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A Dose-Escalation and Pharmacokinetic Study of Subcutaneously Administered Recombinant Human Interleukin 12 and Its Biological Effects in Japanese Patients with Advanced Malignancies

Department of Medicine III, Hamamatsu University School of Medicine, Hamamatsu 431-3192 [R.O., K.O.]; Department of Surgical Oncology, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima 734-0037 [Y. Y.,T. To.]; Department of Urology, Osaka Medical Center for Cancer and Cardiovascular Diseases, Osaka 537-8511 [T. Ki.,T. Ko.]; First Department of Surgery, Nihon University School of Medicine, Tokyo 157-0065 [M. S.]; Department of Dermatology, Saitama Medical School, Saitama 350-0495 [Y. K., S. I.]; Department of Urology, Cancer Institute Hospital, Tokyo 170-8455 [I. F.]; First Department of Surgery, Okayama University School of Medicine, Okayama 700-0914 [A. G.]; Second Department of Surgery, Gifu University School of Medicine, Gifu 500-8705 [Y. S., S. S.]; Department of Surgery, Yamaguchi University School of Medicine, Yamaguchi 755-8505 [S. H., M. O.]; Department of Epidemiology and Biostatistics, School of Health Sciences and Nursing, Faculty of Medicine, University of Tokyo, Tokyo 113-0033 [Y. O.]; Japanese Foundation for Cancer Research, Cancer Institute, Tokyo 170-8455 [S. T.]; and Japan Society for Cancer Chemotherapy, Osaka 550-0002 [T. Ta.], Japan

	ABSTRACT

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A pilot dose-escalation study of recombinant human interleukin 12 (rhIL-12) was conducted in Japanese patients with advanced malignancies. Cohorts of three patients received escalating doses of rhIL-12 that increased from 50 to 300 ng/kg/day s.c. three times a week for 2 weeks followed by 1-week rest. The same dosage and schedule was repeated for two additional courses. Sixteen previously treated patients were registered, and 15 were evaluated. Common toxicities were fever and leukopenia; the abnormality of laboratory tests included elevations in aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, C-reactive protein, and ß2-microglobrin. Dose-limiting toxicity was the grade 3 elevation of aminotransferases, and was observed in two of six patients at the 300-ng/kg dose level after the first course in one patient and after the third course in the other. Leukopenia was observed at all of the dose levels; two of six patients at 300 ng/kg experienced grade 3 leukopenia. Thus, 300 ng/kg was determined to be the maximum acceptable dose. Peak plasma levels of rhIL-12 decreased in the second courses, but the areas under the curve were almost the same in the first and second courses. Biological effects included increases of plasma levels of IFN-, tumor necrosis factor-, IL-6, IL-10, and neopterin. In two patients with renal cell carcinoma, complete response and partial response of metastatic tumors were observed with 50 and 300 ng/kg; the responses lasted for 5 and 3.5 months, respectively. Although immunological response to rhIL-12 varies depending on administration route and schedule and on patients’ physiological conditions, the recommended dose for Phase II studies is 300 ng/kg s.c. three times a week for 2 weeks followed by 1-week rest.

	INTRODUCTION

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IL² -12 is a heterodimeric cytokine with a molecular weight of 70,000, consisting of two nonidentical subunits of M_r 40,000 and M_r 35,000 (1 , 2) . IL-12 plays a pivotal role in controlling cell-mediated immunity through a number of important biological activities such as induction of IFN-; is produced by monocytes, B cells, and other antigen-presenting cells; enhances the growth and cytotoxicity of T and natural killer cells; and is known to accelerate the differentiation of naive T cells (Th0) to Th1 cells and to suppress the differentiation of Th0 cells to Th2 cells (3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16) .

IL-12 has in vivo antitumor activity against a number of murine tumors. Of note, irrespective of whether tumors were at early or late stages of growth, i.p. injections of recombinant IL-12 induced complete tumor regression of s.c. tumors. Furthermore, the IL-12-treated mice exhibited resistance to the rechallenge of the same tumor but not to a second syngeneic tumor. After the IL-12 treatment, CD4+ and CD8+ T cells infiltrated into the tumors, and the prior administration of anti-IFN- monoclonal antibody completely abrogated the antitumor effect. Additionally, IL-12 inhibited the metastases of highly aggressive murine tumors in lungs and lymph nodes (17, 18, 19, 20, 21, 22, 23) . IL-12 also inhibited angiogenesis and basic fibroblast growth factor-induced corneal neovascularization (24) . IL-12 augments cytolytic activity of peripheral blood mononuclear cells from patients with solid tumors and from patients undergoing allogeneic bone marrow transplantation (25, 26, 27, 28) .

