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Next Article 
Blood, Vol. 92 No. 12 (December 15), 1998:
pp. 4491-4508
REVIEW ARTICLE
Emerging Applications of Recombinant Human Granulocyte-Macrophage
Colony-Stimulating Factor
By
James O. Armitage
From the Department of Internal Medicine, University of Nebraska
Medical Center, Omaha, NE.
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INTRODUCTION |
CLINICAL INDICATIONS for use of
recombinant human granulocyte-macrophage colony-stimulating factor
(rHuGM-CSF) have expanded considerably since the drug first became
available in the early 1990s for acceleration of myeloid engraftment in
neutropenic patients. Initial clinical trials of rHuGM-CSF were based
on prevailing knowledge of the biologic effects of endogenous GM-CSF at
the time and therefore concentrated on the drug's myeloproliferative effects in myelosuppressed patients. As additional information accumulated from in vitro research and from results of clinical trials,
it became apparent that rHuGM-CSF had diverse biologic effects and
played a vital role in various functions of the immune system,
including responses to inflammation and infection, as well as in
hematopoiesis. Consequently, a variety of potential clinical uses for
rHuGM-CSF are under investigation, such as prophylaxis or adjunctive
treatment of infection in high-risk settings or immunosuppressed
patient populations, use as a vaccine adjuvant, and use as
immunotherapy for malignancies.
The molecular sequence of endogenous human GM-CSF was first identified
in 1985; within a few years, three different synthetic human GM-CSFs
were produced using recombinant DNA technology and bacterial,1 mammalian,2 and yeast expression
systems.3 Sargramostim is yeast-derived rHuGM-CSF produced
using Saccharomyces cerevisiae; bacterially derived rHuGM-CSF
is produced using Escherichia coli and is termed molgramostim;
and mammalian-derived rHuGM-CSF is produced using Chinese hamster ovary
cells (CHO) and is termed regramostim. These preparations are not
identical and are differentiated by their specific amino acid sequences
and degree of glycosylation.1-3 Sargramostim has an amino
acid sequence identical to that of endogenous human GM-CSF, except that
it contains leucine instead of proline at position 23 and may have a
different carbohydrate moiety. Sargramostim is glycosylated to a lesser
extent than regramostim, and molgramostim is not glycosylated. The
degree of glycosylation of rHuGM-CSF may be an important
characteristic, because it can affect pharmacokinetics, biologic
activity, antigenicity, and toxicity.4-7
This review discusses current knowledge concerning the biologic
effects, pharmacokinetics, and emerging clinical uses of rHuGM-CSF, with a focus on the yeast-derived rHuGM-CSF, sargramostim, the only
form of synthetic rHuGM-CSF commercially available in the United
States. Information on molgramostim, the form of rHuGM-CSF available in
Europe, is also provided. Because literature reports do not always
indicate the form of rHuGM-CSF used, the term "rHuGM-CSF" is used
throughout this review to describe the drug when the expression system
was not identified or when multiple studies using different forms of
rHuGM-CSF reported similar findings.
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PHARMACOLOGY OF SARGRAMOSTIM |
Biologic effects.
GM-CSF was first identified based on its ability to stimulate the
clonal proliferation of myeloid precursors in vitro.8 Endogenous GM-CSF, a heavily glycosylated polypeptide, was the first
human myeloid hematopoietic growth factor to be molecularly cloned,
which allowed the expression of large quantities of the protein. More
than a decade of in vitro and in vivo research using murine GM-CSF and
synthetic rHuGM-CSFs has shown that the name of this CSF is
restrictive, because it describes only one aspect of the numerous
biologic effects that have now been attributed to GM-CSF. Although
GM-CSF plays a vital role in hematopoiesis by inducing the growth of
several different cell lineages, it also enhances numerous functional
activities of mature effector cells involved in antigen presentation
and cell-mediated immunity, including neutrophils, monocytes,
macrophages, and dendritic cells.9-20
The biologic effects of GM-CSF are mediated via binding to receptors
expressed on the surface of target cells. The GM-CSF receptor is
expressed on granulocyte, erythrocyte, megakaryocyte, and macrophage
progenitor cells as well as mature neutrophils, monocytes, macrophages,
dendritic cells, plasma cells, certain T lymphocytes, vascular
endothelial cells, uterine cells, and myeloid leukemia
cells.21-27 Molecular cloning studies have shown that the
GM-CSF receptor is composed of two distinct subunits,  and common
 (c; Fig
1).28 The -subunit binds GM-CSF with low affinity. The
c has no detectable binding affinity for GM-CSF on its
own, but forms a heterodimer with the -subunit that has high
affinity for GM-CSF. Whereas the -subunit is unique to the GM-CSF
receptor, c is shared with the receptors for
interleukin-3 (IL-3) and IL-5.29
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| Fig 1.
