A Phase I Study of Recombinant Human Leukemia Inhibitory Factor in Patients with Advanced Cancer1

2056 Vol. 9, 2056–2065, June 2003 Clinical Cancer Research

Dishan H. Gunawardana,2 Russell L. Basser, genitor cells increased in response to rhLIF. In stage 2, Ian D. Davis, Jonathan Cebon, Paul Mitchell, platelet recovery to baseline levels was earlier for patients ؍ ␮ < Craig Underhill, Trevor J. Kilpatrick, receiving higher doses of rhLIF ( 4.0 g/kg/day; P 0.02). The neutrophil nadir after chemotherapy was less severe in Katrina Reardon, Michael D. Green, Peter Bardy, patients receiving >4.0 ␮g/kg/day of rhLIF. In stages 1 and Pene Amor, David Crump, Siobhan Ng, 2, increases in C reactive protein were seen at higher doses. Roger L. Nation, and C. Glenn Begley Several patients developed evidence of autonomic dysfunc- Centre for Developmental Cancer Therapeutics,3 Parkville, Victoria tion, in particular impotence and episodic hypotension. The 3050 [D. H. G., R. L. B., I. D. D., J. C., P. M., C. U., T. J. K., K. R., dose-limiting toxicities were hypotension and rigors. Phar- M. D. G., S. N.]; Department of Hematology, Institute of Medical and macokinetic studies demonstrated a short half-life (1–5 h) Veterinary Science, Adelaide, South Australia [P. B.]; Amrad Operations, Richmond, Victoria, Australia [P. A., D. C.]; Centre for independent of dose. Pharmaceutical Research, University of South Australia, North Conclusions: We demonstrated a biological effect of Terrace, Adelaide, South Australia [R. L. N.]; and Centre for Child rhLIF on blood progenitor cells, C reactive protein levels, Health Research, University of Western Australia, TVW Telethon and hemopoietic recovery after chemotherapy. Institute for Child Health Research and Western Australian Institute for Medical Research, West Perth, Western Australia [C. G. B.] INTRODUCTION ABSTRACT LIF4 is a cytokine with a broad range of in vitro and Purpose: Leukemia inhibitory factor (LIF) is a pleio- in vivo biological effects. The molecule was first described as tropic molecule of the interleukin 6 family of cytokines. We an inducer of monocytic differentiation in the leukemic cell aimed to examine the safety, pharmacokinetics, and biolog- line M1 (1, 2). LIF is a member of a cytokine family that also ical effects of recombinant human LIF (rhLIF, emfilermin) includes IL-6, IL-11, oncostatin M, ciliary neurotrophic fac- in patients with advanced cancer. tor, and cardiotrophin 1. These cytokines have overlapping Experimental Design: In stage 1 of the study, 34 patients biological activities and act through receptors that share a received rhLIF or placebo (3:1 ratio) at doses of 0.25–16.0 common signaling molecule, the gp130 subunit (3). The other ␮g/kg/day or 4.0 ␮g/kg three times daily for 7 days. In stage subunit of the LIFR complex is the LIFR␤ chain. Expression 2, 40 patients received rhLIF or placebo, either once daily of LIFR␤ determines which cells respond to LIF, because for 14 days commencing the day after chemotherapy (0.25– gp130 is expressed ubiquitously (4). Ligand binding to the 8.0 ␮g/kg/day) or for 7 days commencing the day before receptor complex results in activation of intracellular signal- chemotherapy (4.0 ␮g/kg three times daily). The chemother- ing via the Janus-activated kinase/signal transducers and apy was cisplatin 75 mg/m2 and paclitaxel 135 mg/m2. activators of transcription pathway. Results: In stage 1, platelet counts increased in most Receptors for LIF are expressed on hemopoietic cells patients, including those who received placebo. Blood pro- (macrophages and megakaryocytes), hepatocytes, osteoblasts, preadipocytes, embryonic stem cells, myoblasts, and neuronal cells (5, 6). However, the in vitro biological effects of LIF vary depending on the cell type, so that for example LIF stimulates Received 3/21/02; revised 1/3/03; accepted 1/7/03. differentiation in M1 cells and inhibits differentiation in embry- The costs of publication of this article were defrayed in part by the onic stem cells. In vivo studies in mice have shown that LIF payment of page charges. This article must therefore be hereby marked produces a 2-fold increase in bone marrow megakaryocytes with advertisement in accordance with 18 U.S.C. Section 1734 solely to a dose-dependent increase in platelet numbers (7). Increased indicate this fact. 1 Supported in part by Amrad Operations and the National Health and Medical Research Council, Canberra. P. A. and D. C. were employed by Amrad Operations, whose potential product was studied in the present work. 2 To whom requests for reprints should be addressed, at Department of 4 The abbreviations used are: LIF, leukemia inhibitory factor; IL, inter- Hematology and Medical Oncology, Royal Melbourne Hospital, leukin; LIFR, leukemia inhibitory factor receptor; rh, recombinant hu- Parkville, Victoria 3050, Australia. Phone: 613-9342-7695; Fax: 613- man; ECOG, Eastern Cooperative Oncology Group; tds, three times 9347-7508; E-mail: [email protected]. daily; Epo, erythropoietin; ESR, erythrocyte sedimentation rate; PBPC, 3 Affiliated with: Ludwig Institute Oncology Unit, Austin Repatriation peripheral blood progenitor/stem cell; GM-CFC, granulocyte-macroph- Medical Centre, Heidelberg, Victoria; the Department of Hematology age colony-forming cell; G-CSF, granulocyte colony-stimulating factor; and Medical Oncology, Rotary Bone Marrow Research Laboratories, GM-CSF, granulocyte macrophage colony-stimulating factor; SCF,

