Phase I Study of Recombinant Human Interleukin-3 in Patients With Bone Marrow Failure By Razelle Kurzrock, Moshe Talpaz, Zeev Estrov, Michael G. Rosenblum, and Jordan U. Gutterman Interleukin-3 (IL-3) is a T-cell-derived colony-stimulating factor (CSF) whose primary targets include relatively early, multipotential, hematopoietic progenitor cells. In this trial, we treated 24 patients with recombinant human IL-3 given by a daily 4-hour intravenous infusion for 28 days. The dose levels were 30, 60, 125, 250, 500, 750, and 1,000 tLg/m 2 /d. At least three patients were entered at every dose level. Each participant suffered from bone marrow failure, with the underlying diagnosis being myelodysplastic syndrome (13 patients), aplastic anemia (eight patients), or aplasia after prolonged high-dose chemotherapy (three patients) for multiple myeloma, breast cancer, or acute myelogenous leukemia. Most patients tolerated therapy well, with the most frequent side effects being low-grade fever and headaches. Hematopoietic changes included modest increases in neutrophil counts (eight patients), eosinophil counts (six patients), platelet counts (three patients), and reticulocyte counts (two patients). An increase in blasts occurred in one patient

who had refractory anemia with excess blasts in transformation and was reversible once IL-3 was discontinued. In addition, one patient with chronic myelomonocytic leukemia showed an increase in monocytes (and granulocytes). Progression to acute leukemia did not occur. Pharmacokinetic analyses showed a rapid clearance with a mean half-life of 18.8 minutes at the 60 ig/m'/d dose, and 52.9 minutes at the 250 pLg/m'/d dose. Serum concentrations of 10 to 20 ng/mL of IL-3 were achievable at the 250 Lg/m'/d dose. Our observations indicate that recombinant human IL-3 can be given safely at doses of 1,000 jLg/m'/d or less. In addition, on the basis of preclinical data and the biologic activity observed in this study, further trials of this molecule, alone and in combination with other growth factors, are warranted in patients with pancytopenia. J Clin Oncol 9:1241-1250. © 1991 by American Society of Clinical Oncology.

HEMATOPOIETINS

ing RBCs, neutrophils, basophils, eosinophils, monocytes, platelets, and lymphocytes. Many of

differentiation of neutrophils, monocytes/macrophages, and eosinophils.67 In contrast, the colonies grown in the presence of multi-CSF (also known as IL-3) contain not only neutrophils, macrophages, and eosinophils but also cells of erythroid and megakaryocytic lineages. 2'8 Therefore, a hierarchy of precursor cells exists wherein

are a complex group of

proteins elaborated by human cells to regulate the growth of blood elements."

Pluripotent

stem cells, originating predominantly in the bone marrow, give rise to the large number of circulat-

these hematopoietic cells are short-lived and must

G-CSF and M-CSF support the growth and prolif-

be constantly replenished. In vitro, ongoing gener-

eration of relatively late-committed progenitors,

ation of mature and functional blood cells occurs

and GM-CSF interacts with somewhat earlier

by proliferation and differentiation of progenitor cells, a process that depends on the continuous supply of certain glycoprotein factors, designated

precursor cells that retain the capacity to differen-

colony-stimulating factors (CSFs) or interleukins (ILs).

To date, several different CSFs (granulocyteCSF [G-CSF], macrophage-CSF IM-CSF], granulocyte-macrophage-CSF [GM-CSF], and multi-

CSF), as well as at least 11 different ILs (numbered IL-1 to IL-ll) have been discovered. Each of these

molecules acts on various target cells including those of lymphoid, erythroid, megakaryocytic, or myeloid lineage. For instance, G-CSF and M-CSF are relatively lineage-specific and stimulate the

generation of neutrophils and macrophages, respectively."' GM-CSF stimulates the growth and

tiate into neutrophils, eosinophils, or monocytes.

From the Department of Clinical Immunology and Biological Therapy, MD Anderson Cancer Center, Houston, TX Submitted August 27, 1990; accepted December 28, 1990. Supported in part by Hoechst-Roussel Pharmaceuticals,Inc, Immune Corporation,and the OccidentalPetroleum Corporation Fundfor CancerResearch. Research conducted in part by the Clayton Foundationfor Research. Dr Gutterman is a Senior Clayton Foundation Investigator. Address reprint requests to Razelle Kurzrock, MD, Department of Clinical Immunology and Biological Therapy, BoA 41, MD Anderson CancerCenter, 1515 Holcombe Blvd, Houston, TX 77030. e 1991 by American Society of Clinical Oncology. 0732-183X/91/0907-0001$3.00/0

Journal of Clinical Oncology, Vol 9, No 7 (July), 1991: pp 1241-1250

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1241

1242

KURZROCK ET AL

The pleiotropic effects of IL-3 are based on its ability to support the growth of cells from early progenitors to mature cells of multiple lineages. The medical uses for hematopoietic growth factors may include 9 1" replenishment of iatrogenic or disease-associated hematopoietic dysfunction by raising cell counts from suppressed to normal levels, enhancement of host resistance to infections, and the increased production of effector cells with cytotoxic activities. Ultimately, to accomplish these goals, it is likely that combinations of different CSFs will be tailored to the needs of individual disease states. Recently, the complementary DNA and gene encoding human IL-3 have been cloned, 2' 13 permitting production of large amounts of homogeneous material suitable for therapeutic trials in clinical medicine. Characterization studies have shown that although IL-3 shares some biologic features with other CSFs, the IL-3 gene has a distinct sequence. The study described herein is a phase I trial with the objectives of ascertaining the safety, biologic effects, and pharmacokinetics of recombinant human IL-3 in patients with bone marrow failure. MATERIALS AND METHODS ProductInformation The isolation, manufacturing, characterization, and purification of recombinant human IL-3 were performed by Immunex Corporation (Seattle, WA). Recombinant human IL-3 was produced from yeast using recombinant DNA obtained from the messenger RNA of stimulated human T cells isolated with a probe from gibbon ape IL-3. The product differs from the natural substance by a substitution of aspartic acid for asparagine at positions 15 and 70. This prevents the formation of N-linked glycosylation and results in a more uniform product. The protein molecular weight is 17,000 d. The IL-3 was provided in the form of sterile lyophilized powder. It was reconstituted in sterile water and diluted in isotonic saline containing 0.1% human serum albumin.

