/ml; on day 5, they were 3.8 ± 2.5 μg/ml and 246 ± 49 μg-min/ml, respectively. For all dose groups, there was no significant difference between day 1 and day 5 CNI-1493 pharmacokinetics, nor was there any difference in pharmacokinetics when CNI-1493 was given with IL-2 as compared with administration alone.

CNI-1493 is a synthetic guanylhydrazone compound that was found in preclinical studies to inhibit the release of a variety of inflammatory cytokines (16). It was tested in this Phase I study alone and in combination with high-dose bolus IL-2. In addition to determining the side effect profile and MTD of the compound, the pharmacological activity of CNI-1493 was studied to determine whether there was evidence of suppression of laboratory effects typically associated with high-dose IL-2.

As a single agent given daily for 5 days, CNI-1493 was well tolerated; the MTD was not reached at the highest dose administered in this study, 32 mg/m². Higher doses were not tested as a single agent because it was considered inappropriate to treat the study population, patients with unresectable melanoma or renal cancer, with CNI-1493 alone if the drug were not to be used subsequently in combination with high-dose IL-2. The only toxicities attributed to CNI-1493 alone were occasional injection site phlebitis and grade 1 elevations of serum creatinine. In animal studies, injection-site phlebitis was very common and occasionally marked⁶. In this study, CNI-1493 was given in dilute solution over 0.5 h to avoid contact with a high concentration of drug. This appeared to minimize the problem.

Serum creatinine increased ≥0.5 mg/dl from baseline to just before starting combination therapy with IL-2 in two patients; one of the patients was treated at 25 mg/m² and one was treated at 32 mg/m². During combination therapy with 25 or 32 mg/m² of CNI-1493, nephrotoxicity greater than that expected from IL-2 alone was noted three of six patients. Clinically, reversible nephrotoxicity was the dose-limiting side effect of the combination.

In rat and dog toxicology studies of CNI-1493 alone, there was no biochemical evidence of nephrotoxicity. However, histological abnormalities were noted in rats treated at the highest dose used, 20 mg/kg (equivalent to 120 mg/m²) daily for 14 days. Two types of lesions were noted. The more common was multifocal degeneration and/or regeneration of the tubular epithelium; this was more pronounced in males. The changes were usually seen near the corticomedullary junction and were characterized by small groups of basophilic tubules lined with attenuated epithelium and sometimes with necrotic tubular cells. Minimal interstitial response, consisting of fibrosis and occasional mononuclear inflammatory cells, was also noted around some tubules.

High-dose IL-2 alone causes considerable nephrotoxicity. Increased creatinine is a common adverse event associated with IL-2 administration; creatinine >2.5 mg/dl is reported in 33% of patients receiving high-dose IL-2 (25). Renal dysfunction occurs during high-dose IL-2 administration in association with capillary leak syndrome and prerenal azotemia; it resolves rapidly upon discontinuation of IL-2. Increases in serum creatinine after discontinuation of IL-2 are generally not seen.

A class of anti-inflammatory drugs very different from CNI-1493, NSAIDs are known to cause nephrotoxicity; all patients in this study received NSAIDs as prophylaxis against IL-2 toxicity. The mechanism of NSAID-induced nephrotoxicity is presumed to be the blocking of renal prostaglandin production (26, 27, 28). Patients with underlying cardiovascular abnormalities appear to be more sensitive to the nephrotoxic effect of NSAIDs than individuals with normal cardiovascular systems. The nature of the mechanism of nephrotoxicity in patients receiving high-dose IL-2 and CNI-1493 remains to be determined. It may be that circulatory and renal compromise, which occurs during high-dose IL-2 therapy, and/or interaction with NSAIDs may predispose to nephrotoxicity in patients concomitantly receiving CNI-1493.

IL-2 induces the release of several proinflammatory cytokines, which have their own toxicities (8, 9, 11, 12, 13). Patients receiving the higher doses of CNI-1493 had lower mean and median TNF levels than those receiving the lowest doses. When compared with reference patients treated with high-dose IL-2 alone, TNF levels were suppressed by >90% by either 8 or 16 mg/m² of CNI-1493. Furthermore, although the t_1/2 of CNI-1493 is only slightly more than 1 h, TNF levels remained suppressed after IL-2 doses administered 8 and 16 h later, suggesting that CNI-1493 exerts a pharmacological effect even when it is no longer detectable in the plasma.

CNI-1493 also appeared to exert a dose-dependent protective effect against IL-2-induced hepatocellular toxicity. There was a decrease in peak total bilirubin levels during the first course of combined CNI-1493 and IL-2. Patients who received 8 or 16 mg/m² CNI-1493 had lower peak ALT levels than patients receiving 2 or 4 mg/m²; AST changes followed a similar pattern. No such pattern was seen with alkaline phosphatase changes. These observations suggest a protective effect on liver parenchymal cells, an effect also seen in the rat (20).

The total number of doses of IL-2 administered was not increased by coadministration of CNI-1493. The compound did not appear to inhibit the cardiovascular effects of IL-2, especially the development of hypotension. This is the same situation observed in studies of soluble TNF receptor (29), a compound that has now been demonstrated to have substantial therapeutic efficacy in patients with illnesses related to TNF excess (30, 31). Thus, it appears that inhibition of TNF activity, although effective in certain clinical situations, does not protect against dose-limiting IL-2 toxicity.

In summary, we have demonstrated that CNI-1493, a synthetic guanylhydrazone compound, can inhibit TNF release at well-tolerated doses. In addition, although the compound did not appear to improve overall tolerance to high-dose IL-2, it did seem to ameliorate IL-2-induced hepatotoxicity. Additional studies will be undertaken to elucidate the role of CNI-1493 in illnesses associated with proinflammatory cytokine excess.