Preclinical Pharmacologic Basis for Clinical Use of rhIL-11 as an Effective Platelet-Support Agent

Introduction

Recombinant human interleukin-11 (rhIL-11), also known as oprelvekin(Drug information on oprelvekin) (Neumega), is the first platelet growth factor to be approved by the United States Food and Drug Administration (FDA) for the prevention of severe chemotherapy-induced thrombocytopenia (CIT) in patients with solid tumors, lymphoma, or those who are at high risk of developing thrombocytopenia. In this setting, rhIL-11 has been shown to reduce the postchemotherapy platelet nadir and, thus, shorten the time required for platelet recovery, thereby enabling many patients to receive successive cycles of chemotherapy at full doses.[1]

The ability to attain these benefits, however, is dependent on the appropriate use of rhIL-11, particularly timing its administration within the framework of the patient’s expected platelet response to chemotherapy. These practical considerations, while seemingly simple, require an understanding of both the role of endogenous IL-11 in the process of megakaryocytopoiesis and the pharmacodynamic effects of rhIL-11. The purpose of this review is to provide an overview of the preclinical pharmacology of rhIL-11, the manufactured recombinant human form of IL-11, by providing a rationale for both its clinical efficacy and recommended dosing regimen. A brief background of the role of endogenous IL-11 in the regulation of megakaryocytopoiesis is provided as a prelude.

Role of Cytokines in the Regulation of Megakaryocytopoiesis and Biology of Endogenous IL-11

As reviewed by several authors,[2-5] a number of cytokines and growth factors are known to be involved in the regulation of megakaryocytopoiesis and platelet formation (Figure 1). IL-11 indirectly promotes thrombopoiesis either by stimulating the release of granulocyte-macrophage colony-stimulating factor (GM-CSF, sargramostim(Drug information on sargramostim) [Leukine]) and interleukin-6 (IL-6) or synergizing with interleukin-3 (IL-3).[5] IL-3 stimulates the proliferation of megakaryocyte progenitor cells in vitro and acts synergistically with stem-cell factor (SCF) to stimulate the growth of burst-forming unit–megakaryocytes (BFU-MKs) and colony-forming unit megakaryocytes (BFU-MKs); IL-3 acts synergistically with GM-CSF to stimulate BFU-MK growth. Therefore, the action of IL-3 occurs primarily on the proliferation of immature cells (the early phase of megakaryocytopoiesis).

IL-6 acts primarily on megakaryocytes at more advanced stages of maturity—a process that furthers their maturation. It does so by enhancing the diameter, ploidy, acetylcholinesterase activity, and protein synthesis of megakaryocytes.[2] Synergism with CSF (IL-3) is required for IL-6 to effect an increase in the number of megakaryocyte colonies.[4] In vivo, GM-CSF enhances proliferation of megakaryocytes, but not platelets. However, GM-CSF does not appear to act independently, but rather synergistically with other cytokines, including IL-3, IL-6, IL-11, thrombopoietin, and stem-cell factor. Stem-cell factor acts synergistically with IL-3, GM-CSF, and IL-6 to stimulate megakaryocytopoiesis. The action of stem-cell factor appears to be restricted to the proliferation of megakaryocyte progenitors. Thrombopoietin is a humoral-growth factor that directly promotes proliferation, growth, and maturation of megakaryocytes.[6]

Role of IL-11

IL-11 is a pleiotropic cytokine that is expressed in vivo in a variety of tissues, including the brain (specifically in hippocampal neurons), spinal cord, thymus, spleen, bone (osteoblasts, osteoclasts), bone marrow (megakaryocytes), heart, lung (mucosal epithelial cells), connective tissues (chondrocytes, synoviocytes), small and large intestines (mucosal epithelial cells), kidney, testes (spermatogonia), and uterus (endometrial tissues).[5,7,8] The gene expression and secretion of IL-11 is stimulated by interleukin-1a (IL-1a), transforming-growth factor (TGF)-b1 and TGF-b2, and low platelet count.[5]