Patient Selection and Study Design Twenty-four patients with bone marrow failure were entered in the study. All patients had bone marrow verification of their diagnosis. Patients with primary bone marrow failure (aplastic anemia, myelodysplastic syndrome [MDS]), as well as those with secondary bone marrow failure caused by prolonged high-dose chemotherapy, were eligible. In accordance with the French-American-British classification system," individuals with MDS were designated as having refractory anemia (RA), refractory anemia with excess blasts (RAEB), refractory anemia with excess blasts in

transformation (RAEBT), or chronic myelomonocytic leukemia (CMML). Eligibility criteria included an absolute neutrophil count (ANC) less than 1,500 cells/4L, a Karnofsky performance status > 50%,"5 a life expectancy of at least 3 months, and preserved hepatic (bilirubin < 1.5 mg/dL) and renal (creatinine < 2 mg/dL) function. Patients were required to have discontinued prior therapy at least 4 weeks before entry into the study. Informed consent was obtained from all patients according to institutional policy. Each patient was treated at the MD Anderson Cancer Center with daily 4-hour intravenous (IV) infusions for 28 days. The dose levels were 30, 60, 125, 250, 500, 750, and 1,000 ig/m2 /d. At least three patients were treated at each dose level. Patients were monitored daily. Vital signs including heart rate, blood pressure, temperature, and respiratory rate were recorded before, during, and after the infusion. A physical examination was performed before the initial dose and weekly thereafter while the patient was in the study. A complete blood cell count was performed three times per week. Electrolytes, urinalysis, prothrombin time (PT), partial thromboplastin time (PTT), reticulocyte count, and a chemistry profile including renal and liver function tests were obtained before treatment and then twice weekly. Triglycerides and cholesterol were measured weekly. An ECG and chest roentgenogram were performed before the study and after the final dose. After the initial 28 days of therapy, each patient had an 8-week rest period, during which blood counts were monitored every second to third day. Maintenance courses (28 days) could then be administered (at the same dose and schedule) provided there had been evidence of a hematologic response during the first course. A hematologic response was defined by any one of the following three changes: (1) doubling of neutrophil counts and achievement 9 of an ANC > 1.0 x 10 /L, (2) doubling of platelet counts and achievement of an absolute platelet count > 20 x 10'/L, or (3) an increase in hemoglobin by > 2 g/dL. Symptoms were classified as mild, moderate, or severe. Mild referred to symptoms that caused no change in performance levels and did not require medication for relief. Moderate symptoms were those requiring medication for relief. Severe signified symptoms that were inadequately relieved by medication and caused a greater than 30% drop in performance status. Changes in laboratory parameters were also classified according to their degrees of abnormality. Changes in laboratory values designated severe included the following: bilirubin, > 5 x normal; SGPT, greater than 300 IU; and creatinine, greater than 2.5 x baseline. Treatment was withheld from any patient with toxicity classified as severe until full recovery occurred. Recombinant IL-3 therapy was then reinstituted at the next lower dose level. Treatment was permanently discontinued if severe toxicity recurred. Therapy was also held if any of the following changes were observed: a greater than 25% increase in spleen size, a doubling of bone marrow blasts with the blasts comprising more than 20% of the marrow cells, or a doubling in the WBC count and the count reaching a level greater than 20.0 x 10'/L.

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1243

RECOMBINANT HUMAN INTERLEUKIN-3 ClonogenicAssays Preparation of cells. Heparinized bone marrow cells from myelodysplastic syndrome patients were obtained. The cells were layered over Ficoll-Hypaque (Pharmacia Fine Chemicals, Piscataway, NJ) and centrifuged (400g, 4'C) for 20 minutes to remove neutrophils and RBCs. Adherent cell fractionation. To enrich for hematopoietic progenitor cells, the adherent fraction was first removed. Low-density bone marrow mononuclear cells were incubated in plastic tissue culture dishes or flasks (Falcon Plastics; Becton Dickinson, Oxnard, CA) with 10% fetal calf serum (FCS; Flow Laboratories, McLean, VA) in a-me0 dium for 90 minutes at 37 C. In this modification of the technique described by Shaw et al,'b nonadherent cells were removed by gentle pipetting and repeated vigorous washing of plates with a-medium. The procedure was repeated until no more cells adhered to the tissue culture dishes. Nonadherent cells harvested in this way contained less than 3% monocytes as confirmed by the following techniques: microscopic differential count of up to 200 cells prepared with Wright's stain, nonspecific (a-naphthyl butyrate) esterase staining, and immunocytochemical analysis using CD13 (anti-MY-4) monoclonal antibody (Coulter Immunology, 7 Hialeah, FL). Mixed colony culture assay. The mixed colony-forming unit-granulocyte erythroid monocyte macrophage (CFUGEMM) assay was performed according to the method of Fauser and Messner.'"W In brief, 2 x 10' nucleated nonadherent bone marrow cells were cultured in methylcellulose with Iscove's modified Dulbecco's medium (Gibco, Grand Island, NY), 30% FCS (Flow), erythropoietin 1.0 U/mL (British Columbia Cancer Research Institute, Vancouver, Canada), and either 15% leukocyte-conditioned medium prepared with phytohemagglutinin (PHA-LCM) (a medium containing a mixture of many growth factors released by 20 phytohemagglutinin-stimulated leukocytes) or IL-3 10 to 100 ng/mL (Immunex) or both. One milliliter of the culture mixture was placed in 35-mm Petri dishes (Lux; Nunc Inc, Naperville, IL) and incubated at 37°C with 5% carbon dioxide in air in a humidified atmosphere. All cultures were evaluated after 14 days for the number colonies. Burstforming unit-erythroid (BFU-E) colonies were defined as an aggregate of more than 500 hemoglobinized cells, or three or more erythroid subcolonies. Colony-forming unit granulocyte-macrophage (CFU-GM) colonies consisted of at least 40 granulocyte-macrophage and/or monocytic cells, and CFU-GEMM colonies contained erythroid, granulocyte, and monocyte-macrophage elements. An analysis for cellular composition was performed by plucking individual colonies from cultures with a micropipette, placing the cells on a glass slide, staining with May-Grunwald-Giemsa, and evaluating morphologically under a light microscope.

