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Stem Cells, Vol. 14, No. 5, 517-532, September 1996
© 1996 AlphaMed Press

ORIGINAL PAPER

Thrombopoietic Activity of Recombinant Human Interleukin 11 (rHuIL-11) in Normal and Myelosuppressed Nonhuman Primates

Franklin J. Schlermana, Andrea G. Breea, Michelle D. Kaviania, Sandra L. Naglea, Lori H. Donnellya, Lawrence E. Masona, Robert G. Schauba, Stephan A. Gruppb, Samuel J. Goldmana

a Genetics Institute, Inc., Cambridge, Massachusetts, USA;
b Children's Hospital, Philadelphia, Pennsylvania, USA

Key Words. rHuIL-11 • Nonhuman primate • Megakaryocytopoiesis • Thrombopoiesis • Chemotherapy

Correspondence: Franklin J. Schlerman, Genetics Institute, 2 Burtt Road, Andover, MA 01810, USA.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We have extensively characterized the hematological response of normal and myelosuppressed nonhuman primates to treatment with recombinant human interleukin 11 (rHuIL-11) in vivo. In normal cynomolgus monkeys, rHuIL-11 significantly increased peripheral platelet counts when administered at doses of 10 µg/kg/day to 100 µg/kg/day either by constant i.v. infusion or s.c. injection. As few as four days of rHuIL-11 treatment were sufficient to increase peripheral platelet counts significantly. In addition, extending the treatment period enhanced both the magnitude and the duration of the response. Bone marrow megakaryocytes from animals treated with 100 µg/kg/day of rHuIL-11 were increased in size compared to controls and were ultrastructurally normal. A nonhuman primate myelosuppression model using carboplatin, which causes severe thrombocytopenia with platelet counts of <=20 x 103 platelets/µl, was developed. This novel model was used to evaluate the effectiveness of rHuIL-11 in platelet restoration. rHuIL-11, administered s.c. at a dose of 125 µg/kg/day either concurrently or following chemotherapy, prevented severe thrombocytopenia in addition to accelerating platelet recovery compared to control animals receiving no rHuIL-11. These data demonstrate that rHuIL-11 has potent in vivo thrombopoietic effects when administered to normal and myelosuppressed nonhuman primates, and that rHuIL-11 can be an important therapy to reduce the severity and duration of thrombocytopenia following chemotherapy.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Interleukin 11 (IL-11) is a multifunctional cytokine with diverse hematopoietic activities in vitro. In several assays for primitive hematopoietic and lymphohematopoietic progenitors, recombinant human (rHu)IL-11 has synergistic stimulatory activity when added to other cytokines such as IL-3 and IL-4 [1-3]. In addition, rHuIL-11 has multiple effects on erythropoiesis and megakaryocytopoiesis in vitro. rHuIL-11 stimulated macroscopic erythroid burst colony formation when added in combination with Steel factor or IL-3. The combination of rHuIL-11 and IL-3 stimulated the formation of hemoglobinized colonies even in the absence of added erythropoietin, indicating that rHuIL-11 could act at late stages of erythroid differentiation as well as on progenitors [4]. Stimulation of both BFU-megakaryocyte (BFU-MEG) and colony-forming unit-MEG progenitors by rHuIL-11, as well as stimulation of megakaryocyte endoreplication and maturation, have been reported [5-7]. These effects were seen predominantly in synergy with IL-3, although more recently, synergistic stimulation of megakaryocyte colony formation has been reported for the combination of rHuIL-11 and c-Mpl ligand [8]. These data demonstrate that rHuIL-11 acts at both early and late stages of megakaryocyte development.

Studies in mice and rats have confirmed the hematopoietic activities of rHuIL-11, and have demonstrated that stimulation of megakaryocytopoiesis and thrombopoiesis is the predominant in vivo hematopoietic activity of rHuIL-11 in normal animals [9-11]. In contrast to in vitro assays where rHuIL-11 activity was seen predominantly in synergy with other cytokines, rHuIL-11 was active in vivo when administered as a single agent. In normal mice and splenectomized mice, administration of rHuIL-11 resulted in a marked stimulation of megakaryocytopoiesis and a corresponding increase in peripheral platelet counts [10]. Enhanced stimulation of megakaryocytopoiesis and thrombopoiesis was seen when rHuIL-11 was administered to mice by constant s.c. infusion [12]. This study also showed that rHuIL-11-treated animals had increased numbers of splenic myeloid, erythroid and megakaryocytic progenitors. A similar pattern of megakaryocyte and platelet stimulation has been reported in normal rats [11].