Royal Melbourne Hospital, Parkville, Victoria, Australia; Walter and stem cell factor; Meg-CFC, megakaryocyte colony-forming cell; Cmax, Eliza Hall Institute for Medical Research, Parkville, Victoria, Australia; maximum concentration; AUC, area under the plasma concentration-

and the Department of Hematology and Medical Oncology, Western time curve; Cl, clearance; Vd, volume of distribution; t1/2, half-life; Hospital, Footscray, Victoria, Australia. BFU-E, blast-forming unit (erythroid).

platelet levels have also been observed in primates (8). The action of LIF on nerve cells has also been examined in animal models. Direct application of LIF to sites of nerve transection improved survival of both sensory and motor neurons (9, 10). Nerve transection has been shown to increase expression of LIF and IL-6, and is associated with retrograde axonal transport of LIF (11). LIF has also been shown to retard progression of motor neuron disease in a murine model of this disorder (12). Other biological effects of LIF include induction of acute phase proteins (8), reduced lipoprotein lipase activity, and os- teoblast stimulation (13). Emfilermin is rhLIF produced in Escherichia coli. Admin- Fig. 1 Protocol schema. In stage 1, patients were randomized to re- istration of a single s.c. dose in healthy volunteers found that ceive rhLIF or placebo in a 3:1 ratio, which drug was then administered rhLIF was safe and well tolerated up to doses of 4 ␮g/kg.5 for 7 days. This was followed by a period of at least 7 days and maximum of 28 days observation. Stage 2 consisted of chemotherapy Here we report results of a randomized, blinded, placebo- that, in the majority of patients, was followed by study drug adminis- controlled, Phase I dose escalation study to test the safety and tered daily for 14 days. One cohort of patients (4.0 ␮g/kg tds rhLIF) pharmacokinetics of rhLIF administered before and after chem- received study drug from the day before chemotherapy and for a total of otherapy in patients with advanced cancer. 7 days.

PATIENTS AND METHODS Patients. Eligible patients were those with advanced can- ␮ cer at least 18 years of age, ECOG performance status of 0–2, the 4.0 g/kg tds cohort. Patients continuing from stage 1 to absolute neutrophil count of Ն1.5 ϫ 109/liter, hemoglobin level stage 2 remained on the same dose of rhLIF, with the exception ␮ of Ն90 g/liter, and platelet count of 120–500 ϫ 109/liter. that patients who received 16.0 g/kg/day in stage 1 were given Ն 8.0 ␮g/kg/day in stage 2. Adequate renal (creatinine Cl 1.2 ml/sec) and hepatic (biliru- 2 Յ ␮ Chemotherapy consisted of paclitaxel 135 mg/m given bin 25 mol/liter) function were also required. Exclusion 2 criteria included surgery, radiotherapy, or chemotherapy within over3hbyi.v. infusion, followed by cisplatin 75 mg/m i.v. 4 weeks of study entry, prior irradiation to Ͼ30% of estimated over 1 h, with mannitol 10% i.v. over 15 min. Premedication for red marrow volume, and uncontrolled brain metastases. chemotherapy was 20 mg of dexamethasone given p.o. the night The study was approved by the Institutional Ethics Com- before chemotherapy and again on the morning of chemother- mittees of the participating hospitals. Each patient gave in- apy, 50 mg ranitidine i.v., 12.5 mg promethazine i.v. or equiv- formed consent before treatment. alent, and 24 mg ondansetron i.v. The study drug was commenced the day after chemother- Study Design apy for patients in the once daily cohorts and given for a total of 14 days. Patients receiving 4.0 ␮g/kg tds began treatment with The study design is shown in Fig. 1. rhLIF or placebo the day before chemotherapy, and continued Stage 1. The study was double-blinded with respect to for a total of 7 days. Study involvement was completed after one rhLIF (emfilermin is the International Nonproprietary Name cycle of chemotherapy, but patients were able to receive addi- issued by the WHO for rhLIF produced in E. coli). Patients were tional chemotherapy cycles (but without rhLIF) at the discretion randomized to rhLIF or placebo in a 3:1 ratio. A computerized of the investigator. During the study, the use of cytokines other randomization schedule was used. Patients received study drug than rhLIF such as filgrastim, sargramostim, or Epo was not alone given s.c. once daily for 7 days. At least 4 patients were permitted. treated in each dose cohort. Dosing was escalated in the following manner: 0.25, 0.50, 1.0, 2.0, 4.0, 8.0 and 16.0 ␮g rhLIF per Monitoring and Laboratory Studies kilogram body weight per day. An additional group of patients was treated with rhLIF 4.0 ␮g/kg tds to determine whether more Stage 1. Patients were monitored at least daily during frequent dosing enhanced the biological effects of rhLIF. Pa- administration of study drug for adverse events. Formal clinical tients were observed for at least 7 days after finishing the study assessments were performed at screening and day 15. All of the drug. patients were observed for at least 7 days after the last dose of Stage 2. After completion of stage 1, patients were able study drug. to receive chemotherapy provided no longer than 28 days had Full blood counts and ESR were performed at screening elapsed after the last dose of study drug. Patients that did not and daily for 15 days from the start of administration of study complete stage 1, or that completed stage 1 but did not want to drug. Platelet aggregometry and PBPC analyses were per- ␮ continue, were replaced for stage 2. Patients were also allowed formed in the 4.0 g/kg tds cohort only. Progenitor cell to enter stage 2 directly without treatment in stage 1 to expand assays were performed as described previously (14–16). In brief, blood samples for progenitor cell assays were separated using Ficoll-Paque. Mononuclear cells were examined in triplicate cultures using 104 and 105 viable cells/ml. The cultures were examined at 14 days using a dissection micro- 5 Data on file, Amrad operations. scope. GM-CFCs were stimulated with G-CSF, GM-CSF,