Schematic representation of the GM-CSF receptor (GMR),
which is composed of two distinct subunits, and . Binding of
rHuGM-CSF to GMR leads to formation of the signaling complex and
activation of a Janus kinase (JAK2). Regulation of gene expression by
JAK2 activates transcription proteins STAT1, STAT3, and STAT5.
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The signal transduction pathways that occur after rHuGM-CSF binds to
the GM-CSF receptor are under evaluation. There appear to be at least
two distinct signaling pathways, each involving a distinct region of
c.30 The first, which leads to induction of
c-myc and activation of DNA replication, involves activation of
a Janus kinase (JAK2) that is physically associated with
c.31 Regulation of gene expression by JAK2
appears to be mediated by production of a DNA-binding complex
containing the signal transducer and activator of transcription (STAT)
proteins STAT1, STAT3, and STAT5.32,33 The second pathway
involves activation of ras34 and mitogen-activated
protein kinases,35 with consequent induction of
c-fos and c-jun, which are genes involved in regulation
of hematopoietic differentiation.31
Pharmacokinetics.
Information regarding the pharmacokinetics of rHuGM-CSF after
intravenous or subcutaneous administration is available from studies in
healthy adults,36 adults with malignancy or myelodysplastic syndrome,6,37-40 and children with recurrent or refractory
solid tumors.41,42 Because evidence exists from animal and
clinical studies that the degree of glycosylation of synthetic
rHuGM-CSFs influences pharmacokinetic parameters,4-6 data
regarding the pharmacokinetics of sargramostim and molgramostim are
presented separately.
Studies have determined that the pharmacokinetics of sargramostim are
similar among healthy individuals and patients.39 The
pharmacokinetics of sargramostim are dependent on the route of
administration. Table 1 compares
pharmacokinetic parameters after intravenous and subcutaneous
administration of sargramostim in healthy adult males.36
Peak serum concentrations are higher after intravenous administration;
however, bioavailability (as determined by the area under the
concentration-versus-time curve) of sargramostim is similar between
administration routes. The elimination of sargramostim occurs
principally by nonrenal mechanisms.39 Serum concentrations
are more prolonged after subcutaneous administration than after
intravenous administration.36,42 The magnitude of the
percentage of increase in absolute neutrophil count with a specific
dose of sargramostim is greater after subcutaneous injection than after
2-hour intravenous infusion.39
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Table 1.
Pharmacokinetic Parameters After Intravenous and
Subcutaneous Administration of Sargramostim in Healthy
Adult Males
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The pharmacokinetics of molgramostim (0.3 to 30 µg/kg) also were
studied after subcutaneous and intravenous
administration.37 Maximum serum concentrations and area
under the concentration-versus-time curve increased with dose for both
routes of adminstration, but appeared larger after intravenous
administration in comparison to the same dose administered
subcutaneously. However, rHuGM-CSF concentrations greater than 1 ng/mL
were maintained longer after subcutaneous administration.
Immunoreactive molgramostim was detected in the urine of patients,
ranging from 0.001% to 0.2% of the injected dose, supporting nonrenal
elimation. The half-life after intravenous adminstration ranged from
0.24 to 1.18 hours; the mean half life was 3.16 hours after
subcutaneous adminstration.