The activity of rHuIL-11 has also been studied in a number of murine models of compromised hematopoiesis, including lethally irradiated bone marrow-transplanted mice and moderately to severely myelosuppressed mice [13-18]. In all these models, rHuIL-11 had potent thrombopoietic activity, improving platelet nadirs and accelerating platelet recoveries compared to controls. In addition, several of these studies have confirmed the multilineage potential of rHuIL-11, demonstrating significant improvement in both neutrophil [13, 15] and RBC recoveries [14, 16], as well as stimulation of all lineages of hematopoietic progenitors [14].

Only one report has examined the ability of rHuIL-11 to stimulate hematopoiesis in a large animal model. In this study, rHuIL-11 stimulated both megakaryocytopoiesis and peripheral platelet production in normal dogs. Hematological recovery following sublethal irradiation, however, was not significantly different in rHuIL-11-treated dogs compared to controls, although some trends were noted [19]. To further define the activity of rHuIL-11 in a large animal model, we have extensively characterized the response of normal and myelosuppressed nonhuman primates to treatment with rHuIL-11. We show that rHuIL-11 administration results in a dose-dependent stimulation of platelet production in normal cynomolgus monkeys. In addition, cynomolgus monkeys myelosuppressed with carboplatin and treated with rHuIL-11 showed improved platelet nadirs and had accelerated platelet recoveries compared to controls. These data are consistent with the results of recent human clinical trials [20, 21] and show that rHuIL-11 has potent thrombopoietic activity in both normal and myelosuppressed nonhuman primates.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Forty-six healthy male cynomolgus monkeys (Macaca fascicularis; 38 males, 8 females) weighing from 3 to 9 kg were used for the normal animal studies. The animals were singly housed and cared for according to the guidelines of the American Association for Accreditation of Laboratory Animal Care. All studies were approved by Institutional Animal Care and Use Committees. Monkeys were anesthetized with ketamine hydrochloride (Aveco, Inc.; Fort Dodge, IA) for blood sampling procedures. For myelosuppressed studies, 12 healthy male cynomolgus monkeys weighing from 3 to 6 kg were used. Intravenous fluids, parenteral alimentation, antibiotics and blood products transfusion support were administered through chronic indwelling venous catheters externalized via a modified swivel and tether system (Spaulding Medical Products; Danville, PA) which permits free-ranging motion of the animal throughout the course of treatment.

Recombinant Cytokine
rHuIL-11 purified to homogeneity from Escherichia coli was used in these studies. For constant i.v. infusion (civi), the protein was diluted with saline to appropriate concentrations supplemented with 0.5% heat-inactivated homologous monkey serum. The civi control article was saline for injection (Abbott Laboratories; North Chicago, IL) supplemented with 0.5% heat-inactivated homologous monkey serum. The civi groups were treated for seven consecutive days with saline (n = 2) or rHuIL-11 at a dose of either 10 µg/kg/day (n = 2), 30 µg/kg/day (n = 2), or 100 µg/kg/day (n = 3). For s.c. injections, the protein was administered without dilution and the control article was saline for injection. The s.c. groups were treated with rHuIL-11 at doses of 30 µg/kg given twice daily (BID) for 7 days; 60 µg/kg once daily (QD) for 4, 7 or 14 days; 50 µg/kg BID for 7 days (n = 3/group); or 100 µg/kg QD for 28 days (n = 8, 4 male, 4 female). Control groups were administered saline for 7 days BID or 14 days QD (n = 3/group), or vehicle for 28 days QD (n = 8, 4 male, 4 female). rHuIL-11 was administered to myelosuppressed primates by s.c. injections QD at a dose of 125 µg/kg/day (n = 6). Controls were myelosuppressed, but received no rHuIL-11 (n = 6).

Hematology
In normal animal studies, blood samples were obtained from each animal for complete blood counts at initiation of the study. Additional blood samples were taken three times weekly throughout the study period. Peripheral blood cell counts were obtained using a Baker Series 9000 automated cell counter (Serono Baker; Allentown, PA) using primate-specific discriminator settings. WBC differential counts were performed manually using Diff-Quick (Baxter Healthcare Corporation; Miami, FL) stained blood smears. Reticulocyte counts were quantitated by flow cytometry as described previously [22]. Reticulated platelets were quantitated by methods described previously [23].