Pharmacology Venous serum samples were collected for pharmacology on day 1 before the IL-3 infusion, at 1 hour during the infusion, at the end of the 4-hour infusion, and after the infusion every 3 minutes for 15 minutes, thereafter every 5 minutes until a total of 40 minutes had elapsed after

stopping treatment. Blood samples were centrifuged, and the serum was decanted and stored at OoC until analysis. Serum IL-3 titers were measured by an enzyme-linked immunoadsorbent assay at Immunex Corporation. Serum samples were exposed to mouse monoclonal antibody against IL-3. A rabbit polyclonal antibody against IL-3 was then added. The label used was a goat antirabbit immunoglobulin conjugated to horseradish peroxidase. The lower limit of sensitivity of this assay is 25 pg/mL. RESULTS PatientCharacteristics

Twenty-four patients were treated and all were considered assessable. Their diagnoses included myelodysplastic syndrome (13 patients), aplastic anemia (eight patients), and bone marrow failure after prolonged high-dose chemotherapy (three patients) (Table 1). Chemotherapy had been administered to the three individuals in the latter group for multiple myeloma, breast cancer (without marrow involvement), and acute myelogenous leukemia. Their last dose of chemotherapy was 4 months, 2 months, and 9 months before the time of initiation of IL-3. Details on the three chemotherapy-treated patients are as follows. The patient with multiple myeloma had received melphalan and prednisone therapy intermittently for 2 years and then continuously, without blood count monitoring, for an additional year. At the end of this period, there was no evidence of myeloma, but severe peripheral blood and bone marrow aplasia was noted. Therapy was withheld, and 4 months

later, the patient was referred to us with ongoing bone marrow failure. In the case of the patient with breast cancer, a program of high-dose mitox-

antrone and thiotepa followed by autologous marrow transplant was administered 2 months before referral. The patient was started on IL-3 because

of failure of engraftment, with ongoing severe neutropenia, thrombocytopenia, and anemia. The third patient with acute myelogenous leukemia was first treated in January 1987 with high-dose cytarabine and daunorubicin. She achieved a complete remission after two courses but relapsed in June 1988. Mitoxantrone and etoposide were administered and resulted in severe aplasia without evidence of residual leukemia. Nine months later, she was referred to us because blood and bone marrow tests still showed no evidence of leukemia, but she remained aplastic.

Cytogenetic analysis was performed on bone

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1244

KURZROCK ET AL Table 1. Patient Characteristics

Noof

patient

Characteristic

No.

s entered

24

Age (years) Median Range Performance status 0 1 Sex Female Male Prior therapy None Androgens Immunotherapy Corticosteroids Antithymocyte globulin Cyclosporine A Gammaglobulin Interferon alpha Interferon gamma Hematopoietic growth factors GM-CSF Erythropoietin Chemotherapy Diagnoses Myelodysplastic syndrome RA RAEB RAEBT CMML Aplastic anemia Aplasia after prolonged high-dose chemotherapy* Hematologic parameters Median baseline neutrophil counts (x 10'/L) Range of baseline neutrophil counts (x 10'/L) No. of RBC transfusion-dependent patients No. of platelet transfusion-dependent patients

%

55 23-71

were platelet transfusion-dependent. We generally give RBC transfusions when the hemoglobin falls below 8 g/dL and platelet transfusions when the platelet count dips below 20 x 109/L. Side Effects

22

91.7

The toxicities associated with IL-3 are shown in

2

8.3

Table 2. In general, IL-3 was well tolerated. Patients at all dose levels were febrile. Elevation in

