A feasibility study of roquinimex (Linomide) and alpha interferon in patients with advanced malignant melanoma or renal carcinoma.

Thirty-one patients with advanced renal carcinoma or malignant melanoma were treated in the first feasibility study of alpha-interferon (Roferon) and the new oral immunomodulating agent, Linomide. Linomide 5 mg or 10 mg p.o. daily was given for 2 weeks; alpha-interferon was then added at 3 MU s.c. three times weekly, escalating in each patient by 3 MU per week, if tolerable, up to 12 MJ. The combination was poorly tolerated with nausea, vomiting, somnolence and myalgia commonly reported. Adverse events accounted for treatment withdrawal in ten patients and contributed to withdrawal in four other patients. Treatment with Linomide alone in the first 2 weeks led to a significant increase in white blood cells, neutrophils and platelets. When alpha-interferon was added, the platelet count decreased significantly over the following 6 weeks. Nineteen patients had white cell phenotype and function measured. After 2 weeks of 5 mg Linomide, a transient but significant decrease in the absolute number of activated T-helper cells (CD4+DR+) was observed. No changes in natural killer (NK) cell number or activity were observed. Twenty-two patients were evaluable for response. One with metastatic renal cell carcinoma had a complete response and six had stable disease. This study does not support the use of the combination because significant toxicity was seen without the anticipated immunological benefits.

A feasibility study of roquinimex (Linomide) and alpha interferon in patients with advanced malignant melanoma or renal carcinoma MJ Mackean', D Kerr2, M Lesko3, A Svedberg4, F Hansson4, D Jodrell" and J Cassidylt 'CRC Medical Oncology Department. Beatson Oncology Centre. Glasgow Gll 6NT. UK: 2CRC Institute for Cancer Studies. Clinical Research Block. University of Birmingham. UK: 3Department of Immunology. Westem Infirmary Unit Trust. Glasgow. UK: 4Pharmacia and Upjohn. AB. Lund Research Centre. Lund.

Sweden
Summary Thirty-one patients with advanced renal carcinoma or malignant melanoma were treated in the first feasibility study of a-interferon (Roferon) and the new oral immunomodulating agent, Linomide. Linomide 5 mg or 10 mg p.o. daily was given for 2 weeks; a-interferon was then added at 3 MU s.c. three times weekty, escalating in each patient by 3 MU per week, if tolerable, up to 12 Mi. The combination was poorty tolerated with nausea, vomiting, somnolence and myalgia commonly reported. Adverse events accounted for treatment withdrawal in ten patients and contributed to withdrawal in four other patients. Treatment with Linomide alone in the first 2 weeks led to a significant increase in white blood cells, neutrophils and platelets. When a-interferon was added. the platelet count decreased significantly over the following 6 weeks. Nineteen patients had white cell phenotype and function measured. After 2 weeks of 5 mg Linomide, a transient but significant decrease in the absolute number of activated T-helper cells (CD4+DR-) was observed. No changes in natural killer (NK) cell number or activity were observed. Twenty-two patients were evaluable for response. One with metastatic renal cell carcinoma had a complete response and six had stable disease. This study does not support the use of the combination because significant toxicity was seen without the anticipated immunological benefits.
Keywords: Linomide; alpha interferon; phase trial: metastatic melanoma: advanced renal cell carcinoma Immunotherapv has been used for sexveral years to treat both advanced renal cell carcinoma and metastatic melanoma.
Recombinant a-interferon as a single agent shoxx-s response rates of 10-16%7 in renal cancer (Horoszewxicz et al. 1989: Minasian et al. 1993. with a median response duration of 12.2 months (Minasian et al. 1993). In a review of recombinant a-interferon for metastatic melanoma. the response rate was 22% and median response duration 22.6 months (Kirkw-ood. 1991 ). Although phase II trials of combinations of interferon with other agents including interleukin 2 (IL-2) and 5-fluorouracil (5 FU) give higher response rates. these have not proven superior in randomized studies and further exploration of combinations of immunomodulators is justified (Oliver. 1994).
Linomide is an oral quinoline-3-carboxamide derixvatix e w ith immunomodulatince activitv ( Stalhandske et al. 1982). In preclinical studies. Linomide enhances both the number and actixvitv of natural killer (NK) cells due to recruitment of new target-binding cells from precursor cells in the bone marrow (Kalland et al. 1985a: Kalland. 1990). An increase in the number of active monocvtes and T-cells has also been demonstrated w-ith Linomide (Stalhandkse et al. 1986: Larsson et al. 1987. In clinical pilot studies of patients with solid tumours (Bergh et al. 1997) and acute myeloid leukaemia patients after autologous bone marrow transplantation (Bengtsson et al. 1992). increased numbers of circulating phenotxpicafly actixated Received 16 April 1997Revised 31 December 1997Accepted 19 May 1998 Correspondence to: MJ Mackean NK cells and monocvtes and increased NK cell function in vitro have been obser ed x uith Linormide. Linomide also has in xi-xo antitumour activitv against a vaniety of experimental tumours im mice and rats. includina renal carcinoma and both B 16F10 and Harding-Passes mouse melanomas (Kalland et al. 1985b: Kalland. 1986: Haminin et al. 1988. Linomide prolonged survival in mice transplanted with B16 melanoma. reduced the number of spontaneous and ix. induced metastases and suppressed the g!rowth of established lung metastases (Harmngn et al. 1988). Two minor responses in renal cancer were seen in the pilot study of Linomide alone (Bergh et al. 1997).
The combination of Linomide and a-interferon has not previously been evaluated. Potential svnergsy betw een Linomide and a-interferon could be mediated bv interferon independent enhancement of NK cell cytotoxicitx bv Linormide (Kalland et al. 1985a.) This study u-as designed as a feasibilitv and immunopharmacology study in patients with advanced malirnant melanoma or renal cell carcinoma. Its aim was to identifA a tolerable dose of Linomide when civen in combination with a-interferon. Toxicitx was assessed. immunological parameters in peripheral blood were studied in one centre and objective tumour response was evaluated when appropriate.