Plasma Protein Analysis
Plasma samples were analyzed by the Center for Blood Research Laboratories, Boston, MA for the presence of an acute phase response. Plasma proteins were measured by nephelometry and agarose electrophoresis. Plasma fibrinogen concentrations, analyzed in our lab, were quantitated using Dade® Data-Fi® Fibrinogen Determination Reagents (Baxter Diagnostics Inc.; Deerfield, IL).

Megakaryocyte Size Determination and Electron Microscopy
Eight male and eight female cynomolgus monkeys were s.c. administered either vehicle (n = 8) or rHuIL-11 (n = 8) QD at a dose of 100 µg/kg/day for 28 consecutive days. At necropsy, samples of femoral bone marrow were collected and bone marrow smears were prepared and stained with Wright-Giemsa. Additional samples were fixed in 3% glutaraldehyde, cacodylate buffer (pH 7.3-7.4). Samples were processed and embedded on Araldite-Jembed resin.

Megakaryocyte size was evaluated in three individual blocks/animal (24 total blocks/group) from vehicle and rHuIL-11-treated groups. Blocks were sectioned at 0.5 µm, mounted and stained with toluidine blue and viewed on a Nikon Microphot upright light microscope (Nikon; Melville, NY) using a Plan Apo 100x oil objective. A total of 97 vehicle-treated and 100 rHuIL-11-treated megakaryocytes were sized using a Présage image analysis system (Advanced Imaging Concepts, Inc.; Princeton, NJ). Perimeter and area measurements were collected for cytosolic membrane-bound portions of the megakaryocytes. Absence of nuclei or partial representation of megakaryocytes were used as exclusion criteria.

A cross-sectional area was also determined by computer-assisted image analysis of intact megakaryocytes from bone marrow smears. Megakaryocytes were imaged using a Nikon Microphot upright light microscope and a 40x oil objective. A cross-sectional area of 16 megakaryocytes per animal (128/group) was quantitated using a Présage system. The cell perimeter of intact megakaryocytes was traced with the use of a digitizing tablet and stylus (Wacom Technology Corp.; Vancouver, WA) and the cross-sectional area was calculated.

Electron microscopy was performed on a subset of the same samples of femoral bone marrow collected on day 28 and prepared as described above. Ultrathin sections (60-70 nm) were cut using a Reichert Jung Ultracut E ultramicrotome (Leica; Deerfield, IL), and sections were picked up on 50 or 200 mesh thin-bar copper grids coated with Formvar (Ted Pella, Inc.; Redding, CA). Sections were stained with uranyl acetate and lead citrate for viewing on a Philips 300EM transmission electron microscope (Philips Electronic Instruments Co.; Mahwah, NJ).

Chemotherapy Treatment and Monitoring Protocols for Myelosuppressed Studies
Myelosuppression was induced in 12 animals by i.v. administration of carboplatin (Paraplatin®, Bristol Laboratories; Princeton, NJ) at a dose of 25 mg/kg/day on days 1, 2 and 3. rHuIL-11 (125 µg/kg/day) was administered QD by s.c. injection beginning on either day 1 (n = 3) or day 4 (n = 3) and continuing until platelets were at or above 50 x 103 cells/µl for two consecutive days. A standard care control group was administered carboplatin but received no rHuIL-11 (n = 6). Animals were examined daily for clinical signs. Peripheral blood counts with differentials were obtained daily. Reticulocyte counts, reticulated platelets, serum chemistries, electrolytes and plasma fibrinogen concentrations were monitored throughout the study. Prophylactic antibiotics were administered when ANCs were <500 cells/µl. Whole blood or platelet transfusions were administered as necessary based on clinical and hematologic parameters.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Hematologic Effects of rHuIL-11 Administration to Normal Cynomolgus Monkeys
Animals receiving a civi of rHuIL-11 at doses of 10, 30 and 100 µg/kg/day for seven days (Fig. 1AGo) showed dose-dependent increases in peripheral platelet counts compared to saline-treated control animals. Platelet counts were initially elevated by day 8 in all groups while platelet counts peaked on days 12-14. The magnitude of the platelet increases ranged from 90% to 162% above baseline, with mean platelet counts approaching 1 x 106 cells/µl in the 100 µg/kg/day group. Platelet counts returned to baseline levels by day 20 or 21 in all groups. rHuIL-11 was also active when administered s.c. BID at 30 µg/kg (60 µg/kg/day) and 50 µg/kg (100 µg/kg/day) for seven days (Fig. 1BGo). Peripheral platelet counts increased with similar kinetics to that seen following civi administration of rHuIL-11. The increase in peripheral platelet counts was of similar magnitude in both groups (123% at 60 µg/kg/day and 156% at 100 µg/kg/day).




