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

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

ABSTRACT: Preclinical studies have shown that rhIL-11, also known as oprelvekin (Neumega), stimulates early and later stages of megakaryocytopoiesis (including proliferation and differentiation of megakaryocyte precursors and maturation of megakaryocytes), to produce an increase in peripheral platelet count. Because of these effects, rhIL-11 must be administered to patients with cancer sufficiently in advance of the platelet nadir (within 6 to 24 hours postchemotherapy) to allow adequate time for megakaryocyte maturation and platelet formation. Therefore, the maximum platelet response coincides with the time when the platelet nadir would normally be experienced. In myelosuppressed, nonhuman primates, optimal platelet response occurred following 14 days of treatment at a dose equivalent to the 50-µg/kg daily dose recommended in humans; lower doses and shorter durations were less effective. These data support the current dosing recommendation in humans, which states that rhIL-11 dosing continues until platelet recovery to ³ 50,000/µL has been achieved for 2 consecutive days or for a total of 10 to 21 days in each cycle. The nonhematopoietic effects of rhIL-11 include a renal effect, resulting in plasma-volume expansion, as well as potential beneficial clinical effects in damaged or inflamed intestinal mucosa, including potential mitigation of mucositis and a rationale for future studies in inflammatory bowel disease. [ONCOLOGY 14(Suppl 8):12-20, 2000]

Introduction

Recombinant human interleukin-11 (rhIL-11), also known as 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 [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) 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.

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