PATIENTS AND METHODS
Thirty-one patients were entered in the study wvith histoloaically xerified malignant melanoma or renal cell carcinoma. Elicibilitv Present address: *ICRF Medical Oncolons Department Western General Hospital. Edinburgh. UK: IANCHOR Medical Oncoloes Depanment Aberdeen Royal Infirmar. Aberdeen. UK criteria included advanced disease not amenable to surgery or radiotherapy. WHO performance status 0 or 1. age 18-75. no surgery. hormonal therapy. chemotherapy or radiotherapy within 4 weeks of start of study, and no prior immunotherapy. Written informed consent from all patients was obtained in accordance with local ethical committee guidelines. Patients with CNS metastases. active severe infection, congestive heart disease. severe asthma andlor chronic bronchitis. previous cardiac disease or other malignancy. raised bilirubin or creatinine. or liver metastases were specifically excluded. Patients were not allowed concomitant medication with corticosteroids. H, antagonists. or non-steroidal anti-inflammatory drugs (NSAIDs) in view of their effects on the immune system. Ciprofloxacin w'as not permitted because of a previous reported skin reaction with Linomide. nor acetvlsalicvlic acid because it competes with Linomide for binding to albumin.
All patients had full medical history. examination. weight. clinical chemistry and haematology taken before start of treatment and at 2. 3. 4. 5. 6 and 8 weeks. Oral Linomide. once a day. was given alone for the first 2 weeks of therapy. Subcutaneous a-interferon was then added. three times a week, at a dose of 3 MU and escalating in each patient by 3 MU per week up to 12 MU. if tolerated. The dose of Linomide was 5 mg in the first cohort of patients and 10 mg in the second. There was no intrapatient escalation of Linomide. The study plan was to treat ten patients with each dose of Linomide until they had completed 5 weeks of treatment (2 weeks of Linomide and 3 weeks of Linomide and a-interferon).
Nineteen patients had blood sampling for immunopharmacology twice during the week before starting, therapy. then at 2. 4. 6 and 8 weeks of therapy and 3 weeks after cessation of therapy. Blood sampling was performed 24 h after the last dose of Linomide and 48 h after the last dose of interferon. Analyses were bv FACS-scan flow cvtometry for enumeration of NK-like cells. monocytes and T-cells. Functional studies of the NK cell activitx with K562 and Daudi were performed at baseline. 2 and 8 weeks only (Lesko et al. 1989). Response was assessed by WHO criteria (Miller et al. 1981) every 8 weeks bx bidimensional measurement of palpable lesions or imaging. Adxerse events were graded according to WHO cnteria at each xisit. Patients were initially treated for 8 weeks. but could continue with treatment if there was no disease progression. Time to