The activity of IL-11 was initially identified in an immortalized primate bone marrow stromal cell line, called PU-34. IL-11 mediates its effects on bone marrow megakaryocytes through direct association between its unique IL-11 receptor and glycoprotein (gp)130. Once activated, the gp130 molecule transmits a signal to the target cell.[7] Receptors for IL-6 (as well as oncostatin M, leukemia-inhibitory factor, and ciliary-neurotrophic factor) also use the gp130–signal-transduction pathway, which may be a shared mechanism through which IL-11 and IL-6 enhance megakaryocyte proliferation.[7,9]

Mediation of thrombopoiesis by thrombopoietin via its receptor, cMpl (which does not utilize the gp130 signal transduction pathway), suggests the involvement of additional pathways (in addition to the pathway for IL-11) in the regulation of megakaryocytopoiesis.[10] IL-11 and thrombopoietin have been shown to act synergistically to stimulate megakaryocyte colony growth and polyploidization in vitro.[11] These data suggest mutual augmentation, but not interdependence (as discussed later)[8] of these two different pathways in promoting megakaryocytopoiesis. However, the clinical relevance of these in vitro findings is unknown.

The human IL-11 complementary DNA was cloned from a human, fetal lung-fibroblast cell line.[12] rhIL-11 is a protein produced in Escherichia coli by recombinant DNA methods. It differs from naturally occurring IL-11 sheerly by the absence of the amino-terminal proline residue. The following is a discussion of the preclinical pharmacologic profile of rhIL-11.

Megakaryocytopoietic Effects of rhIL-11

In Vitro Studies

Collectively, data from in vitro studies indicate that rhIL-11 promotes the proliferation, differentiation, and maturation of megakaryocytes and exerts a direct stimulatory action on cells at all phases of megakaryocytopoiesis—from primitive progenitors to mature megakaryocytes. In these studies, the effects of rhIL-11, itself a multifunctional cytokine, appeared to result from a synergistic action with other cytokines, primarily IL-3[8,13-14] and/or stem-cell factor,[8,13] rather than an independent effect.[8,14] This may reflect a complex interaction between endogenous cytokines that takes place within the bone marrow microenvironment in vivo.

rhIL-11 synergizes in vitro with IL-3 or stem-cell factor to stimulate primitive, multilineage hematopoietic progenitor cells and the proliferation of lineage-committed myeloid and erythroid progenitor cells from murine bone marrow.[13] In combination with IL-3 or stem-cell factor, rhIL-11 has been shown to stimulate a marked proliferative response in primitive, pluripotential progenitors (pre-CFCmulti)—a response that resulted in a tenfold increase in the number of multilineage (composed of erythroid, megakaryocytic, and other myeloid cells) hematopoietic, progenitor cells (CFCmulti). This response was dependent on the presence of IL-3, in whose presence rhIL-11 stimulated an increase in the number of pure erythroid BFU–erythroid (E)-derived colonies (regardless of the presence of erythropoietin(Drug information on erythropoietin)) and also supported the maturation of late-stage erythroid progenitors (CFU-E).

Additionally, in studies in mouse or human bone marrow cells, rhIL-11 and IL-3 synergistically produced increases in the number and size of megakaryocyte colonies and in megakaryocyte ploidy.[14,15] By contrast, in cultured, human T–cell-depleted bone marrow mononuclear cells, rhIL-11 alone failed to stimulate the growth of megakaryocyte colonies. Yet, in the presence of IL-3, rhIL-11 produced a synergistic increase in the number and size of the megakaryocyte colonies.[14] This same response was observed in highly purified bone marrow CD34+ cells (ie, early progenitor cells).

The in vitro megakaryocytopoietic effects of rhIL-11 in human (and murine) bone marrow are not mediated through (or dependent on) input from thrombopoietin.[8] In human bone marrow cultures, the addition of an antibody to neutralize thrombopoietin activity did not prevent the synergistic stimulation by rhIL-11 in combination with IL-3 or SCF of the formation of colonies that were derived from BFU-MK (P < .01 for IL-3; P < .02 for SCF) and CFU-MK (P < .001 for IL-3; P < .05 for SCF) or the generation of CD41+ (megakaryocytes) from CD34+ cells in liquid cultures containing rhIL-11, with or without IL-3.[8] However, rhIL-11 can act synergistically with thrombopoietin to promote megakaryocyte colony formation from murine bone-marrow–derived mononuclear cells.