and SCF. Erythroid colonies (BFU-E) were stimulated with recombinant LIF.5 Five-ml of blood were collected in a hepa- GM-CSF, IL-3, IL-6, SCF, and Epo. Meg-CFCs were stim- rinized tube, centrifuged within3hofcollection, and the aliquot ulated with G-CSF, GM-CSF, SCF, IL-3, IL-6, Epo, and of plasma was then frozen at Ϫ20°C until the time of assay. megakaryocyte growth and development factor, and stained Samples were analyzed in ascending dose order, as each cohort for quantitation of MEG-CFC as described previously (14– completed the study. The limit of detection using this method 16). CD 34 assays were performed as described previously was 0.05 ng/ml. (17). Briefly, mononuclear cells were separated and analyzed on Coulter profile II flow cytometer (Hialeah, FL). A two- Statistical Analysis color method was used in which lineage-specific antibodies The data were analyzed using SigmaStat (Jandel Scientific (CD3, CD2, CD14, CD19, and CD20; all from Coulter) were Software, San Rafael, CA) or Prism (GraphPad Software, San labeled with tricolor dye (Caltag, San Francisco, CA) and Diego, CA). Data are expressed as medians and ranges for used to separate these cells from lineage-negative CD 34ϩ continuous data, and frequency and percentage for categorical cells. The cutoff for positive cells was determined from the data, unless otherwise specified. Dose cohort size was deter- negative control antibody profiles, and percentages and ab- mined empirically to allow efficient dose escalation and not to produce sufficient statistical power to allow comparisons be- solute numbers of cells were determined as described (17). tween individual dose cohorts. CRP and fibrinogen were measured at least four times a week during the study period as another measure of possible biological activity of rhLIF. Serum lactate dehydrogenase levels RESULTS and other biochemical parameters including urea, electrolyte, Patients and creatinine levels, and liver function tests were measured Fifty-two patients in total were enrolled in stages 1 and 2. weekly. The most common tumor types were non-small cell lung cancer Neurological assessment included a standardized neurolog- and carcinoma of unknown primary. In each of stages 1 and 2, ical symptom questionnaire on motor, sensory, and autonomic 2 patients were withdrawn after enrolment before receiving disturbances (18). Patients also had neurological examinations, study drug. The reasons for withdrawal were inadequate major creatine kinase measurements, and assessments of autonomic organ function (therefore, that patient was deemed ineligible), function including postural changes in blood pressure and heart acute myocardial infarction, reaction to paclitaxel chemotherapy in stage 2, and withdrawal of consent. These 4 patients are not rate response. Assessments were performed at baseline, at com- included in the safety or biological activity analyses. The base- pletion of rhLIF treatment, and again 1 week later. Neurological line characteristics of the patients that received at least one dose assessment was performed independently of adverse event re- of study drug are shown in Table 1. There were no significant porting. differences between the placebo and rhLIF cohorts in terms of Assays for antibodies to rhLIF were performed weekly. age, sex, performance status, and extent of prior treatment. Each assay required 5 ml of blood that was collected in a The number of patients in each dose cohort is shown in heparinized tube. The blood was centrifuged, and the aliquot of Table 2. Twenty-six patients who completed stage 1 were plasma was frozen and transported to Amrad Operations, where treated in stage 2. An additional 16 patients were enrolled a specific ELISA was performed. directly into stage 2, of whom 14 received treatment. Stage 2. Formal clinical assessment was performed at screening before the commencement of stage 2 and on day 22 Peripheral Blood Counts ␮ (day 15 for the 4.0 g/kg tds cohort). Adverse events were Stage 1 (rhLIF/Placebo Alone before Chemotherapy). ␮ assessed daily in the 4.0 g/kg tds cohort, and at least twice Platelet levels rose over the period of monitoring in most pa- weekly in the other cohorts. Full blood counts and ESR were tients, including those treated with placebo. We have observed checked at least three times weekly during the period of rhLIF this effect on platelets in patients receiving placebo in previous administration. Biochemical analyses were performed at screen- studies (19). Fig. 2 shows the peak level of platelets versus ing, and on days 16 and 22 (twice weekly to day 15 in the 4.0 baseline for patients receiving placebo, lower doses of rhLIF ␮g/kg tds cohort). Neurological assessments and antibody as- (0.25–2.0 ␮g/kg/day), doses of 4.0–16.0 ␮g/kg/d rhLIF and 4.0 says were as for stage 1. ␮g/kg tds rhLIF. There were no significant differences between these four groups (P ϭ 0.60; ANOVA). There were also no significant changes in platelet aggregometry, hemoglobin, or Pharmacokinetic Analysis neutrophil counts (data not shown). In stage 1, samples for measurement of plasma LIF con- Stage 2 (rhLIF with Chemotherapy). The median time centration were taken on days 1, 2, 5, 6, and 7. On days 1 and of platelet recovery to baseline after administration of chemo- 7, 12–13 samples were taken over a 24-h period. In stage 2, therapy was shorter in the patients receiving “high” doses of samples were taken on days 2, 8, 9, 14, and 15 for the once daily rhLIF (with Ն4.0 ␮g/kg/day arbitrarily defined as the high dose cohorts and days 1, 5, 6, 7 and 8 for the 4.0 ␮g/kg tds cohort. and including the 4.0 ␮g/kg tds cohort; Fig. 3). Recovery to Multiple samples over a 24-hour period were taken on days 2 baseline levels was 3 days earlier in patients that received high and 8 for once-daily cohorts and days 1 and 7 for the 4.0 ␮g/kg doses of rhLIF when compared with patients that received doses tds cohort. The plasma concentration of LIF was measured using Յ2.0 ␮g/kg/day or placebo. This grouping of patients was a validated ELISA assay that detected both endogenous and justified, because there was no significant difference in either