RESULTS
The characteristics of the 31 patients entered are show-n in Table 1.
Two patients were ineligible because of lack of histological X enfication and an insufficient interval from prior hormonal therapy. Both are included in the intention to treat and safety analvses. but not the immunopharmacologv analysis.
Toxicity Table 2 summarizes toxicity experienced by each patient with Linomide alone and the change in toxicitr with the addition of interferon. All patients experienced some kind of toxicity during treatment. Nausea and vomiting was common with Linomide alone (61 %). but was generally mild. The addition of a-interferon appeared to enhance this toxicity in 11 out of 27 patients. especially on the 10-mg Linomide dose. A pattem of fever. rigors.   There were no treatment-related deaths. One patient died on study from progressive disease. Nine patients experienced serious adverse ex ents possibIx related to Linomide. fi e of w'hom discontinued Linomide: two with confirmed. exudatixve pericarditis. one A ith a pleural effusion combined A ith gastrointestinal disturbances. one with Guillian-Barre syndrome and the fifth with lethargy and leg pain. Of the remaining four patients A ho continued Linomide. two had anaemia. one gastrointestinal disturbance and one suspected. non-exudative pericarditis.
Treatment duration and doses of a-interferon Median treatment duration 'Aas 8 w'eeks. including, 6 weeks of ainterferon A ith Linomide (range 1-81 A eeks). Treatment was discontinued in 13 patients because of disease progression after a median of 8 w-eeks (rangye 2-19). and in ten patients because of toxicitv after a median of 7 weeks (rance 1-12). Of the remaininc2 eiaht patients. four stopped therapy because of 'completion of study' (after a median of 33 weeks. range . and four at patient's or clinician's request with toxicity as a contributing ex ent after a median of 9 A eeks treatment (ranoe 2-1 ).
The first cohort of 17 patients received 5 mg of Linomide. Of these. three did not start a-interferon. txwo because of toxicitx and one of progressix e disease. After the first eirht patients. the maximal dose of a-interferon as reduced from 12 to 9 MU because 12 MU was not tolerated. Ox erall. w ith 5 mc of Linomide. 11 patients reached a dose of at least 9 MU of a-interferon three times a week and Awere treated for a median of 11 weeks.
The second cohort of 14 patients receixed 10 mr of Linomide. one of 'Ahom did not start a-interferon because of disease progression. Of the 13 patients receixing a-interferon. 12 received the maximal planned dose of 9 MU of a-interferon with a median treatment duration of 10 w-eeks. Five patients subsequently required dose reductions of a-interferon and three required dose reductions of Linomide ow'inc to side effects.
Objective responses Response w'as ex aluable in 22 patients (13 on 5 mg and nine on 10 mg of Linomide). There was one complete remission after 36 w'eeks treatment in a 57-y-ear-old male with lunc metastases from renal cell carcinoma in the 10-mg group. He continued treatment for 56 weeks and stopped because of completion of study. but progressed 17 'Aeeks later. Six patients (three renal and three melanoma) had stable disease. 'ith a median time to progression of 18 weeks (ranae 12-81 weeks). Oxerall median time to progression was 9 weeks (range 2-81 weeks) for the 5-mg, group and 12 weeks (3-73 weeks) for the 10-mg group.