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USE IN ENHANCING HEMATOPOIETIC RECOVERY AFTER CANCER CHEMOTHERAPY
AND BONE MARROW TRANSPLANTATION (BMT) |
rHuGM-CSF is classified as a multilineage CSF because it stimulates the
proliferation and differentiation of hematopoietic progenitor cells of
neutrophil, eosinophil, and monocyte colonies.43 Parenteral
administration of rHuGM-CSF induces a dose-dependent increase in
peripheral blood neutrophil counts.19,44 Sargramostim alters the kinetics of myeloid progenitor cells within the bone marrow,
causing rapid entry of cells into the cell cycle and decreasing the
cell-cycle time by as much as 33%.45 The leukocyte
response to rHuGM-CSF is reflected in peripheral blood
principally as an increase in segmented neutrophils, but also
involves an increase in monocytes and eosinophils.19,46-48
Leukocyte differentials generally demonstrate a shift to the left;
myelocytes, promyelocytes, and myeloblasts may be present. When
rHuGM-CSF is discontinued, leukocyte counts gradually decrease to
pretreatment levels.19,43
The myeloproliferative effects of rHuGM-CSF are also the result of its
interaction with other cytokines. rHuGM-CSF functions in conjunction
with erythropoietin and IL-3 to promote the proliferation and
differentiation of erythroid and megakaryocytic progenitors, respectively.8,49 The addition of thrombopoietin to early acting cytokines, such as rHuGM-CSF, increases the overall in vitro
megakaryocyte expansion compared with thrombopoietin alone and also
generates different subpopulations of CD41+ megakaryocyte
progenitors, with much less coexpression of CD42b and CD34 and slightly
more coexpression of c-kit.50 In addition, the
overall number of CD34+ cells increases approximately
fivefold with the combination of thrombopoietin and early acting
cytokines. A trial in sublethally irradiated nonhuman primates showed
that coadministration of sargramostim and thrombopoietin augmented
megakaryocyte, erythrocyte, and neutrophil recovery compared with
either cytokine alone.51
Enhancing neutrophil proliferation is an important aspect of rHuGM-CSF
function; however, effects of this multilineage growth factor on other
cells of the immune system, including monocytes and macrophages, have
been identified. Administration of rHuGM-CSF not only increases the
number of circulating monocytes, but also increases the function of
monocytes and macrophages, including oxidative metabolism,
cytotoxicity, and Fc-dependent phagocytosis.19,52,53 rHuGM-CSF enhances dendritic cell maturation, proliferation, and migration.20,54,55 In addition, class II major
histocompatibility complex (MHC) expression on macrophages and
dendritic cells is increased by rHuGM-CSF, enhancing the function of
antigen-presenting cells.56
Combined, these effects of rHuGM-CSF not only increase hematopoietic
cell counts, but also enhance immune function. The ability of rHuGM-CSF
to accelerate myeloid recovery and to prevent infection has resulted in
multiple approved indications for sargramostim and molgramostim in
their respective countries. The drugs are used in patients after
autologous BMT (AuBMT), peripheral blood progenitor cell (PBPC)
transplantation, induction therapy for acute myelogenous leukemia
(AML), engraftment delay or failure after BMT, and chemotherapy-induced
neutropenia. These uses are well established and have been recently
reviewed.57-61 Research has expanded in some of these
settings to investigate new uses of rHuGM-CSF, including use in
combination with granulocyte colony-stimulating factor (G-CSF) for PBPC
mobilization, to prime leukemic cells before or during chemotherapy for
AML, and as an adjunct to increase chemotherapy dose intensity.
PBPC mobilization in combination with G-CSF.
There has been increasing interest in combining rHuGM-CSF with other
cytokines, especially G-CSF, as a means of improving mobilization
without having to administer chemotherapy. This is especially true in
the allogeneic transplant setting, where a nontoxic mobilization
regimen that allows for collection of a sufficient number of cells to
promote engraftment in a minimum number of leukaphereses is most
critical. Lane et al62 evaluated the PBPC mobilization
efficacy of G-CSF at 10 µg/kg/d (n = 8), sargramostim at 10 µg/kg/d
(n = 5), or sargramostim plus G-CSF each at 5 µg/kg/d (n = 5) in
normal donors. The median CD34+ cell yield with the
combination regimen and with G-CSF was significantly higher than for
rHuGM-CSF alone (101 × 106, 119 × 106, and 12.6 × 106, respectively;
P < .01 for both comparisons).
An analysis of CD34+ cell subsets showed some interesting
differences between the different mobilization regimens. A higher proportion of cells in the combination regimen were
CD34+CD38 and
CD34+CD38 HLA-DR+
(Table 2). Pluripotent progenitor cells are
characterized as CD34+CD38 and are
likely responsible for long-term hematopoietic reconstitution after
transplantation. These cells can be further subdivided according to the
presence or absence of HLA-DR, with HLA-DR+ cells giving
rise to lymphoid and myeloid precursors.63 The greater
percentage of this subpopulation of cells mobilized by the combination
regimen translated into a higher overall number of
CD34+CD38HLA-DR+ cells in
leukapheresis products than products from subjects mobilized with
either G-CSF or rHuGM-CSF alone (1.41 × 106, 0.36 × 106, and 0.12 × 106,
respectively; P < .05 for all comparisons). Moreover, the
plating efficiency of colony-forming unit-granulocyte-macrophage
(CFU-GM) and burst-forming unit-erythroid (BFU-E) was
higher in cells stimulated by rHuGM-CSF than in those stimulated by
G-CSF. Whether this would correlate with more rapid engraftment is not
known, although the investigators have reported that PBPCs mobilized by
the combination regimen successfully engrafted after allogeneic PBPC
transplantation.64 Ali et al65 also compared
mobilization of PBPCs in normal donors using rHuGM-CSF at 5 µg/kg/d
plus G-CSF at 10 µg/kg/d (n = 15) versus G-CSF at 10 µg/kg/d alone
(n = 35). They found a statistically insignificant increase in
CD34+ cells in the leukapheresis products from donors
mobilized with the combination of cytokines; however, the number of
CD3+ cells in the leukapheresis product was significantly
lower with the combination regimen than with G-CSF alone, ie, 160 versus 328 × 106/kg.