Table 1 Baseline characteristics of all patients that received study drug All patients (n ϭ 48) Stage 1 (n ϭ 34) Stage 2 (n ϭ 40) Characteristic Placebo % rhLIF % Placebo % rhLIF % Placebo % rhLIF % Number of patients 12 25% 36 75% 8 24% 26 76% 10 2% 30 75% Age, years Median 62 59 62 60 63 58.5 Range 41–74 29–76 41–74 29–76 48–74 29–76 Sex Male 6 50% 25 69% 4 50% 19 73% 5 50% 21 70% Female 6 50% 11 31% 4 50% 7 27% 5 50% 9 30% Prior chemotherapy 3 25% 8 22% 1 13% 4 15% 3 30% 7 23% Prior radiotherapy 2 17% 6 17% 1 13% 3 12% 1 10% 5 17% ECOG performance status Median 1 1 1 1 1 1 Range 0–20–20–20–10–20–2 Tumor type NSCLCa 213 212 29 Unknown Primary 4 9 3 6 3 9 Breast 2 1 0 0 2 1 Mesothelioma 0 3 0 2 0 3 Renal 2 1 1 1 1 0 Soft tissue sarcoma 0 3 0 2 0 3 Stomach 1 1 1 0 1 1 Otherb 151314 a Non-small cell lung cancer. b Includes ovary, bladder, colon, endometrium, and prostate.

the time to platelet recovery or the depth of the platelet nadir Table 2 Number of patients that received study drug by cohort between patients who received placebo or Յ2.0 ␮g/kg/day ϭ ϭ rhLIF. The baseline characteristics of the “low” and high dose Stage 1 (n 34) Stage 2 (n 40) groups are shown in Table 3. There were some differences with Dose (␮g/kg/d) rhLIF Placebo rhLIF Placebo regard to age, sex, and prior chemotherapy. The median baseline 0.25 3 1 3 1 platelet count of the high dose group was higher than that of the 0.50 3 1 3 1 1.0 3 1 3 1 low dose group, although this difference did not reach statistical a ϭ 2.0 3 1 3 (1) 1 significance (P 0.09; t test). The median time of platelet 4.0 3 1 3 (1) 1 (1) recovery to baseline was 12 days after chemotherapy in the high 8.0 3 1 6 (2) 2 dose rhLIF group. On this day, platelet levels in patients receiv- 16.0 3 1 —— ing high doses of rhLIF were significantly higher than those in 4.0 ␮g/kg tds 5 1 9 (6) 3 (3) Total 26 8 30 (10) 10 (4) the combined low dose plus placebo group (P ϭ 0.02; t test). a The levels remained significantly higher for the next 2 days. The ( ), patients enrolled directly into stage 2, who did not receive treatment in stage 1. median time to platelet recovery in the other group was 15 days. No patients required platelet transfusion during stage 2. Consistent with the effect on platelet recovery, rhLIF also hastened neutrophil recovery. After chemotherapy, neutrophil Peripheral Blood Progenitor Cells counts fell additionally in patients who received placebo or low The results of CD34 and PBPC assays are shown in Fig. 5. dose rhLIF when compared with those who received Ն4.0 A Ͼ10-fold increase in GM-CFC and BFU-E levels was seen in ␮g/kg/day (Fig. 4). The median neutrophil count remained 2 of the patients receiving rhLIF 4.0 ␮g/kg tds. This was above 2.0 ϫ 109/ml in patients receiving the high doses of associated with an 8-fold and 3-fold increase in Meg-CFC rhLIF. At day 14 after chemotherapy, the difference between the levels, and a 10-fold and 1.2-fold increase in CD34-positive high dose group and the group that received either low dose or cells. Consistent with the increases in progenitor cell counts, the placebo was significantly different (P ϭ 0.02; t test). This platelet levels in these 2 patients increased ϳ2-fold and 1.5-fold, significant difference was maintained on day 15 (P ϭ 0.01; t respectively, during stage 1 (see Fig. 2D). These 2 patients were test). Only a few patients had neutrophil counts measured on day relatively young (52 and 57) and had a good performance status 16, and neutrophils counts recovered to Ն2.0 ϫ 109/ml in the (0 and 1). Neither patient had received chemotherapy, radiother- placebo group by day 17. Episodes of febrile neutropenia were apy, or hematologic growth factors before the study. infrequent with this chemotherapy regimen and did not allow a comparison of this end point for the different groups. Biochemical Assays There was no significant difference in hemoglobin levels at No significant changes in renal or liver function tests different doses of rhLIF versus placebo. (including lactate dehydrogenase) were demonstrated.