6

25.0

18

75.0

8

33.3 33.3 41.6

8 10 6

4 3 2 1 1 5

20.8

4 1 3

12.5

13

54

1 4 3 8

33.3

3

12.5

0.46 0.024-1.4

temperature coincided with the infusion and re-

solved about 2 hours later. At higher dose levels, most participants also had headaches requiring acetaminophen or codeine for relief. There were no neurologic side effects. Gastrointestinal problems (nausea or vomiting) were rarely seen. Five patients, at doses of 125, 250, 500, 500, and 750 ýig/m2/d, respectively, required dose reduction or discontinuation of therapy because of the following side effects during the initial 28-day course: (1) increase in bone marrow blasts (Table 3, patient no. 7); (2) new onset of atrial fibrillation in a man (patient no. 12) with a history of, but no ECG documentation of, previous episodes; (3) dyspnea and pleural effusion (patient no. 15) of which the relationship to IL-3 administration is unclear because of the concomitant occurrence of neutropenic sepsis; (4) severe nausea, vomiting, and headaches (patient no. 14) that appeared by the second day of therapy and resolved within 48 hours after its discontinuation; and (5) increased monocyte counts in a patient (patient no. 20) with chronic myelomonocytic leukemia. In addition, patient no. 3 with MDS had therapy temporarily interrupted because of an increase in the WBC

21

86

16

66

*Underlying disease was acute myelogeno us leukemia, breast cancer, and multiple myeloma.

marrow metaphases from each of t he subjects and showed the following: normal dip loid karyotype, 16 patients; insufficient metaphase s, two patients; and chromosomal abnormalities, six patients. The chromosomal anomalies included trisomy 8 (three patients), i(17q) (one patient), mo nosomy 5 and 7 (two patients), and t(5q-;12p+) ( one patient), All patients were cytopenic. Th e median baseline ANC was 0.460 x 10'/L (r ange, 0.024 to 1.4 x 109/L). Twenty-one patientts (86%) were RBC transfusion-dependent; 16 patients (66%)

count to 20.0 x 109/L, and patient no. 9 refused therapy after 23 days for personal reasons. Finally, one man (patient no. 16) with MDS, who had an increase in platelet counts and no significant side effects during the first course of IL-3, developed recurrent urticaria during maintenance treatment. In this patient, pruritic urticarial lesions appeared on his trunk, starting on day 12 of his second course. These lesions recurred daily with the infusion and resolved several hours later. There was no concomitant dyspnea or wheezing. Basophil counts and histamine levels (measured during an episode of urticaria) did not increase over pretherapy levels. Diphenylhydramine partially relieved the rash and itching; however, the symptoms worsened daily, and on day 15, it was decided to discontinue IL-3 therapy.

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1245

RECOMBINANT HUMAN INTERLEUKIN-3 Table 2. Side Effects Associated With Recombinant Human IL-3 Dose of p.g/m'/d (no.of patients) Side Effects

30-60 (6)

125-250 (7)

500-750 (8)

1,000 (3)

Fever (> 38.3°C) Chills Headache Nausea Vomiting Bone pain Dyspnea Elevated hepatic transaminase Atrial fibrillation Peripheral edema Urticaria

100* 100 17 17 0 0 17 0 0 0 0

100 100 50 14 0 0 0 0 14 14 0

100 100 75 [12.5] 25 [12.51 0 [12.5] 50 0 [12.5]t 12.5 0 12.5 0 [12.51

100 100 100 0 0 0 0 0 0 0 0

*Numbers refer to the percentage of patients with grade I-II toxicity; numbers in brackets refer to the percentage of patients with severe toxicity. tThis patient had concomitant sepsis, which may have accounted for her dyspnea. tThis patient had a history of symptoms suggestive of previous episodes of transient atrial fibrillation.

Renal toxicity, as assessed by urinalysis, and serum BUN and creatinine, were not observed. Hepatic toxicity was also not seen, except in a single patient at the 500 pg/mZ dose level who showed an increase in SGOT from 20 to 170 IU;

SGOT returned to baseline within 1 week after discontinuing IL-3. There were no changes in quantitative immunoglobulin levels (IgG, IgM, IgA). In the nine patients with available histamine levels before and after the study (day 14 and/or

Table 3. Hematologic Effects of IL-3 Patient/ Diagnosis

ANC

1/AA 2/MDS 3/MDS 4/AA 5/MDS 6/MDS 7/MDS 8/CHEMO 9/CHEMO 10/AA 11/AA 12/MDS 13/MDS 14/CHEMO 15/MDS 16/MDS 17/AA 18/AA 19/AA 20/MDS 21/MDS 22/MDS 23/MDS 24/AA

0.2 - 0.8 0.9- 16.8 0.5- 1.8 0.6 -2.3 0.9-2.5 -2.4 13.7 0.9- 2.8 0.1 - 0.6 -

AEC

Pit

Corrected Retic

Bone Marrow Blasts

-

-

-

-

-

-

-

-

-

-

-

-

-

-

9-24 -

-

0.7 0.03 0.05 - 0.58 0.02 - 0.3 0.05 - 0.30 0 -5.4 0.06 - 2.5

10-54 87- 147 73 -140 -

0.07 - 1.4 1.7 - 6.0 -

Dose 2 (gg/m )

Cell

5-s30 -

-

-

-

-

-

-

-

-

-

-

-

-

-

30 30 30 x 16 d; 30 x 12d 60 60 60 125x 14d;60x 14d 125 125 x 23 d 250 250 250 x 250 500 x 500 x 500 500 500 750 750 x 750 1,000 1,000 1,000

1d 5d; 250 x 23 d 3 d; 250 x 25 d

4d

Comment

-

-

AMC: 1.6 -s 0.16 -

AMC: 33 - 55

NOTE. All patients were treated for 28 consecutive days unless otherwise noted. ANC, AEC, AMC and pit counts are x 10'/L; reticulocyles, bone marrow blasts, and cellularity are percent; numbers are shown only for patients who had a consistent trend in count changes on > 3 counts. Abbreviations: AA, aplastic anemia; AEC, absolute eosinophil count; AMC, absolute monocyte count; cell, cellularity; CHEMO, high-dose chemotherapy-induced aplasia; d, day; Retic, reticulocyles; Pit, platelets.