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  		Figure 1. Mean platelet counts from rHuIL-11-treated cynomolgus monkeys. A) Animals were administered rHuIL-11 by either civi for 7 days at doses of 10 µg/kg/day ({blacksquare}) n = 2, 30 µg/kg/day (•) n = 2, 100 µg/kg/day ({blacktriangleup}) n = 3, or B) by s.c. injection BID at 30 µg/kg (60 µg/kg/day {blacksquare}), 50 µg/kg (100 µg/kg/day •), or saline control 0.5 ml (1.0 ml/day {blacktriangleup}). The data represent mean ± SD in the 100 µg/kg/day-treated civi group and all s.c.-treated groups. Mean platelet counts in the civi control group remained at baseline levels throughout the study period.

 
Effects of rHuIL-11 on Megakaryocyte Size and Maturation
Analysis of the size distribution of megakaryocytes from control and rHuIL-11-treated animals was performed on sections prepared from megakaryocytes, as well as on whole megakaryocytes from bone marrow smears (Table 1Go). In both cases, there was a dramatic increase in the numbers of very large megakaryocytes in rHuIL-11-treated animals compared to controls (Table 1Go). As expected, the cross-sectional area of intact megakaryocytes calculated from bone marrow smears was greater than corresponding areas calculated from megakaryocyte sections. The mean cross-sectional area calculated from intact megakaryocytes was 1615 ± 826 in rHuIL-11-treated animals versus 914 ± 496 in controls (p < 0.01; Student's t-test). The mean cross-sectional area calculated from megakaryocyte cross-sections was 582 ± 226 in rHuIL-11-treated animals versus 362 ± 160 in controls (p < 0.01; Student's t-test).


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  		Table 1
 
Electron microscopic analyses of megakaryocytes from rHuIL-11-treated animals were performed on a subset of the same samples analyzed in the sizing experiment above and confirmed the increase in size of megakaryocytes from rHuIL-11-treated monkeys (Fig. 2Go). Although the megakaryocytes in rHuIL-11-treated animals were larger, they were ultrastructurally normal, mature megakaryocytes. The megakaryocytes of both vehicle-treated and rHuIL-11-treated animals exhibited landmarks of normal maturation, including segmented nuclei containing condensed chromatin, highly developed demarcation membrane systems and mature granule formation (Fig. 2Go).



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  		Figure 2. Megakaryocytes from the bone marrow of cynomolgus monkeys treated with vehicle (n = 8, 4 male, 4 female [A, C]) or rHuIL-11, 100 µg/kg/day (n = 8, 4 male, 4 female [B, D]), administered s.c. for 28 days, appear normal. Characteristic multilobed nucleus (N) with condensed chromatin is shown (A, B). The demarcation membrane system (DM) and alpha granules (arrows) are regularly dispersed throughout the cell (A, B, C and D). The internal channels (arrowheads) of the DM are regularly spaced and the alpha granules lie within the DM zones. Short segments of endoplasmic reticulum and polyribosomes are seen. Magnification is x 9100 in panels A and B; the white bar represents 2 microns. Magnification is x16,250 in panels C and D; the white bar represents 1 micron.

 
Effects of rHuIL-11 on RBC Counts
Mild, transient anemia characterized by a decrease in RBC parameters was seen in all groups receiving rHuIL-11. In the groups receiving rHuIL-11 by civi, RBC count nadirs ranged from 11% below baseline in the 10 µg/kg/day group to 22% below baseline in the 100 µg/kg/day group (Fig. 3AGo). In the groups receiving rHuIL-11 BID by s.c. administration, the anemia was similar in magnitude ranging from 14% below baseline in the 50 µg/kg (100 µg/kg/day) group to 18% below baseline in the 30 µg/kg (60 µg/kg/day) group (Fig. 3BGo).




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  		Figure 3. Mean percent change from baseline RBC counts from rHuIL-11-treated and saline-treated cynomolgus monkeys. A) Animals were administered rHuIL-11 either by civi for seven days at doses of 10 µg/kg/day ({blacksquare}) n = 2, 30 µg/kg/day (•) n = 2, 100 µg/kg/day ({blacktriangleup}) n = 3, or saline control {blacklozenge}) n = 2 or B) by s.c. injection BID at 30 µg/kg (60 µg/kg/day {blacksquare}), 50 µg/kg (100 µg/kg/day •), or saline control 0.5 ml (1.0 ml/day {blacktriangleup}). The data represent mean ± SD in the 100 µg/kg/day-treated civi group and all s.c.-treated groups.