Fig. 2 Peak level of platelets versus baseline in stage 1. The (A) placebo (n ϭ 8), (B) 0.25–2.0 ␮g/kg/day rhLIF (n ϭ 12), (C) 4.0–16.0 ␮g/kg/ day rhLIF (n ϭ 9); and (D) 4.0 ␮g/kg tds rhLIF (n ϭ 5) groups are shown. The 2 patients in D referred to in Fig. 5 are shown (E and f).

ing symptom in 3 of these patients. No one receiving placebo reported this symptom in stage 1. Two patients that received active rhLIF reported onset of impotence in stage 1 that persisted throughout stage 2. An additional 3 patients reported it developing in stage 2, and 1 of these patients received placebo. The impotence had not resolved by the completion of stage 2 in any of the patients. Generally these patients received higher doses of rhLIF: it was reported by patients receiving Ն2.0 ␮g/kg/day. Because there were in total 25 males in the active therapy group and 6 in the placebo group, the percentage developing impotence during the study was similar for the two Fig. 3 Platelet counts after chemotherapy. Chemotherapy was admin- groups (16% in active group and 17% in the placebo group). The istered on day 0. The patients receiving low dose rhLIF (E, n ϭ 22, Յ2.0 ␮g/kg/day and placebo) versus high dose rhLIF (f, n ϭ 18, Ն4.0 impotence was not consistently associated with hypotension. ␮g/kg/day and 4.0 ␮g/kg tds) are shown. The small cohort sizes pre- However, 1 patient, who received 8.0 ␮g/kg/day of rhLIF de- cluded detection of any significant difference between the individual veloped both impotence and postural dizziness. Other symptoms ␮ ␮ cohorts (4.0, 8.0 g/kg/day, 4.0 g/kg tds) when compared directly with consistent with autonomic dysfunction were reported: postural the placebo cohort. Note the significantly enhanced platelet recovery for the Ն4.0 ␮g/kg/day cohort versus the low dose cohort. dizziness was noted in 6 patients, 2 of whom received placebo, and nocturnal diarrhea occurred as a new symptom in 1 patient who also developed postural dizziness and received 1.0 ␮g/kg/ day rhLIF. Fibrinogen and ESR were not altered significantly by rhLIF administration (data not shown). CRP levels were elevated during stages 1 and 2 for patients Adverse Events receiving doses of rhLIF Ն4.0 ␮g/kg/day. Whereas baseline Stage 1. The adverse events shown in Table 4 are those CRP levels were highest in patients receiving Ն4.0 ␮g/kg/day attributed, by the blinded investigators, to study drug in Ն2 rhLIF, these patients also had the largest median rise in CRP. patients, or that were deemed severe. The dose-limiting toxici- CRP levels in patients receiving Ն4.0 ␮g/kg/day rhLIF were ties at 16.0 ␮g/kg/day were hypotension and rigors. The most significantly higher than those in the placebo group over the common adverse events were fevers and rigors, hypotension, course of stage 1 (Fig. 6; P ϭ 0.008; Wilcoxon test) and stage headache, dizziness, and local reactions at the rhLIF injection 2 (data not shown; P Ͻ 0.001). In stages 1 and 2, CRP levels in sites. The fevers and rigors occurred within2hofrhLIF patients receiving Ն4.0 ␮g/kg/day rhLIF rose to a peak 24 h administration and usually resolved spontaneously within 1 h. after the first dose. No atherosclerotic complications of elevated Musculoskeletal pain occurred on the day of rhLIF administra- CRP were observed during the study period. tion and in some cases persisted throughout the day. Hypotension occurred in 5 patients receiving rhLIF. It was Neurological Evaluation not noted in any placebo patients. There were 3 episodes of There were no consistent motor or sensory changes found. severe hypotension. These occurred in patients receiving 2.0, However, some changes in autonomic function were docu- 4.0, and 16.0 ␮g/kg/day of rhLIF. The episodes occurred within mented. Seven male patients reported impotence in response to 1 h of administration of study drug. Two patients were treated the neurological symptom questionnaire, but it was a pre-exist- with i.v. normal saline with resolution of the event within a few