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1246

KURZROCK ET AL

day 28), no elevations were seen. There was a trend toward an increase in triglyceride levels between days 1 and 28 of the study, with the median change in values being 20 mg/dL (range, -59 to + 183 mg/dL); however, this change did not reach statistical significance (P = .06, signed-rank test). There were no significant changes in cholesterol levels.

17.0 16.5

15.0 14.5 3.5

c-i x

3.0 2.5 2.0 1.5 1.0 0.5

Responses

0.0

Eight patients exhibited an increase in neutrophil counts, six patients an increase in eosinophil counts, three patients an increase in platelet counts, and two patients an increase in reticulocytes (Table 3). There were no significant changes in lymphocyte or basophil counts. Hematologic responses were generally observed within 2 to 4 weeks after initiation of IL-3, and disappeared within 2 weeks after the 28-day course. However, in one patient (Table 3, patient no. 22), the increase in granulocyte (neutrophil and eosinophil) count peaked by the second week of therapy. By the end of the fourth week, the neutrophil count had decreased to baseline levels, whereas the eosinophil count remained high. Several subjects showed unexpected hematologic changes. In one individual with MDS (Table 3, patient no. 3) and an initial WBC count of 2.0 x 109/L (ANC, 0.46 x 109/L), the increase in blood counts first manifested as an increase in peripheral blood blasts without a concomitant change in bone marrow blasts. Over the next few days, a stepwise maturation pattern was seen with increasing promyelocytes and myelocytes, followed later by metamyelocytes, then by bands, and finally neutrophils (Fig 1). When the WBC count reached 20 x 10'/L (day 16), IL-3 was held. After 4 days, the WBC count had decreased to 4.0 x 109/L. IL-3 was restarted and given for the additional 12 days needed to complete a 28-day course. The WBC count increased to 7.4 x 109/L and remained in the range of 4.0 x 109/L (ANC, 2.5 x 109/L) for 1 month of the rest period. There was no improvement in platelet counts (platelet counts, 10 to 20 x 109/L) or in hemoglobin. One patient (Table 3, patient no. 10) with severe aplastic anemia had a dramatic trilineage recovery, the beginning of which first manifested on day 27 of IL-3 treatment. The bone marrow function has continued to improve for 10+ months, with platelet counts reaching 140 x 109/L without further IL-3 administration. How-

DJO

l Ir

vLdy

h)

L

0E "^ia Day 16

Fig 1. The absolute numbers of myeloid elements in the peripheral blood of patient no. 3, who received 30 ig/m'/d of IL-3, are depicted. Bone marrow blasts were 5.8% at baseline and 1.6% on day 15 (data not shown). (0) Blasts, (0) promyelocytes and myelocytes; (0) metamyelocytes; (i) bands, (0) polymorphonuclear cells.

ever, this woman had been treated with antithymocyte globulin 3 months before IL-3, and delayed responses to this therapy are known to occur. Patient no. 12 with CMML received only a single 250 ±ig/m 2 dose of IL-3 because of the development of atrial fibrillation. During the subsequent 2 weeks, a significant alteration in his counts was noted, with a decrease in peripheral monocytosis from 49% to 4%, and an increase in ANC (0.6 2.3 x 109/L) and in platelet counts (87 - 147 x 10'/L). There had been no evidence of cycling in his blood counts in the 6 months before receiving IL-3. We were not able to administer a subsequent course of IL-3 for 3 months, and, at that time, administration of 28 days of escalating doses (60 to 250 Cýg/m 2/d) resulted in neither cardiac arrhythmias nor in improved blood counts. Progression of MDS to acute leukemia did not occur. However, one patient (Table 3, patient no. 20) with the CMML subtype of MDS showed an increase in absolute monocyte count (33 - 55 x 10'/L) in addition to an increase in neutrophils (2.4 -- 13.7 x 109/L). The monocytosis necessitated discontinuation of therapy after day 4. There was no increase in blasts. In addition, one patient (Table 3, patient no. 7) with RAEBT (based on the presence of Auer rods in the bone marrow cells) showed an increase in bone marrow blasts from 9% to 24% after 2 weeks of IL-3 administration (125 lig/m2/d). Withholding therapy was associated with a return of bone marrow blasts to baseline levels within 1 week. Treatment was restarted with a 50% dose reduction (60 plg/m 2/d), and after the additional 14 days needed to com-

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1247

RECOMBINANT HUMAN INTERLEUKIN-3

13.4 to 23.5 minutes at the 60 Lg/m 2 dose (Table 6 and Fig 2). Clearance at this dose level closely fits an open one-compartment mathematic model. At the 250 Rg/m 2 dose level, clearance of IL-3 was biphasic in two patients and monophasic in one patient. The mean terminal phase t/ 2 was 53 + 13 minutes. The change in plasma t/ 2 from the 60 to the 250 [Lg/m 2 dose level appeared to be dosedependent. The volume of distribution (Vd) ranged from 7.3 L to 108 L and did not appear to be related to dose. There was substantial variability in the apparent Vd in individual patients on day 1 versus day 28 of treatment, as well as in different patients at the same dose level.