These findings suggest that IL-12 may have potent antitumor activities in humans, and Phase I clinical studies of two rhIL-12 products have been conducted by i.v. or s.c. administrations in patients with advanced tumors (29, 30, 31, 32, 33, 34, 35) . The incidence and degree of adverse reactions depended highly on the dosing protocol of the drug, and some objective tumor responses were observed in patients with renal cell carcinoma or melanoma (31, 32, 33 , 35) and with cutaneous T-cell lymphoma (34) .

In an animal tumor study in Japan, a stronger antitumor effect of IL-12 was observed by three-times-a-week administration than by a once-a-week schedule (21) . Therefore, we conducted a dose-escalation study of rhIL-12 injected s.c. three times a week for 2 weeks followed by 1-week rest, repeating this course for at least two more courses.

	PATIENTS AND METHODS

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Patients.
Eligibility criteria included the following: adult patients with histologically confirmed malignancies that were refractory to conventional therapy; Eastern Cooperative Group performance status 0, 1, or 2 (36) ; adequate bone marrow function defined as leukocyte count 3,000/µl, hemoglobin 9.5 g/dl, and platelet count 100,000/µl; adequate hepatic function defined as bilirubin 1.5 mg/dl, and aspartate aminotransferase and alanine aminotransferase 2 times the normal upper limit; and adequate renal function defined as creatinine equal to 2 mg/dl. Exclusion criteria included the following: history of allergic reactions to biological products; positive skin reaction to a test dose of rIL-12; uncontrolled infection; uncontrolled diabetes mellitus; overt autoimmune disease; seropositivity to hepatitis C virus or hepatitis B surface antigen; concomitant use of corticosteroids; and history of interstitial pneumonia, pulmonary fibrosis, peripheral neuropathy or other central nervous system disease and/or inflammatory disease of the colon. Pregnant women, nursing mothers, and patients with lymphoid malignancy were also ineligible. Patients who had received antitumor drugs within the preceding 4 weeks or who had ever received any other investigational drugs were also ineligible.

Study Design.
The study was an open-label, nonrandomized, multicenter Phase I dose-escalation study. The protocol was approved by each institutional review board, and all of the patients gave written informed consent. The study was conducted in accordance with the new Good Clinical Practice guideline from the International Conference for Harmonization. rhIL-12 (Yamanouchi Pharmaceutical Co. LTD., Tokyo, and Genetics Institute, Inc, Cambridge, MA) was s.c. injected three times a week for 2 weeks followed by 1-week rest. At least three courses of treatment were repeatedly given every 3 weeks, and tumor response was evaluated at the end of each course.

At least three patients were enrolled at each dose level of 50, 100, 200, and 300 ng/kg. Intrapatient dose escalation was not allowed during the first two courses but was permitted after the third course.

Toxicity was assessed using the National Cancer Institute common toxicity criteria with slight modification. A grade 3 or greater toxicity was regarded as DLT. Grade 4 neutropenia and grade 4 lymphopenia were not regarded as DLTs when they decreased to grade 3 or less within a week after the withdrawal of rhIL-12. If any patient developed a DLT at a particular dose level, then an additional three patients were enrolled at that dose level. Dose escalation was halted when 2 patients at a particular dose level experienced any DLT, and the dose level was designated as the maximum tolerated dose.

Pharmacokinetics.
Serial blood samples were collected before and 3, 6, 8, 10, 24, and 48 h after the first injection of the first course and after the sixth injection of the second course. rhIL-12 in the serum and urine was analyzed by ELISA with lowest detection limits of 2 pg/ml and 20 pg/ml, respectively.

Immunobiology.
Plasma for IFN-, TNF-, IL-6, and IL-10 and sera for neopterin concentrations were collected before and 24 h after the first and the sixth injections of rhIL-12 of each course. Four cytokines were measured by ELISA using murine monoclonal antibodies of commercially available ELISA kits: BMS228 for human IFN- (Bender MedSystems, Vienna, Austria), Quantikine HSTA50 for human TNF- (R&D Systems, Minneapolis, MN), Quantikine HS600 for human IL-6 (R&DS;), and Human IL-10 US KHC0103 for human IL-10 (BioSource International, Inc., Camarillo, CA). Neopterin was assayed according to the method reported by Hausen et al. (37) . Sera for anti-rhlL-12 antibody and anti-Chinese hamster ovary protein antibody were also obtained before the start of therapy and 3 weeks after the end of the final course. These antibodies were measured by ELISA.