An important observation of this study was the expression of the IL-11 receptor in human bone–marrow–derived CD41+ CD14- cells and enhancement by rhIL-11 of phosphorylation of gp130 (the receptor signaling subunit) and STAT3 (a transcription factor that is part of the IL-11 signaling pathway[5] in human bone marrow megakaryocytes). No IL-11 receptors were detected on platelets. These combined data support a direct action of rhIL-11 on human megakaryocytes.

In Vivo Studies

rhIL-11 Promotes Differentiation and Proliferation of Progenitor Cells

rhIL-11 (in conjunction with other endogenous-growth factors) has the ability to promote the proliferation of primitive progenitor cells by provoking progenitor cells to leave the quiescent G0 stage and enter the active G1/S-phase of the cell cycle, thereby shortening cell-cycle time. Injection of mice with rhIL-11 resulted in a dose-dependent increase in absolute numbers of femoral bone marrow progenitor cells, including multipotential progenitor cells (CFU-GEMM [colony-forming unit–granulocyte-erythroid-macrophage-megakaryocyte]), CFU-BM (bone marrow), and BFU-E, that reached statistical significance at the 4- and 8-mg doses (P < .05 vs vehicle-treated controls).[16] A time-sequence study showed that rhIL-11 (8 mg) also increased the cycling rates of these progenitors, resulting in a significantly (P < .005) higher percentage of progenitors in S-phase compared with controls.

rhIL-11 Increases Platelet Count by Promoting Maturation of Megakaryocytes

Injection of rhIL-11 intraperitoneally for 5 days into healthy mice resulted in an increase in the peripheral platelet count,[17] thereby confirming in vitro evidence of the maturational effects of rhIL-11 on megakaryocytes.[14,15] In normal and splenectomized mice, increases in peripheral platelet count induced by treatment with rhIL-11 for 7 days was shown to correspond with an increase in megakaryocyte progenitors and in endoreduplication of bone marrow megakaryocytes (maturational effects).[18] Peripheral platelet counts were increased by 30% to 40%, and these increases were maintained throughout a 14-day dosing period.

In the latter study, increased endoreduplication was reflected by statistically significant increases (P < .01) in the percentage of megakaryocytes with a modal ploidy of 32N in normal and splenectomized mice on days 3 (28%) and 7 (26%) of treatment with rhIL-11, compared with the percentage in saline-treated control mice (day 3, 13%; day 7, 16%).[18] At baseline, 10% to 15% of megakaryocytes in the study population were at 32N. This shift in ploidy distribution was accompanied by corresponding decreases from baseline (15% to 20%) in the percentage of 8N megakaryocytes (9% in rhIL-11–treated mice vs 17% in controls). A similar shift in ploidy distribution was seen in splenectomized mice with similar relative increases in 32N megakaryocytes on days 3 and 7 of treatment.

With respect to the effect of rhIL-11 on megakaryocyte progenitors, bone marrow assay showed a threefold increase in megakaryocyte–colony-forming cells (MK-CFC) in normal mice on day 7 of rhIL-11 treatment, and a twofold increase in splenectomized mice on days 3 and 7 of treatment—relative to MK-CFC levels in controls.[18] No significant changes in red blood cell or white blood cell counts or white blood cell differential were observed, thereby suggesting that the predominant in vivo hematopoietic effect of rhIL-11 on normal bone marrow is stimulation of megakaryocytopoiesis and thrombopoiesis.

This study demonstrates that the administration of rhIL-11 alone stimulates early and later stages of megakaryocytopoiesis to produce an increase in platelet count—a process that requires several days to manifest itself.[18] The clinical implication is that to derive a maximum effect following the myelotoxic insult of chemotherapy, the timing of rhIL-11 administration must take into account the kinetics of the megakaryocytopoietic process. Additional data supporting the early postchemotherapy administration of rhIL-11 in clinical practice are provided below.