plete a 28-day course, the bone marrow blasts remained stable. ClonogenicAssays Two patients (Table 3, patient no. 22 and 23) were studied before, during, and after IL-3 treatment. Their colony counts are shown in Tables 4 and 5. Before treatment, a dose-dependent increment in CFU-GM colony numbers was observed in patient no. 22 (Table 4). At IL-3 doses of 10 ng/mL, a mean of 49.5 CFU-GM colonies were counted; at doses of 100 ng/mL, a mean of 62.5 CFU-GM colonies were counted. The effect of IL-3 on BFU-E was similar but not significant because of the small number of erythroid colonies. A dose-response stimulatory effect was also observed on marrow samples obtained from the patient when undergoing treatment. Moreover, with increasing duration of in vivo IL-3 treatment, the total number of CFU-GM colonies increased by threefold to fourfold at every concentration of IL-3. However, the number of BFU-E colonies decreased during the course of therapy. Clinically, an increment in granulocyte counts was observed, whereas there was no change in reticulocytes. In patient no. 23 (Table 5), BFU-E colonies were scarce and did not change with either the addition of IL-3 in vitro or with in vivo treatment. In vitro exposure to IL-3 increased CFU-GM colony numbers above those of the controls, but there was no dose-response effect. Further, treatment of this patient with IL-3 did not enhance colony numbers. Clinically, this patient showed no increase in reticulocytes or platelets. An increase in neutrophils, albeit minor, was noted by the fourth week of therapy.

DISCUSSION The purpose of this phase I study of recombinant human IL-3 was to determine the biologic effects, toxicity, and pharmacokinetics of this molecule in patients suffering from bone marrow failure. On the basis of this trial, it is apparent that, at doses of 30 to 1,000 pLg/m 2/d, IL-3 is tolerated well and is without evidence of serious irreversible toxicity. Many patients experience lowgrade fever and headaches (Table 2), but these are generally ameliorated by acetaminophen. Our pharmacokinetic studies indicate that the clearance of IL-3 is rapid, with a mean terminal t'/2 of less than 1 hour (Table 6 and Fig 2). The short tV2 suggests that this material should be given by prolonged IV infusion rather than by bolus injection. Alternatively, subcutaneous or intramuscular injection may be effective as these routes of administration often result in prolonged serum levels and many cytokines have been successfully administered in these ways.21, 22 We studied the in vitro and ex vivo effect of IL-3 on the two patients with MDS who were treated at the highest dose level (1,000 pg/m/md) (Tables 4 and 5). With increasing time on therapy, one

Pharmacology The clearance of IL-3 from plasma was extremely rapid, with a half-life (t½/ 2 ) ranging from

Table 4. Mean Number of Hematopoietic Colonies and Blood Counts of Patient No. 22 Before, During, and After IL-3 Treatment at 1,000 L±g/m'/d

Control

PHA-LCM

IL-3 10 ng/mL

IL-3 20 ng/mL

IL-3 50 ng/mL

IL-3 100 ng/mL

Absolute Counts PMN Eos Pit Retic

BFU-E CFU-GM BFU-E CFU-GM BFU-E CFU-GM BFU-E CFU-GM BFU-E CFU-GM BFU-E CFU-GM Pretreatment Day 13 Day28

0 0 0

5 1.5 0

4 2.5 0

20 36.5 107.5

10.5 3 0

49.5 110.5 121

13.5 3.5 2.5

58 128 171

13.5 4.5 4.5

60 157 199

15 7 3.5

62.5 244 277

(xl0'/L) 0.90 0.06 2.20 2.70 0.40 2.50

NOTE. The mean number of duplicate counts are presented. Abbreviations: Eos, eosinophil; Pit, platelet; PMN, polymorphonuclear cell; Retic, reticulocyte.

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(%) 15 15 9

0.1 0.1 0.1

1248

KURZROCK ET AL Table 5. Mean Number of CFU-GM Colonies and Blood Counts of Patient No. 23 Before, During, 2 and After IL-3 Treatment at 1,000 pg/m /d Absolute Granulocyte

Pretreatment Day 11 Day28

Control

PHA-LCM

IL-3 10 ng/mL

2 1 1

14 9 10.5

19 16 12

IL-3 20 ng/mL

IL-3 50 ng/mL

IL-3 100 ng/mL

Neut

Eos

Pit (x 10'/L)

Retic (%)

24 19.5 14

22.5 20.5 20.5

24 27 19

.100 .200 .600

.007 .024 .007

7 9 6

< 0.1 < 0.1 < 0.1

NOTE. The mean of duplicate counts are presented; 0-6 BFU-E colonies were found in dishes containing PHA-LCM or IL-3 with no correlation to dose or CFU-GM numbers. Abbreviation: Neut, neutrophil.

patient showed a progressive increase in CFU-GM colony numbers, whereas the second patient exhibited no IL-3-related effect. CFU-GM colony counts from the first patient also showed a dose-related increase after in vitro exposure to IL-3, whereas those from the second patient did not. It is possible that these differences reflect biologic heterogeneity in the underlying disease processes. For instance, the second patient had a more advanced disease state (RAEB) than the first (RA), and was also more severely pancytopenic at the outset. A concern often voiced regarding the application of hematopoietic growth factors to patients with hematologic malignancies is the potential to stimulate the neoplastic clone. In this study, 13 participants had an MDS, and one had prolonged aplasia after remission induction for acute myelogenous leukemia. Twelve of these individuals showed no clinical evidence of IL-3-induced stimulation of the malignancy. In one patient with RAEBT, an increase in bone marrow blasts occurred while on therapy; however, whether these represented benign or malignant blasts is not known, and the blast count returned to baseline

after therapy was withheld for 1 week. Only one patient (diagnosis, CMML) demonstrated evidence of disease progression. Of interest, there was no enhancement of monocytosis in a second IL-3-treated patient with this disorder, and a third patient had a significant decrease in monocytes accompanied by an increase in platelet and neutrophil counts. IL-3 therapy was associated with an increase in granulocyte counts in 13 of 24 patients. However, in five of these individuals, the increase reflected an increase in eosinophils rather than neutrophils, and in a sixth patient, an initial increase in both neutrophils and eosinophils in the first 2 weeks of treatment was followed by a decline of neutrophils, but not of eosinophils, to baseline levels. In this regard, Saito et al23 have previously demonstrated that, in suspension cultures of human bone marrow cells exposed to IL-3, neutrophilic and eosinophilic cells develop within 2 weeks; by 3 weeks, however, the majority of nonadherent cells are of eosinophilic lineage. Despite preclinical studies242 showing that IL-3 stimulates mast cell proliferation and evokes an increase in histamine levels in animals, we did not see any increase in