Table 3 Baseline characteristics of lowa and highb dose groups in stage 2 Low (n ϭ 22) High (n ϭ 18) Age, years Median 62.5 54.5 Range 47–74 29–70 Sex Male 12 14 Female 10 4 Prior chemotherapy 7 3 Prior radiotherapy 3 3 ECOG performance status Median 1 1 Fig. 4 Median neutrophil counts after chemotherapy. Chemotherapy Range 0–20–2 was administered on day 0. Counts are shown from day 8 onwards for Tumor type patients receiving placebo (E; n ϭ 10), 0.25–2.0 ␮g/kg/day (Œ; n ϭ 12) Non-small cell lung cancer 7 4 and Ն4.0 ␮g/kg/day of rhLIF (f; n ϭ 18; includes 4.0 ␮g/kg tds). Note Unknown primary 5 7 the significantly enhanced neutrophil recovery for the Ն4.0 ␮g/kg/day Breast 3 0 cohort versus the combined placebo and 0.25–2.0 ␮g/kg/day cohorts. Soft tissue sarcoma 0 3 Other 7 4 a Low dose group received placebo or rhLIF Յ 2.0 ␮g/kg/day. b High dose group received rhLIF Ն 4.0 ␮g/kg/day or 4.0 ␮g/kg three times daily. DISCUSSION This study examined the safety and pharmacokinetics of emfilermin (rhLIF) when given before and after chemotherapy. A number of parameters were monitored to determine the biological activity of rhLIF. hours. The hypotension in the third patient resolved on lying On the basis of previous studies (7, 8), rhLIF was predicted down for 10 min. to influence the platelet count. When rhLIF was administered Five patients did not complete stage 1. The reasons were alone, platelet counts increased over time in the majority of withdrawal of consent, disease progression, severe hypotension patients. However, a similar increase was also seen in patients ␮ (16.0 g/kg/day cohort), and intolerable rigors (2 patients in 4.0 receiving placebo, and the difference between the placebo and ␮ g/kg tds cohort). active drug groups was not significant. The rise in platelets in all Stage 2. The adverse events attributed to rhLIF were of the groups was probably a result of the regular venesection similar in type, frequency, and severity to the events in stage 1. that was required, a response we have seen before (19) and One patient receiving rhLIF developed deep vein thrombosis. illustrating the importance of placebo cohorts in these studies. ϫ 9 The platelet count at the time was 333 10 /liter. Of more interest were the results after chemotherapy. In stage 2, Three patients did not complete stage 2. The reasons were recovery of platelets to baseline levels after chemotherapy was withdrawal of consent by 1 patient, sepsis, and renal impairment significantly more rapid in patients receiving high doses of ␮ unrelated to the study drug and hypotension (2.0 g/kg/day rhLIF. For this analysis, patients were divided into a low (in- cohort). cluding placebo) and a high dose group. This division was based There were no deaths related to the study drug. on the lack of evidence of a biological effect of rhLIF in any of the parameters tested at the lower doses. Furthermore, the com- Pharmacokinetics parison of patients who received low (apparently inactive) doses Absorption of rhLIF from the s.c. injection site was rapid, of rhLIF with those receiving active doses would only serve to with maximum plasma concentration reached within2hand reduce the significance of a genuine effect of rhLIF. However, within 10–20 min in some patients. The other major pharma- there were differences in the baseline characteristics of the low

cokinetic parameters measured were Cmax, AUC, Cl, Vd, and and high dose groups, particularly with regard to age and prior t1/2. These are shown in Table 5 for stage 1. The parameters chemotherapy that would tend to favor the high dose group. A measured in stage 2 were similar to those in stage 1. Cl and Vd significant advantage in terms of platelet recovery was demon- appeared to be inversely related to the dose, with results at the strated for those patients who received high doses of rhLIF. 4.0 ␮g/kg/day dose being similar to the 4.0 ␮g/kg tds dose. Thus, although when administered alone rhLIF did not have a However, calculation of these two parameters assumed that detectable effect on platelet counts, the thrombopoietic action absorption from the s.c. injection site was complete, which may was more apparent after the hematopoietic stress imposed by

not have been the case. t1/2 appeared to be independent of dose chemotherapy. It is noteworthy that the effects seen with rhLIF and was relatively short at ϳ2 h. The relationship between dose are quite consistent with those seen with the related cytokines of rhLIF and plasma concentration is shown in Fig. 7. This IL-6 (20) and IL-11 (21), as well as others. In particular, IL-11 demonstrates that, in stage 1, when the 16.0 ␮g/kg/day cohort has been demonstrated to be useful clinically despite a relatively (Fig. 7B) was compared with the 8.0 ␮g/kg/day cohort (Fig. 7A), modest effect on platelets, comparable with the effect seen with Ͼ the Cmax was 10-fold higher and the AUC 4-fold higher. Thus, rhLIF here, and in contrast to the dramatic rise in platelets after for this dose increment, a doubling of dose more than doubled administration of thrombopoietin (22–25). Given the small num- the resulting concentration. bers of patients in the study and the differences between patient