Investigations are ongoing to determine optimal doses and sequence of
administration of the cytokines in combination.66,67 In a
follow-up study to that reported by Lane et al,62 healthy volunteers received either sargramostim at 10 µg/kg/d for 3 or 4 days
followed by G-CSF at 10 µg/kg/d for 2 days.66 In
comparison to the results of single-agent G-CSF for 4 days and
combination rHuGM-CSF and G-CSF for 5 days, sequential administration
failed to demonstrate any differences in the extent of mobilization as measured by CD34+ cells. In addition, the proportion of the
CD38 subset, which contains the more primitive
hemotopoietic cells, was higher with the combination of sargramostim
and G-CSF for 5 days.
Molgramostim also has been studied in combination or in sequence with
G-CSF for mobilization of PBPCs.67 The combination of the
two cytokines resulted in dramatic and sustained increases in the
number of CFU-GM per kilogram collected per harvest, with administration of G-CSF to patients already receiving molgramostim increasing the hematopoietic progenitor cell content nearly 80-fold. A
randomized trial comparing combination therapy versus G-CSF or
molgramostim (10 µg/kg) alone is ongoing. Additional trials are
required to determine the optimal scheduling of cytokine adminstration as well as apheresis scheduling.
Priming effect before or during chemotherapy for AML.
Myeloid leukemic cells and their precursors have GM-CSF receptors, and
there is in vitro evidence that the proliferation and differentiation
of these cells is supported by exposure to rHuGM-CSF.68-71 Thus, recruitment of chemoresistant resting leukemic cells into sensitive phases of the cell cycle by rHuGM-CSF may enhance the antileukemic effect of chemotherapy. The rHuGM-CSF-induced increases in leukemic cells in S phase and intracellular phosphorylation of
cytarabine have been shown to promote drug-induced cell
kill.72 In contrast to enhancing cytarabine cytotoxicity,
Lotem and Sachs73 found that the typical features of
apoptosis were prevented by rHuGM-CSF and G-CSF in a murine leukemic
cell line. The growth factors also inhibited apoptosis induced by
cytarabine, but the overall clonogenic cell reduction was not reduced.
Because contradictory laboratory data exist, it has not been possible
to predict the clinical benefit of cytokine priming before results of
studies in patients with AML.
In a multicenter, randomized trial of 114 patients (17 to 75 years of
age) with newly diagnosed AML, Büchner et al74
compared use of chemotherapy alone with use of chemotherapy
administered in conjunction with sargramostim priming. Sargramostim at
250 µg/m2 was administered once daily by subcutaneous
injection starting 24 hours before chemotherapy and continuing until
neutrophil recovery occurred after the induction courses, consolidation
course, and first two maintenance courses. Overall, 79% of
sargramostim-treated patients and 84% of controls achieved disease
remission; persistent leukemia was observed in 4% and 18% of
patients, respectively. In patients younger than 60 years of age,
complete remissions were achieved in 82% of sargramostim-treated
patients and 73% of controls, with fewer relapses in the
sargramostim-treated patients during the first 6 months (3% and 22%,
respectively).74
Similar studies in patients with newly diagnosed AML receiving
induction chemotherapy have been conducted with
molgramostim.47,75-77 Patients received molgramostim at 250 µg/m2 or 5 µg/kg/d starting either on days 1 to 3 before chemotherapy or with induction chemotherapy. Although a trend
toward benefit in disease-free survival was observed, the use of
rHuGM-CSF during induction therapy of AML does not appear to have a
significant impact on treatment outcome. The use of rHuGM-CSF for
priming remains an intriuging therapeutic approach for AML. Because
negative effects on the course of AML were rarely observed, additional studies are being conducted to determine the benefits and risks of
growth factors administered before or concurrently with chemotherapy regimens in the treatment of AML. However, the definitive results of an
ongoing ECOG trial are awaited before use of rHuGM-CSF for priming
effects can be recommended outside of a clinical trial.