Table 6. IL-3 Pharmacokinetic Summary Patient No. 5* (day 1) 5* (day 28) 6* (day 1) 6* (day 28)

Dose 2 (p.g/m ) 60 60 60 60

t'/h (min) -

Mean ± SEM 7 12 13 11

125 250 250 250

14

500

11.3 5.8 4.5 Mean ± SEM -

t'213 (min)

Vd (liters)

Cxt (ng/mL x min)

18.2 20.1 23.5 13.4 18.8 ± 2.1 101 64 68 26.7 52.9 + 13.2 17.3

18.5 101 106 41 66.6 t 21.8 30 20.3 7.32 107.9 45.17 - 31.6 13.7

180 36.6 37.7 56.4 77.7 ± 34.4 536 643.6 1,613 165.3 807.3 ± 165.3 1,512.4

NOTE. Blood for pharmacokinetic evaluation was obtained on days 1 and 28 of the IL-3 infusion. Abbreviation: Cxt, concentration x time.

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1249

RECOMBINANT HUMAN INTERLEUKIN-3

A Eq•

5 MINUTES

Fig 2. Clearance of IL-3 from plasma after a 4-hour intravenous infusion of either (0) 60 pg/m2 (n = 2) or (0) 250 pg/m' (n = 3). Values shown are means - SEM. Lines shown are the least squares best fit lines for the data points.

either basophils or histamine levels in any of our patients, although one person did develop urticaria during a second course of IL-3. Two of 24 patients showed an increase in reticulocyte counts; three showed an increase in platelets. However, the majority of patients did not exhibit bilineage or trilineage responses. Preclinical studies suggest that although IL-3 supports progenitors at an early stage of hematopoietic development in vitro, other factors are needed for the process of terminal differentiation.26 Monkey trials have also indicated that IL-3 administered alone has little effect as compared with sequential IL-3 followed by GM-CSF therapy.2 7 These studies predict that optimal responses to IL-3 may only be achieved when this agent is given in conjunction with other growth factors. In this regard, synergy has been shown between IL-3 and IL-6,28 G-CSF,29 erythropoietin, 30 IL-1,31 M-CSF, 32 IL-4,3 and GMCSF.27 The response of one of our patients with MDS may be relevant, as he showed an initial worrisome increase in peripheral blood blasts. Over the next few days, a stepwise maturation

pattern, with increased promyelocytes, myelocytes, metamyelocytes, bands, and, finally, neutrophils, occurred (Fig 1). Several investigators3 4 have suggested that some malignant hematologic cells constitutively express hematopoietins, and it is conceivable that endogenous growth factor expression may have influenced the response pattern in this individual. Finally, the effects of IL-3 may be more pronounced in other settings. For example, Gillio et a135 have shown that administration of IL-3 following intensive myelosuppressive therapy dramatically enhances myeloid recovery and ablates the predicted period of severe neutropenia in monkeys. The implications of these findings for cancer patients who have been treated with chemotherapy or with bone marrow transplantation warrants investigation. In summary, our data indicate that recombinant human IL-3 can be safely given to patients with bone marrow failure. Doses of 30 to 1,000 pug/m2/d by 4-hour IV infusion have few side effects other than mild fever and headaches. This molecule is rapidly cleared with a t/2 of less than 1 hour. IL-3 administration was associated with some hematopoietic stimulatory activity at all dose levels. However, the responsive lineage varied from patient to patient, perhaps reflecting the multiple disease categories included in this phase I trial. There was no evidence of progression to acute leukemia in any patient with a preleukemic disorder. As demonstrated by Ganser et al, 36 ,37 IL-3 can also be administered by subcutaneous injection, and hematopoietic responses are seen with few side effects. Based on the preclinical data and the available clinical results, phase II trials should be designed to further explore prolonged IV infusions or subcutaneous injections of IL-3 alone, and to combine this molecule with other growth factors.