 
Quantitation of Reticulated Platelets and Reticulocytes
As a relative measure of thrombopoiesis following rHuIL-11 administration, reticulated platelets were quantitated by flow cytometry. Animals were administered rHuIL-11 BID by s.c. injection at a dose of 50 µg/kg (100 µg/kg/day) for seven days. Absolute reticulated platelets (ARP) were increased by day 6, peaked on day 10 and then began to decrease on day 13 (Fig. 4AGo). Consistent with previous studies of rHuIL-11 treatment in mice [12], the peak increase in reticulated platelets preceded the peak increase in peripheral platelets (day 13). Both ARP counts and peripheral platelet counts were near baseline levels on day 17.




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  		Figure 4. A) Mean platelet counts ({blacksquare}) compared to ARP counts (solid bar) and B) mean RBC counts ({blacksquare}) compared to mean absolute reticulated RBC counts (solid bar) in cynomolgus monkeys dosed with rHuIL-11 BID at a dose of 50 µg/kg (100 µg/kg/day) for seven days by s.c. injection. Data represent mean ± SD, (n = 3).

 
Reticulocytes were also quantitated to determine if the decrease in RBC counts was associated with any changes in erythropoiesis. RBC counts were initially decreased on day 3 and reached a nadir on day 8. Reticulocytes remained at baseline levels until day 8, at which point they began to increase. On day 10, reticulocyte counts peaked and by day 13, they were decreased as RBC counts returned to baseline levels (Fig. 4BGo).

Effect of Dose Schedule on the Platelet Response to rHuIL-11
We next compared the hematologic effects of QD dosing to BID dosing of rHuIL-11 in nonhuman primates. rHuIL-11 was administered by s.c. injection BID at 30 µg/kg (60 µg/kg/day) for seven days, or QD at 60 µg/kg/day for seven days. In both groups, platelets were initially elevated on day 7 or 8, reached a peak on day 12 and returned to baseline levels by days 22 or 23. The effect of rHuIL-11 was similar in both groups, with platelet counts increased 156% above baseline in the group dosed BID and 104% in the group dosed QD (Fig. 5AGo).




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  		Figure 5. Mean platelet counts and mean percent change from baseline platelet counts from rHuIL-11-treated cynomolgus monkeys. rHuIL-11 was administered BID at: A) 30 µg/kg (60 µg/kg/day •) or QD at 60 µg/kg/day ({blacksquare}) for seven days by s.c. injection; and B) QD at 60 µg/kg/day for either 4 ({blacksquare}), 7 (•) or 14 ({blacktriangleup}) days by s.c. injection. Platelet counts in the saline control remained constant throughout the study. Data represent mean ± SD, (n = 3/group).

 
The effect of different durations of dosing was investigated by administering rHuIL-11 at a dose of 60 µg/kg/day by s.c. injection for either 4, 7 or 14 days (Fig. 5BGo). Significant increases in peripheral platelet counts were noted in all groups with peak platelet counts occurring on days 10, 12 and 15 in the 4-, 7- and 14-day treatment groups, respectively. There was a trend toward platelet increases of greater magnitude with longer dosing, resulting in peak platelet counts of 70%, 100% and 130% for 4, 7 and 14 days of dosing, respectively. The duration of the increase in platelet count was greater in the group that received rHuIL-11 for 14 days compared to the groups that received rHuIL-11 for either four or seven days.

Effect of rHuIL-11 Administration on Peripheral WBCs
Although neither s.c. nor civi administration of rHuIL-11 resulted in any significant change in the absolute WBC counts, differential cell counts showed a transient increase in the mean ANC occurring only on day 6 of treatment. In animals treated s.c. with rHuIL-11 at a dose of 100 µg/kg/day for seven days, as well as those treated by civi at 100 µg/kg/day for seven days, mean neutrophil counts were increased 100%-200% above baseline on day 6. Increased mean ANCs were not observed in animals treated with rHuIL-11 at doses lower than 100 µg/kg/day. No changes in ANC were seen in saline control animals (data not shown).