Immunopharmacology
Nineteen patients had immunopharmacology blood samples tak-en at baseline (14 in the 5-mg, group. five in the 10-mg group). Statistical tests were performed on 13 patients between baseline and 2 weeks and on seven patients between baseline and 8 weeks.
wAith follow-up blood samples in the 5-mg, group only. No such tests were performed on the 10-mg group ow'ing to limited numbers (n = 5). Results are show'n in Table 3 for the 5-mgy Linomide group. There was a statisticallx sicnificant decrease in the absolute number of activated T-helper cells (CD4+DR-) after 2 weeks of 5 ma Linomide. but these changes were not sustained after 8 weeks of combined treatment. No change in the absolute and relatix e number of monocvtes (CD 14-). activated monocytes (CD 1I4DQ-). or NK cells (CD56-CD3+) A-as noted xxith treatment.
In 12 patients tested. there w as no effect on the NK-and lymphokine-activated killer (LAK) cell activity during treatment.
There was a sianificant increase in the number of WBCs and neutrophils during the first 2 weeks of treatment with Linomide in both dose groups and in monocy-tes in the 5-mg Linomide group. but this 'as not sustained to 8 w'eeks 'Ahen a-interferon '-as added. The number of ly mphocytes showed a significant decrease after 8 weeks in the 10-mg Linomide group alone. The number of platelets increased significantly after 2 Aseeks. but then decreased significantly after 8 weeks of treatment in both dose groups. DISCUSSION This is the first time a-interferon and Linomide hax-e been used together. We obserxed no change in the absolute and relatixe number of NK cells after Linomide alone. nor 'Ahen a-interferon A as added. This A-as in contrast to prexious preclinical and clinical studies (Kalland et al. 1985a: Kalland. 1990: Bengtsson et al. 1992: Bergh et al. 1997. wxhich haxe shown an increase in absolute and relative numbers of NK cells '-ith an increase in functional actixitx durinn Linomide treatment. We saw a statistically significant decrease in the number of actix ated T-cells (CD4-DR-) after 2 weeks of treatment xxith Linomide alone. not sustained after 8 wxeeks of combined treatment. In two previous clinical studies xxith Linomide (Bengtsson et al. 1992: Bergh et al. 1997). no effect had been seen on activated T-cells. There was no change in monocytes during treatment in this study compared with increases in absolute and relative numbers of monocytes previously reported (Bengtsson et al. 1992;Bergh et al. 1997).
Prechinical studies have denmsed that enhanced NK cell activity during Linormide treatnent is due to recruitment of new cells from precursor cells in the bone marrow. rather than an increase in the cytotoxic activity of pre-existing cells (Kalland. 1990). We saw no change in LAK and NK cell cytotoxicity. The apparent lack of effect on immunological parameters in this study. particularly NK cell numbers and activity. may be a result of the gross impaiment of the immunological system in patients with advanced cancer. Indeedk patients with metastatic disease often have abnormalities in NK cell function and/or NK cell numbers (Whiteside et al. 1994).
The frequency of adverse events during the first 2 weeks of treatment with Linomide alone appears to be the same in both dose groups. When a-interferon was added. the frequency and severity of toxicity in each group increased. Combined treatment was poorly tolerated. with 45% of the patients stopping therapy because of toxicity as a primary (ten patients) or contributing reason (four clinician/patient request). This is much higher than the 6% withdrawal rate for toxicity reported with a-interferon alone at doses greater than 9 MU three times weekly (Minasian et al. 1993). The similar adverse event profile of Linomide and a-interferon (fever. fatigue. myalgia. flu-like symptoms. nausea and vomiting) probably explains the low tolerance of the combination in these patients.
In an earlier study. single-agent Linomide 15 mg given twice weekly achieved two minor responses in renal cell carcinoma (Bergh et al. 1997). The disappointing observation of only one response in 22 evaluable patients (4.5%) in the current study makes it unlikely that the true response rate is over 20% (P = 0.048. onesided test). The lack of added efficacy with the combination may be due to the absence of the anticipated immunological benefits. particularly an increase in number and functional activity of NK cells. This observation. together with the poor tolerability, leads us to conclude that the combination of a-interferon and Linomide does not merit further exploration using these doses and this schedule.

ACKNOWLEDGEMENT
This study was supported by Pharmacia and Upjohn AB. Clinical Trial Number T12231 18.