Fig. 5 CD34 assays and progenitor cell numbers in patients in 4.0 ␮g/kg tds cohort from stage 1. Four patients received rhLIF and 1 patient received placebo. The 2 patients referred to in the text are shown (F and f). A, CD34 counts; B, GM-CFC (note logarithmic axis); C, BFU-E; D, Meg-CFC.

Table 4 Adverse events in 34 patients in stage 1 rhLIF (n ϭ 26) Placebo (n ϭ 8) Local Injection site reaction/rash/pain 8 (31)a 0 (0) Systemic Musculoskeletal 4 (15) [2]b 0 (0) Fever 6 (23) 0 (0) Rigors 9 (35) [2] 0 (0) Hypotension 5 (19) [3]c 0 (0) Dermatological Pruritus 2 (8) 0 (0) Neurological Fig. 6 Stage 1, median CRP levels. The groups shown received pla- Dizziness 6 (23) 0 (0) cebo (E; n ϭ 8), 0.25–2.0 ␮g/kg/day (Œ; n ϭ 12) and Ն4.0 ␮g/kg/day Headache 5 (19) 0 (0) rhLIF (f; n ϭ 14; includes 4.0 ␮g/kg tds cohort). Paresthesia 4 (15) 1 (13) Impotence 3 (12) 0 (0) Gastrointestinal Nausea/vomiting 4 (15) 0 (0) Other gastrointestinal 8 (31) [1]d 4 (50) groups, a randomized study designed to look specifically at the a ( ), percentage of patients. clinical benefit of rhLIF after chemotherapy would be required b [ ], no. of patients experiencing severe adverse events. to confirm the effect on platelet recovery. c One life-threatening episode included in severe group. When administered alone rhLIF produced no significant d Severe episode of constipation. change in neutrophil counts. However, the neutrophil nadir after chemotherapy was significantly less severe in patients receiving Ն4.0 ␮g/kg/day of rhLIF. This effect on neutrophil recovery was not seen with other thrombopoietic agents and was not a rigors. An increase in blood progenitor cell levels has been seen prominent feature of the preclinical studies. It may be related to previously with numerous cytokines including G-CSF (26, 27), the fact that fewer patients had received prior chemotherapy in GM-CSF (28), megakaryocyte growth and development factor the group that received Ն4.0 ␮g/kg/day of rhLIF. (15), and SCF (14). The kinetics of PBPC mobilization seen Peripheral blood progenitor cell levels were measured in with rhLIF were similar to those seen with SCF (rather than the 4.0 ␮g/kg tds cohort in stage 1. Two of the 5 patients G-CSF), although the mechanism is unknown. receiving active rhLIF at this dose had Ͼ10-fold increases in Because LIF and related cytokines are known to be in- levels of megakaryocyte, granulocyte-macrophage, and eryth- volved in the acute phase response, several markers of this roid precursors, and this was associated with an increase in response were tested. There was a significant increase in CRP in platelet counts. Two other patients in this cohort did not receive response to rhLIF. This was evident although the baseline levels a full course of rhLIF therapy, which was ceased because of of CRP were higher in the high dose cohorts. However, no

Table 5 Stage 1: pharmacokinetic parameters for rhLIF by dose cohorta ⅐ Cmax (ng/ml) AUC (ng min/ml) Cl (ml/min/kg) Vd (liter/kg) t1/2 (min) Dose (␮g/kg) n Day 1 Day 7 Day 1 Day 7 Day 1 Day 7 Day 1 Day 7 Day 1 Day 7 1 3 0.07 Ϯ 0.01 0.11 Ϯ 0.01 b b bbbbbb 2 3 0.13 Ϯ 0.05 0.28 Ϯ 0.23 b b bbbbbb 4 3 0.32 Ϯ 0.06 0.59 Ϯ 0.20 88.5 Ϯ 29.5 86.8 Ϯ 8.0 48.6 Ϯ 15.6 46.3 Ϯ 4.2 11.5 Ϯ 6.3 6.8 Ϯ 3.5 193 Ϯ 167 104 Ϯ 59.8 8 3 0.86 Ϯ 0.11 1.21 Ϯ 0.04 390 Ϯ 82.5 284 Ϯ 76.8 21.2 Ϯ 5.0 29.8 Ϯ 8.8 8.4 Ϯ 0.9 4.7 Ϯ 0.5 279 Ϯ 34.0 115 Ϯ 24.6 16 3 9.62 Ϯ 2.16 c 1882 Ϯ 424 c 8.79 Ϯ 1.98 c 1.83 Ϯ 1.13 c 142 Ϯ 67.8 c 4 tds 5 0.44 Ϯ 0.16 0.55 Ϯ 0.06 103 Ϯ 34.8 128 Ϯ 44.2 40.1 Ϯ 7.6 34.5 Ϯ 14.8 8.4 Ϯ 4.2 5.7 Ϯ 1.3 143 Ϯ 50.7 130 Ϯ 64.8 a Values shown are mean Ϯ SD. b Values below limit of assay. c Unable to calculate because of incomplete data set.