Effect of rHuIL-11 on Plasma Proteins
An acute phase response was noted in animals s.c. treated with rHuIL-11 BID at a dose of 30 µg/kg (60 µg/kg/day) for seven days. Plasma samples analyzed for acute phase reactants showed increases in haptoglobin, C3 and {alpha}1-antitrypsin, and decreases in albumin and transferrin (Fig. 6Go). Plasma fibrinogen, an acute phase reactant that is important in the maintenance of hemostasis, was also measured in animals s.c. administered rHuIL-11 BID at a dose of 50 µg/kg (100 µg/kg/day) for seven days. Mean plasma fibrinogen concentrations increased from a baseline of 187 ± 51 mg/dl on day 1 to a peak of 747 ± 129 mg/dl on day 6. This represents a 299% increase above baseline which returned to baseline concentrations, 178 ± 18 mg/dl, by day 15.



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  		Figure 6. Acute phase response in cynomolgus monkeys treated with rHuIL-11. Mean percent change from baseline of acute phase proteins from nonhuman primates administered rHuIL-11 BID at 30 µg/kg (60 µg/kg/day) for seven days by s.c. injection. Plasma proteins include: albumin, {alpha} 1-antitrypsin, haptoglobin, transferrin and C3. Data represent mean percent change from baseline ± SD, (n = 3).

 
Hematologic Effects of rHuIL-11 Administration in Myelosuppressed Cynomolgus Monkeys
To determine if rHuIL-11 could prevent severe thrombocytopenia following chemotherapy, we developed a nonhuman primate model of myelosuppression using carboplatin. Cynomolgus monkeys were administered carboplatin at a dose of 25 mg/kg/day on days 1, 2 and 3 (total dose of 75 mg/kg). rHuIL-11 was s.c. administered QD at a dose of 125 µg/kg/day, starting either concurrent with initiation of chemotherapy on day 1 (n = 3) or one day following the final dose of chemotherapy on day 4 (n = 3). This dose was chosen based on pharmacokinetic modeling determining the equivalent dose in cynomolgus monkeys to the dose of 50 µg/kg/day in humans, a dose of rHuIL-11 that has proven to be effective in cancer patients receiving chemotherapy (J. Kaye, personal communication). Dosing of rHuIL-11 continued until platelets were at or above 50 x 103 cells/µl for two consecutive days. A control group (n = 6) received carboplatin but no rHuIL-11. This regimen produced a severe thrombocytopenia with platelet counts at or below 20 x 103 cells/µl in five out of six control animals (median 3 days; range 0-6 days). Administration of rHuIL-11, beginning either on day 1 (Fig. 7AGo) or on day 4 (Fig. 7BGo), significantly improved mean platelet nadirs with only one animal experiencing severe thrombocytopenia of 20 x 103 platelets/µl or less (median 0 days; range 0-2 days). rHuIL-11 treatment also accelerated the recovery of platelet counts. Following myelosuppression, mean platelet counts of controls were at or below 50 x 103 cells/µl for a median of 5.5 days (range 3 to 8 days). Platelet counts of the animals receiving rHuIl-11 starting on day 1 were at or below 50 x 103 cells/µl for a median of 3 days (range 2-5 days; Fig. 7AGo) and animals receiving rHuIL-11 starting on day 4 were at or below 50 x 103 cells/µl for a median of 2 days (range 0-3 days; Fig. 7BGo).




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  		Figure 7. Mean platelet counts from myelosuppressed cynomolgus monkeys. All animals were myelosuppressed by administration of carboplatin (as described in Materials and Methods). A) rHuIL-11 was administered at a dose of 125 µg/kg/day by s.c. administration starting on day 1 (•, n = 3, mean ± SE) or B) day 4 (•, mean ± SE, n = 3) versus standard care control receiving no rHuIL-11 ({blacksquare}, n = 6, mean ± SE). Platelet recovery following myelosuppression was statistically significant with day 1 (p <= 0.05, days 14-21) and day 4 (p <= 0.05, days 13-22) rHuIL-11 treatment compared to standard care controls (Student's t-test). Data represent one cycle of chemotherapy.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The data presented in this report confirm, in a large animal model, that the predominant hematological effect of in vivo rHuIL-11 administration to normal animals is stimulation of thrombopoiesis. Cynomolgus monkeys appear to be more responsive to rHuIL-11 than were mice with platelet increases of greater magnitude seen at lower doses. In cynomolgus monkeys treated with rHuIL-11, there was typically a small early decrease in platelet counts seen on days 2-3, followed by an increase in platelets usually seen in five to seven days. Peak platelet counts were seen one to six days after dosing was stopped. Recent data from human clinical studies have shown that rHuIL-11 treatment resulted in an increase in plasma volume of about 20% that was apparent within several days [24]. This increase in plasma volume likely accounts for the small (about 15%), but reproducible, dip in platelet counts seen early following rHuIL-11 treatment. Quantitation of reticulated platelets, a method shown to be a relevant measure of thrombopoietic activity in mice [25], dogs [26], nonhuman primates [27] and humans [23], showed a twofold increase in new platelets between days 3 and 6. These data, coupled with the observation that peripheral platelet counts begin to rise after day 3 of treatment, suggest that rHuIL-11 stimulation of thrombopoiesis occurs early after treatment, even though it is not apparent in the periphery for five to seven days. The observation that the platelet count usually does not peak until several days after rHuIL-11 treatment stops likely reflects the time required for the more immature megakaryocytes, which were stimulated during treatment, to completely mature and produce platelets. Consistent with this interpretation, reticulated platelets peaked on day 10, three days after rHuIL-11 treatment had stopped.