The study data indicated that splenic megakaryocytopoiesis does not contribute to the increase in peripheral platelet count induced by rhIL-11 treatment in vivo. However, observation of a more rapid decline in peripheral platelets to control levels in splenectomized mice compared with normal mice (day 10 vs day 15) suggests that following completion of rhIL-11 dosing, splenic megakaryocytopoiesis may help sustain the rhIL-11-induced platelet response.[18]

The maturational effect of rhIL-11 on megakaryocytopoiesis has also been demonstrated in larger animals. In normal dogs, subcutaneous injections of rhIL-11 at doses of 30, 60, 120, and 240 mg/kg/d for 14 days resulted in increases in posttreatment peripheral platelet counts by magnitudes ranging from 1.4- to 3.1-fold, compared with pretreatment levels (P = .01 vs baseline for peak platelet increases at doses of 120 and 240 mg/kg/d).[19] rhIL-11 treatment was associated with an increase in the proportion of megakaryocytes with a ploidy number of 32N/64N on day 7 and on day 14 of treatment compared with megakaryocytes from control dogs (P £ .01 for days 7 and 14). Peripheral blood neutrophil, total white blood cell or differential counts were not affected by rhIL-11 treatment. These observations in dogs confirm the prominent megakaryocytopoietic effects of rhIL-11 seen earlier in mice.[18]

In normal, nonhuman primates (cynomolgus monkeys), continuous intravenous infusion of rhIL-11 at doses of either 10, 30, or 100 mg/kg/d for 7 days produced a dose-dependent increase in the platelet count that was apparent on day 8 and peaked on days 12 to 14 (Figure 2).[20] The extent of the increases in platelet count from baseline ranged from 90% to 162%. Administration via the subcutaneous route (60 and 100 mg/kg/d for 7 days) resulted in similar platelet response profiles. The peak increase in peripheral platelet count was preceded by a peak increase in reticulated platelets, thereby confirming a maturational thrombopoietic effect.

This study also showed a trend toward improved platelet response with longer duration of treatment. By increasing the treatment duration (60 mg/kg/d subcutaneously) to 14 days, higher peak platelet counts were achieved. This platelet increase was also sustained for a longer period compared with the response achieved with shorter durations of treatment (4 or 7 days) (Figure 3). These data are consistent with the recommendation of a 10- to 21-day dosing period for rhIL-11 in humans (or until the post-nadir platelet count is ³ 50,000/µL for 2 consecutive days).

Bone marrow megakaryocyte counts taken from nonhuman primates treated with intravenous rhIL-11 100 mg/kg/d were significantly greater than those from untreated controls (P < .01). These megakaryocytes were also ultrastructurally normal. Maturation of normal megakaryocytes was evidenced by microscopic visualization of landmarks of normal maturation, including segmented, nuclei-containing, condensed chromatin; highly developed demarcation membrane systems; and mature granule formation.[20] In this species, platelets produced by treatment with rhIL-11 have also been shown to have a normal lifespan and to be structurally and functionally normal.[21]

The capacity of rhIL-11 to increase the megakaryocyte population in human bone marrow (when used for the FDA-approved indication of prophylaxis of chemotherapy-induced thrombocytopenia and in accordance with the recommended dose regimen) has been demonstrated in an immunohistologic and morphological study of bone marrow specimens. The specimens were taken from rhIL-11-treated women with advanced breast cancer who had no apparent bone marrow involvement.[22] Subcutaneous administration of rhIL-11 at doses of 50 or 75 mg/kg/d for 14 consecutive days during a prechemotherapy period resulted in a statistically significant twofold increase in the frequencies (numbers) of morphologically identified megakaryocytes (from 0.5 ± 0.1% to 1.0 ± 0.3%; P < .001), and a dose-related increase in peripheral blood-platelet counts. Increases in proliferation of marrow cells (P < .01) and ploidy of megakaryocytes (P < .012) were also observed. At the 75 mg/kg/d dose, rhIL-11 therapy induced a three- and tenfold increase in the number of CFU-MK progenitor cells in two patients, respectively. These data confirm that the in vivo megakaryocytopoietic effects of rhIL-11 shown in nonhuman species also occur in human patients with cancer.