	RESULTS

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Patient Characteristics.
Sixteen patients were registered, but 1 patient did not receive the study drug because of rapid deterioration of the clinical condition, and, thus, 15 patients were evaluated (Table 1) . All of the patients had previously been treated with chemotherapy or cytokines. The ECOG performance status was 0, 1, and 2 in 12, 2, and 1 patient, respectively, which indicated that clinical conditions of studied patients were relatively good.

Toxicity.
A common adverse event was fever of up to grade 2, which was noted in 12 patients including all of the 9 patients receiving 200 ng/kg or more (Table 2) . Fever resolved without treatment or with treatment by nonsteroidal anti-inflammatory drugs and did not prevent the continuation of rhIL-12. Less common adverse events included pain, redness and itching at the injection sites, chest discomfort, anxiety/nervousness, headache, stomatitis, and tachycardia, each of which was noted only in one patient. No dry skin, hypotension, or eosinophilia was reported.

Estimation of MAD.
Grade 3 elevation of aminotransferases was observed in two of six patients in the 300 ng/kg group. In one of the two patients (Fig. 1 , ), the aminotransferase levels started to rise on day 5 in the first course and reached grade 3 on day 8, and, despite the continuation of the drug, the levels came down until day 12 (the final administration day in the first course) and returned to the normal levels on day 19; in the second course, however, only grade 1 elevation of aminotransferases was observed. In the other of the two patients (Fig. 1 , •), grade 3 elevation of aminotransferases was noted in the third course. The levels were normal before the third course but were elevated to grade 3 on day 19 of this course and returned to the normal level on day 26. The serum concentrations of IL-12 in this patient were not different between the first and second courses, although we did not measure the concentration after the third course. Of six patients in the 300 ng/kg group, two each received two, three, and five courses, respectively.

View larger version (16K): [in this window] [in a new window]   Fig. 1. Serum alanine aminotransferase levels during the s.c. injections of 300 ng/kg of rhIL-12. Arrows, the days on which rhIL-12 was injected. Symbols, individual six patients who received 300 ng/kg rhIL-12.

Pharmacokinetics.
A pharmacokinetic study was performed in nine patients who received 200 or 300 ng/kg (Table 3) because the detection limit of rhIL-12 was apparently not high enough to study the smaller-dose schedules. After the first s.c. injection, the serum rhIL-12 levels reached the maximum values (C_max) at 7.8–24 h later and then slowly decreased until 48 h, at which time rhIL-12 was still detectable (Fig. 2) . Although there was a considerable variation among patients, no clear dose dependency was found either in C_max or in the AUC from 0 to 48 h (AUC_{0–48 h}) between the 200- and 300-ng/kg groups. There was almost no difference in C_max after the first injection of 300 ng/kg in the first course and after the sixth injection in the second course (mean, 61.4 and 56.5 pg/ml, respectively). Mean serum half-life of rhIL-12 was 24.8 h after the first injection of 300 ng/kg in the first course and was prolonged to 62.4 h after the sixth injection in the second course. However, there was almost no difference between the AUC_{0–48 h} after the first injection of 300 ng/kg in the first course and the AUC_{0–48 h} after the sixth injection in the second course (mean, 1669.5 and 1993.6 pg·hr/ml, respectively).

View larger version (15K): [in this window] [in a new window]   Fig. 2. Serum rhIL-12 concentrations after injections of 300 ng/kg rhIL-12. , mean concentrations after the first injection in the first course; , mean concentrations after the sixth injection in the second course; bars, SD.

Biological Parameters.
Plasma or serum concentrations of IFN-, TNF-, IL-6, IL-10, and neopterin after the s.c. injections of 50, 100, 200, and 300 ng/kg rhIL-12 are shown in Table 4 . There were great variations among patients irrespective of the dose. As shown in Fig. 3 , plasma IFN- concentrations were highest after the first dose and tended to decrease after the subsequent doses. The peak levels after the first injection at each dose level were not clearly in dose-dependent, but at 300 ng/kg, the peak levels after the subsequent doses tended to be higher than those of lower doses. IFN- levels of the two patients who showed antitumor responses to rhIL-12 were not higher than those of nonresponders at the same dose level. Plasma TNF- was detectable at all of the dose levels (Table 4 and Fig. 4 ). The concentrations tended to be higher in the 300 ng/kg group but again were not clearly dose-dependent. More than 10 pg/ml TNF- were seen in only three patients, two of whom showed antitumor responses to rhIL-12. Plasma IL-6 and IL-10 and serum neopterin concentrations showed the similar patterns. There was no clear dose dependency, but the concentrations tended to be higher in the 300-ng/kg group. The pretreatment level of IL-10 was very high in one of three patients in the 50 ng/kg group (191.0, 1.8, and 3.5 pg/ml, respectively). This patient had extensive metastatic colon cancer including in the liver and pelvis, showed progressive disease despite two cycles of IL-12 treatment, and died 3 months later. IL-6, IL-10, and neopterin levels of the two patients who showed antitumor responses were not higher than those of nonresponders at the same dose level. No anti-rhlL-12 nor anti-Chinese hamster ovary protein antibodies were detected in sera obtained 3 weeks after the end of the final course in any of the patients.