Bone marrow megakaryocytes from rHuIL-11-treated animals appeared to be fully mature and ultrastructurally normal, displaying typical landmarks of maturation including highly developed demarcation membrane system, mature granule formation and normal budding of platelets from the megakaryocyte surface. These observations contrast the unusual megakaryocyte morphology reported from rHuIL-6-treated nonhuman primates [28], and suggest that the physiological process of normal megakaryocyte maturation was stimulated in rHuIL-11-treated animals. Consistent with this hypothesis was the observation that the mean cross-sectional area of bone marrow megakaryocytes from rHuIL-11-treated animals was significantly increased compared to controls. Additional studies have shown that peripheral platelets from rHuIL-11-treated nonhuman primates responded normally to thrombin as assayed by p-selectin expression. Following seven days of rHuIL-11 adminstration at 125 µg/kg/day, the EC50, the concentration that produces 50% maximal effect, for thrombin-induced upregulation of p-selectin expression was 25.5 units/ml compared to 25.1 units/ml in saline-treated contols. At this time point, platelet counts were approximately twofold higher than controls (M. Kaviani, manuscript in preparation). This result is consistent with in vitro data showing that rHuIL-11 did not stimulate human platelet aggregation directly, nor did it enhance ADP-induced platelet aggregation (K. Ault, personal communication).

There was a transient anemia associated with rHuIL-11 administration. Anemia following rHuIL-11 treatment has not been generally noted in rodents, although a small decrease in RBC was seen in a study where rHuIL-11 was given by s.c. infusion to mice [12]. The anemia seen in nonhuman primates was characterized by a decrease in RBC parameters which was apparent two days after dosing began and reached a nadir after six to nine days. In a normal human volunteer study, analyses of RBC mass and plasma volume following rHuIL-11 treatment have shown that the anemia was not associated with a significant decrease in RBC mass, but rather correlated with an increase in plasma volume, suggesting that the anemia is dilutional [24]. In this study, quantitation of reticulocytes showed that there was no apparent inhibition of erythropoiesis following rHuIL-11 treatment. In addition, a reticulocytosis occurred on days 8-13 and preceded the return of normal RBC counts. These data are consistent with the conclusions of the human study, showing that the transient anemia following rHuIL-11 treatment was not associated with any inhibition of erythropoiesis, and that the recovery from the anemia appeared normal [21].

Several studies were performed to analyze different dose schedules for administration of rHuIL-11. In all these studies, rHuIL-11 was given by s.c. injection since this route appeared as effective as civi administration. After determining that QD dosing was effective (Fig. 5AGo), we treated animals with rHuIL-11 for 4, 7 or 14 days. There were several trends in the data. The magnitude of the increase in platelet counts appeared incrementally higher with longer dosing periods, while the peak platelet count occurred later with longer duration dosing. The duration of the increase in platelets was similar in the animals dosed for four and seven days, but was clearly longer in the group dosed for 14 days. These data are consistent with the other reports that indicate rHuIL-11 can act to regulate megakaryocyte progenitors [5, 7, 29, 30], as well as stimulate maturation of nondividing megakaryocytes [6, 30] and show that the in vivo platelet response to rHuIL-11 is maintained with longer periods of dosing. More recent data have extended these observations and shown that platelet counts in cynomolgus monkeys treated with rHuIL-11 at 100 µg/kg/day for 13 weeks continued to increase throughout dosing, reaching peak platelet counts of greater than 2.3 x 106 platelets/µl, an increase of 350% above baseline (T. Ohata, Yamanouchi Pharm. Co. Ltd., personal communication).