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KURZROCK ET AL

WEHI-3 growth factor activity, mast cell growth factor activity, P cell-stimulating activity, and histamine producing cell-stimulating activity. J Immunol 131:282-287, 1983 9. Vadhan-Raj S, Keating M, LeMaistre A, et al: Effects of recombinant human granulocyte-macrophage colonystimulating factor in patients with myelodysplastic syndromes. N Engl J Med 317:1545-1552, 1987 10. Eschbach JW, Egrie JC, Downin MR, et al: Correction of the anemia of end-stage renal disease with recombinant human erythropoietin. N Engl J Med 316:73-78, 1987 11. Hammone WP, Price TH, Souza LM, et al: Treatment of cyclic neutropenia with granulocyte colony-stimulating factor. N Engl J Med 320:1306-1311, 1989 12. Yu-Chang Yang Y, Clarletta AB, Temple P, et al: Human IL-3 (multi-CSF): Identification by expression cloning of a novel hematopoietic growth factor related to murine IL-3. Cell 47:3-10, 1986 13. Dorssers L, Burger H, Bot F, et al: Characterization of a human multilineage colony-stimulating factor cDNA clone identified by a conserved noncoding sequence in mouse interleukin-3. Gene 55:115-124, 1987 14. Bennet JM, Catovsky D, Daniel MT, et al: Proposal for the classification of the myelodysplastic syndromes. Br J Haematol 51:189-198, 1982 15. Karnofsky DA: Meaningful clinical classification of therapeutic response to anti-cancer drugs. Clin Pharmacol Ther 2:709-712, 1961 16. Shaw GM, Levy PC, LoBuglio AF: Human monocyte antibody-dependent cell-mediated cytotoxicity to tumor cells. J Clin Invest 62:1172, 1978 17. Estrov Z, Grunberger T, Chan HSL, et al: Juvenile chronic myelogenous leukemia: Characterization of the disease using cell cultures. Blood 67:1382-1386, 1986 18. Fauser AA, Messner HA: Granuloerythropoietic colonies in human bone marrow, peripheral blood, and cord blood. Blood 52:1243-1246, 1978 19. Messner HA, Fauser AA: Culture studies of human pluripotent hemopoietic progenitors. Blut 41:327-330, 1980 20. Aye MT, Niho Y, Till JE, et al: Studies of leukemic cell populations in culture. Blood 44:205-210, 1974 21. Kurzrock R, Rosenblum MG, Sherwin SA, et al: Pharmacokinetics, single-dose tolerance, and biological activity of recombinant gamma interferon in cancer patients. Cancer Res 45:1866-1872, 1985 22. Kurzrock R, Rosenblum MG, Quesada JR, et al: Phase I study of a combination of recombinant interferon alpha and recombinant interferon gamma in cancer patients. J Clin Oncol 4:1677-1683, 1986 23. Saito H, Hatake K, Dvorak AM, et al: Selective differentiation and proliferation of hematopoietic cells induced by recombinant human interleukins. Proc Natl Acad Sci USA 85:2288-2292, 1988 24. Metcalf D, Begley CG, Johnson GR, et al: Effects of purified bacterially-synthesized murine multi-CSF (IL-3) on hematopoiesis in normal adult mice. Blood 68:46-57, 1986

25. Mayer P, Valent P, Schmidt G, et al: The in vivo effects of recombinant human interleukin-3: Demonstration of basophil differentiation factor, histamine-producing activity, and priming of GM-CSF-responsive progenitors in nonhuman primates. Blood 74:613-621, 1989 26. Sonoda Y, Yang Y-C, Wong GG, et al: Analysis in serum-free culture of the targets of recombinant human hemopoietic growth factors: Interleukin-3 and granulocyte/ macrophage-colony-stimulating factor are specific for early developmental stages. Proc Natl Acad Sci USA 85:43604364, 1988 27. Krumwieh D, Seiler FR: In vivo effects of recombinant colony-stimulating factors on hematopoiesis in cynomolgus monkeys. Transplant Proc 21:2964-2967, 1989 28. Leary AG, Ikebuchi K, Hirai Y, et al: Synergism between interleukin-6 and interleukin-3 in supporting proliferation of human hematopoietic stem cells: Comparison with interleukin-la. Blood 71:1759-1763, 1988 29. Ikebuchi K, Clark SC, Ihle JN, et al: Granulocyte colony-stimulating factor enhances interleukin-3-dependent proliferation of multipotential hemopoietic progenitors. Proc Natl Acad Sci USA 85:3445-3449, 1988 30. Migliaccio G, Migliaccio AR, Visser JWM: Synergism between erythropoietin and interleukin-3 in the induction of hematopoietic stem cell proliferation and erythroid burst colony formation. Blood 72:944-951, 1988 31. Warren DJ, Moore MAS: Synergism among interleukin-1, interleukin-3, and interleukin-5 in the production of eosinophils from primitive hemopoietic stem cells. J Immunol 140:94-99, 1988 32. Chen BD-M, Clark CR: Interleukin-3 (IL-3) regulates the in vitro proliferation of both blood monocytes and peritoneal exudate macrophages: Synergism between a macrophage lineage-specific colony-stimulating factor (CSF-1) and IL-3. J Immunol 137:563-570, 1986 33. Rennick D, Yang G, Muller-Sieburg C, et al: Interleukin-4 (B-cell stimulatory factor-1) can enhance or antagonize the factor-dependent growth of hemopoietic progenitor cells. Proc Natl Acad Sci USA 84:6889-6893, 1987 34. Oster W, Cicco NA, Klein H, et al: Participation of the cytokines interleukin 6, tumor necrosis factor alsph, and interleukin 1-beta secreted by acute myelogenous leukemia blasts in autocrine and paracrine leukemia growth control. J Clin Invest 84:451-457, 1989 35. Gillio AP, Gasparetto C, Laver J, et al: Effects of interleukin-3 on hematopoietic recovery after 5-fluorouracil or cyclophosphamide treatment of cynomolgus primates. J Clin Invest 85:1560-1565, 1990 36. Ganser A, Lindemann A, Seipelt G, et al: Effects of recombinant human interleukin-3 in patients with normal hematopoiesis and in patients with bone marrow failure. Blood 76:666-676, 1990 37. Ganser A, Seipelt G, Lindemann A, et al: Effects of recombinant human interleukin-3 in patients with meylodysplastic syndromes. Blood 76:455-462, 1990

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Phase I study of recombinant human interleukin-3 in patients with bone marrow failure.

Interleukin-3 (IL-3) is a T-cell-derived colony-stimulating factor (CSF) whose primary targets include relatively early, multipotential, hematopoietic...
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