View this table: [in this window] [in a new window]   Table 4 Plasma concentrationa of INF- (pg/ml), TNF- (pg/ml), IL-6 (pg/ml), IL-10 (pg/ml), and neopterin (pmol/ml) before, and 24 h after, the first and sixth s.c. injections of rhIL-12 at each course

View larger version (16K): [in this window] [in a new window]   Fig. 3. Mean plasma IFN- concentrations before, and 24 h after, injections of 50 ng/kg (), 100 ng/kg (), 200 ng/kg (), and 300 ng/kg (•) rhIL-12 at the first and sixth dosing in each treatment course. Arrows, the days on which rhIL-12 was injected.

View larger version (17K): [in this window] [in a new window]   Fig. 4. Mean plasma TNF- concentrations before, and 24 h, after injections of 50 ng/kg (), 100 ng/kg (), 200 ng/kg (), and 300 ng/kg (•) rhIL-12 at the first and sixth dosing in each treatment course. Arrows, the days on which rhIL-12 was injected.

	DISCUSSION

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This is the first report on IL-12 treatment in Japanese patients as well as on the simultaneous measurements of serum levels of IL-12, IFN-, TNF, IL-6, IL-10, and neopterin after s.c. injections of IL-12. For this rhIL-12 product, the first Phase I dose-escalation study was conducted in the United States by a single-test dose followed 14 days later by cycles of five consecutive daily i.v. administration every 3 weeks, establishing a maximum tolerated dose of 500 ng/kg (30) . The subsequent Phase II study with 500 ng/kg by five consecutive daily i.v. administration, however, resulted in unexpected serious adverse events (29 , 31) . Some patients could not tolerate more than two successive doses, and 2 of 17 patients died. Thorough investigations including animal studies revealed that a single injection of rhIL-12 2 weeks before a consecutive dosing has a profound abrogating effect on the IL-12-induced IFN- production and toxicity (31) . Recently, Rakhit et al. (38) reported that the down-regulation of serum IFN- levels correlated with decreased mRNA expression in mice. This unique biological effect necessitated the careful evaluation of the administration schedule for the safe clinical development of this highly promising cytokine.

In Japan, we had already started this dose-escalation study by s.c. administration before the above unexpected toxicities were observed by i.v. administration in the Phase II study in the United States. We believed that it would be better to administer most cytokines by s.c. injection, which would give lower but more sustained plasma concentrations of cytokines than would i.v. administration. We also thought that, unlike cytotoxic antitumor drugs, for cytokines to exert their biological effects, maximum tolerable doses would not be needed, but sustained biologically active plasma concentrations would be required.

Subcutaneously-injected rhIL-12 was well tolerated up to 300 ng/kg given three times a week for 2 weeks followed by 1-week rest as one course. Grade 3 elevation of aminotransferases was observed in two of six patients receiving 300 ng/kg. The grade 3 elevation was noted after the first course in one patient and after the third course in the other, and thus only one of six experienced the grade 3 elevation in the first course. The elevation of these hepatic enzymes was transient, showing the highest peak 24 h after the initial dosing and decreasing before the start of the next dosing. The peak levels generally became lower despite the continuation of rhIL-12. Even at the 50-ng/kg dose level, one patient with metastatic renal carcinoma showed a complete tumor response. Another patient with metastatic renal carcinoma showed a good partial response at the 300-ng/kg dose level. Therefore, we estimated 300 ng/kg s.c. given three times a week for 2 weeks to be the MAD, and no further dose escalation was attempted. Rook et al. (34) also reported tumor regression in five of nine patients with cutaneous T-cell lymphoma by s.c. injection of either 50, 100, or 300 ng/kg twice weekly in their Phase I dose escalation study with the same IL-12 product. Thus, 300 ng/kg of rhIL-12 given s.c. three times a week seems to be a clinically relevant dose for Phase II studies of this rhIL-12 product. Using other rhIL-12 products, Bajetta et al. (32) , Motzer et al. (33) , and Portielje et al. (35) also observed antitumor effects in patients with metastatic melanoma or advanced renal cell carcinoma by using up to 1.5 µg/kg given s.c. weekly for three weeks as one course.