Fig. 7 Plasma concentration of rhLIF. A, mean plasma concentration during first 24 h after the initial dose of study drug on day 1. Dose levels shown are placebo (E; n ϭ 6), 1.0 ␮g/kg/day (f; n ϭ 2), 2.0 ␮g/kg/day (Œ; n ϭ 3), 4.0 ␮g/kg/day (; n ϭ 3), 8.0 ␮g/kg/day (‚; n ϭ 3), and 4.0 ␮g/kg tds (Ⅺ; n ϭ 5). For the 4.0 ␮g/kg tds cohort the second dose was administered at the 6 h time point, before plasma collection. B, mean plasma concentration during first 24 h after the initial dose of study drug on day 1 for dose level 16.0 ␮g/kg/day (F; n ϭ 3).

increase in ESR or fibrinogen was seen. In clinical studies both and 2 patients required therapy with i.v. fluids. Whereas IL-6 and IL-11 have produced increases in CRP and fibrinogen hypotension was documented as a part of adverse event (20, 21). rhLIF has been shown to increase acute phase proteins assessment, the independent neurological evaluation showed in rhesus monkeys. However, the dose levels tested in the postural dizziness to be a common symptom. Of the 6 pa- primate study were 2, 10, and 50 ␮g/kg/day for 14 days, and the tients affected by postural dizziness, 4 received rhLIF. Three increases seen in that study were much smaller at the 2 ␮g/kg/ patients reported onset of impotence and 1 patient onset of day dose level than at higher doses (8). Therefore, it may be that diarrhea after receiving chemotherapy; 1 of the patients who the doses tested in our study were high enough to induce an experienced impotence received placebo. Whereas neurotox- increase in CRP, but not high enough to increase fibrinogen and icity is a recognized side effect of both chemotherapy drugs ESR. CRP is known to activate the complement system (29) and used in this study (31–33), the symptoms in patients receiv- may have a role in the pathogenesis of arterial inflammation ing rhLIF before chemotherapy suggest that these effects leading to atherosclerosis (30). Whereas nonspecific inflamma- were mediated by rhLIF. tory effects such as fever and local injection site reactions were In addition to hypotension, the main adverse effects of seen during the study, no clinical evidence of arterial inflam- rhLIF in this study were fevers and rigors, hypotension, head- mation was observed. Complement levels were not measured, ache, dizziness, and local reactions at the rhLIF injection sites. and there was no long-term follow-up for delayed atheroscle- Whereas 2 of the 5 patients in stage 1 who received 4.0 ␮g/kg rotic events after study completion. tds were withdrawn because of rigors, all 9 of the patients On the basis of published data (9, 10), we anticipated an treated at this dose level in stage 2 completed the study. The effect of rhLIF on the nervous system. Despite this, we were local reactions and constitutional effects of rhLIF are similar to surprised when both neurological assessment and adverse those reported for related cytokines (20, 21).

event reporting revealed impotence as an effect frequently The t1/2 of rhLIF was relatively short, ranging from ap- associated with rhLIF therapy. Additional symptoms consist- proximately 1–5 h, and was independent of dose. The observa- ␮ ent with an autonomic effect of rhLIF were hypotension, tion of the t1/2 in stage 1 led to the 4.0 g/kg tds schedule being postural dizziness, and nocturnal diarrhea. The episodes of added to the protocol. This schedule resulted in more sustained hypotension were usually noted within the first hour after plasma concentrations, with the biological effects described rhLIF administration, and usually resolved rapidly and spon- above, but no accumulation of rhLIF, presumably because of the

taneously. There were three episodes of severe hypotension, short t1/2. However, in apparent contrast, there was a dispropor-

tionate increase in Cmax and a 4-fold increase in AUC with dose 14. Begley, C. G., Basser, R. L., Mansfield, R., Thomson, B., Parker, escalation from 8.0 ␮g/kg/day to 16.0 ␮g/kg/day. We assumed W. R., Layton, J., To, B., Cebon, J., Sheridan, W. P., Fox, R. M., and this reflected increased bioavailability of rhLIF, although de- Green, M. D. Enhanced levels and enhanced clonogenic capacity of creased Cl could have contributed to this result. The increase in blood progenitor cells following administration of stem cell factor plus granulocyte colony-stimulating factor to humans. Blood, 90: 3378– Cmax at lower dose levels was more linear. 3389, 1997. In conclusion, we demonstrated a biological effect of rhLIF 15. Rasko, J. E., Basser, R. L., Boyd, J., Mansfield, R., O’Malley, C. J., on blood progenitor cells, CRP levels, and on platelet and Hussein, S., Berndt, M. C., Clarke, K., O’Byrne, J., Sheridan, W. P., neutrophil recovery after chemotherapy. Administration was Grigg, A. 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