Severe thrombocytopenia resulting from intensive cytotoxic drug and radiation therapy remains the dose-limiting factor in many chemotherapy and radiation treatment protocols. This has led to testing of several factors, including rHuIL-3, rHuIL-6 and c-Mpl ligand in both preclinical and clinical studies to assess their thrombopoietic potentials [28, 31-41]. We have demonstrated previously that rHuIL-11 could prevent severe thrombocytopenia and accelerate platelet recovery following carboplatin and sublethal irradiation in mice [14]. Interestingly, the ability of rHuIL-11 to improve both platelet and RBC recovery in this model is similar to the activity of c-Mpl ligand in comparable models [39, 41]. Although these studies provided proof of principal for the thrombopoietic activity of rHuIL-11 following chemotherapy, murine models are not always predictive of larger animal species. We therefore developed a nonhuman primate model of severe myelosuppression induced by chemotherapy.

Cynomolgus monkeys were treated with 75 mg/kg of carboplatin administered over three days. This regimen produced a severe thrombocytopenia with platelet nadirs of 20 x 103 platelets/µl or less in five out of six control animals. In contrast, administration of rHuIL-11 prevented severe thrombocytopenia of 20 x 103 platelets/µl or less in five out of six treated animals. In addition, recovery of platelet counts to >100 x 103 platelets/µl was accelerated in all rHuIL-11-treated animals compared to controls. These data are consistent with the results of human clinical trials, where rHuIL-11 administration has improved platelet nadirs following chemotherapy in a phase I clinical study [20], as well as significantly reducing the requirement for prophylactic platelet transfusions in a randomized double-blind phase II clinical trial of patients with severe thrombocytopenia (J. Kaye, personal communication). In these human clinical studies, administration of rHuIL-11 was begun on the day following chemotherapy. In this study, we have shown that initiating treatment with rHuIL-11 on day 1, concurrent with carboplatin chemotherapy, was equally effective in preventing severe thrombocytopenia, as was rHuIL-11 treatment on day 4, after chemotherapy was complete. In addition, the duration of severe neutropenia was similar in rHuIL-11-treated animals and control animals, with median recovery to 500 cells/µl of 10.5 days (range 3-14) and 12.5 days (range 6-16), respectively. The extent of severe anemia requiring transfusion was also similar, with two out of six controls and three out of six rHuIL-11-treated animals requiring RBC transfusions following chemotherapy.

There were changes in plasma proteins in rHuIL-11-treated animals indicative of an acute phase response. This is consistent with previous in vitro and in vivo studies that have shown that rHuIL-11 could stimulate acute phase reactants [42, 43]. Although acute phase reactants are often associated with a response to inflammatory states accompanying infection, injury or trauma, rHuIL-11-treated animals showed no clinical signs that could be associated with injury or infection, nor was there any fever. Burstein et al., noting that several acute phase reactants such as fibrinogen and C4 binding protein have procoagulatory activity, have suggested that the concurrent augmentation of the acute phase response and thrombocytopoiesis may represent a redundant mechanism to augment hemostasis in response to injury [6]. In this context, plasma fibrinogen concentrations were increased in response to rHuIL-11 both in normal animals, as well as in carboplatin myelosuppressed animals. Peak plasma fibrinogen increased 294% above baseline in rHuIL-11-treated carboplatin myelosuppressed animals, compared to 91% in controls.

In this report, we have extensively characterized the hematological response of nonhuman primates to treatment with rHuIL-11. In normal animals, rHuIL-11 effectively increased peripheral platelet counts when administered either by civi or s.c. injection. Laboratory analyses demonstrated that rHuIL-11 administration resulted in larger megakaryocytes and stimulation of thrombopoiesis. More importantly, we have developed a reproducible model of severe thrombocytopenia using carboplatin treatment and demonstrated that rHuIL-11 administration significantly improved the platelet nadirs, as well as shortened the duration of severe thrombocytopenia following chemotherapy. In addition, rHuIL-11 administration increased plasma fibrinogen, which may help to maintain hemostasis. Thus, rHuIL-11 can be an important therapy to reduce the extent and severity of thrombocytopenia following chemotherapy. Further studies in future phase III clinical trials are planned.


   Footnotes
 
Provisionally accepted March 18, 1996.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Received February 8, 1996; accepted for publication June 10, 1996.




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