Common adverse effects other than elevated hepatic enzymes included fever, leukopenia, and elevations of C-reactive protein and ß2-microglobulin. These effects were also transient and moderate in severity. Only one patient experienced mild stomatitis. Atkins et al. regarded stomatitis as well as elevation of hepatic enzymes as DLTs at the 500-ng/kg dose level (30) . The discrepancy may be explained by the fact that the blood levels of IFN- in the 300-ng/kg group in our s.c.-injected patients were only one-tenth of the blood levels of their i.v.-administered patients at the 250-ng/kg dose level. Motzer et al. and Portielje et al. reported that the DLT of their rhIL-12 product given s.c. was the elevation of aminotransferases, while observing a few occurrences of grades 1 and 2 mucositis (33 , 35) .

Unlike in the case of i.v. administration, the pharmacokinetic study revealed no spiking high plasma levels of rhIL-12 by s.c. schedules as seen in this study and other Phase I studies with different rhIL-12 products (32 , 33 , 35) . After the i.v. administration of 250 ng/kg in the Phase I study with the same IL-12 product as the present study, the mean C_max was 6,440 pg/ml, the mean plasma half life was 7.2 h, and the mean AUC_0- was 64,276 pg·h/ml (30) . In contrast, in our s.c. injection of 300 ng/kg, the mean C_max was only 61 pg/ml, but the mean plasma half life was 25 h with detectable rhIL-12 even at 48 h, and the mean AUC_0– was 2,346 pg·h/ml. Thus, the s.c. dosing gave a lower peak and less AUC but more sustained plasma levels than the i.v. dosing. Of note, the plasma concentrations after the sixth dosing in the second course were similar to those after the first dosing in the first course.

As reported, rhIL-12 was a potent inducer of IFN-. Although there was a great variation among patients, the plasma levels of IFN- tended to be higher after the first injection in the first course, become lower with subsequent administrations, and again showed high peaks after the initial dosing of each course after the 1-week rest period. The peak levels were apparently less than those obtained in the i.v. administration. TNF-, IL-6, IL-10, and neopterin were also induced in similar patterns. However, these data should be interpreted carefully for the following reasons: the number of patients in each group was small; there are great variations in clinical conditions and disease stages among patients; the ELISA assay measures not functional but total cytokine levels; and the stability of functional cytokines in stored serum is unknown.

Regarding the antitumor effect, a complete response and a partial response of metastatic tumors were observed in two of eight patients with renal cell carcinoma at 50-ng/kg and 300-ng/kg doses, respectively. This observation confirmed that rhIL-12 had a definite antitumor effect on human cancer as reported previously (31, 32, 33, 34, 35) and warrants additional clinical studies.

Our present study and other previous studies have shown that in vivo immunological response to rhIL-12 vary considerably, depending on the administration routes and schedules, and probably on physiological conditions of patients. Therefore, several regimens with different dosages and schedules should be investigated further. As far as the administration schedule used in this study is concerned, the recommended dose level for Phase II studies is 300 ng/kg.

	ACKNOWLEDGMENTS

 
We express our sincere gratitude to other participating physicians in this dose-escalating study for their cooperation, and to Nobuhiro Nakamura, Kaoru Tsuda, Yoko Amada, Yuko Makino, and Hideo Saotome for their help in the preparation of the manuscript as well as to Dr. Matthew L. Sherman for his critical review of the manuscript.

	FOOTNOTES

 
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.

¹ To whom requests for reprints should be addressed, at Department of Medicine III, Hamamatsu University School of Medicine, 3600 Handacho, Hamamatsu 431-3192, Japan. Phone: 81-53-435-2265; Fax: 81-53-434-2910. E-mail: ohnoryu{at}hama-med.ac.jp

² The abbreviations used are: IL, interleukin; rhIL-12, recombinant human IL-12; DLT, dose-limiting toxicity; MAD, maximum acceptable dose; TNF, tumor necrosis factor; LDH, lactic dehydrogenase, AUC, area under the curve.

Received 2/23/00; revised 4/ 6/00; accepted 4/ 7/00.

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