Adjunctive use to increase chemotherapy dose intensity.
Adjunctive use of rHuGM-CSF may allow an increase in the dose intensity
of combination chemotherapy regimens including drugs with a primary
toxicity of myelosuppression; however, the benefits associated with
rHuGM-CSF in patients receiving dose-intensive chemotherapy may be
limited to early courses of therapy, because late-cycle
thrombocytopenia is not prevented.78-83 The ability of
sargramostim to support a multiple-cycle high-dose chemotherapy regimen
was evaluated in a phase III, double-blind, randomized trial of 56 patients with lymphoma or breast cancer. Patients received
submyeloablative doses of cyclophosphamide, etoposide, and cisplatin
(DICEP) and were randomized to receive sargramostim (250 µg/m2) or placebo subcutaneously every 12 hours.83 Sargramostim-treated patients had a significantly
decreased duration of neutropenia after the first course of
chemotherapy in comparison to patients who received placebo (10 v 12 days; P = .01), but the difference did not achieve
statistical significance after the second or third courses.
Sargramostim-treated patients experienced a statistically significant
1.5-day delay in platelet recovery during the second course. There was
no difference between the groups in numbers of hospitalizations for
febrile neutropenia or the incidence of bacteremia, although any
potential difference might have been obscured, because prophylactic
oral ciprofloxacin was administered to all patients during neutropenia.
However, the duration of hospitalization for neutropenic fever was
shorter for sargramostim-treated patients in the first course.
Importantly, the primary endpoint of this study was duration of
neutropenia during the first course of therapy. The study was stopped
because of a significant difference in duration of neutropenia;
therefore, the small number of patients potentially obscures other
clinical benefits of sargramostim therapy.83
The feasibility of EAP (etoposide, doxorubicin, cisplatin) dose
escalation using molgramostim at 10 µg/kg/d starting on day 4 and
continuing unitl recovery of granulocyte count was studied by Ford et
al.78 Intolerable myelosuppression, including grade 4 neutropenia or thrombocytopenia lasting at least 7 days, occurred in 4 of 5 patients receiving escalated doses of the EAP regimen. At the
lowest doses of each agent, 3 of 6 patients had intolerable myelosuppression. The investigators concluded that molgramostim did not
permit dose escalation of EAP.78 In contrast, molgramostim at 5 µg/kg/d allowed dose escalation of 5-fluorouracil (5-FU) with
leucovorin to 425 mg/m2/d, with further 5-FU dose
escalation according to individual tolerance.79
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USE IN INFECTIOUS DISEASE |
Modulation of host defense against bacterial and fungal infections.
There are several mechanisms by which rHuGM-CSF may enhance host
defense mechanisms against bacterial and fungal infection. Exposure of
neutrophils to rHuGM-CSF in vitro and in vivo has been shown to enhance
expression of cell surface adhesion molecules, such as -integrins,
as well as receptors for the Fc portion of IgG, and receptors for
activated complement components.84,85 Other effects of
rHuGM-CSF on neutrophils include enhanced chemotaxis,17 phagocytosis,10 leukotriene B4 synthesis, release of
arachidonic acid,86,87 and superoxide anion
generation.44,88 The upregulation of neutrophil surface
antigens combined with the induction of phagocyte migration and
increased phagocytic activity contribute to a role for rHuGM-CSF in
host defense. Sargramostim also prolongs neutrophil survival from 96 hours to at least 216 hours by preventing apoptosis.89
Finally, sargramostim induces the expression of class II MHC molecules
on neutrophils, which could potentially allow neutrophils to act as
antigen-presenting cells much like B cells, macrophages, and dendritic
cells.90,91
As a result of its multilineage activity, similar functional effects of
rHuGM-CSF have been observed in monocytes and macrophages. Administration of rHuGM-CSF increases the level of expression of a
number of receptors found on macrophages, such as CD11a, CD11b, and
CD11c, that augment adhesion-dependent phenomena and Fc RII (CDw32)
receptors that bind Ig during phagocytosis.92-94 Upregulation of these receptors would be expected to aid the phagocytic ability of macrophages. Additonally, rHuGM-CSF enhances
antibody-dependent cell cytotoxic activity, respiratory burst, and
superoxide anion generation by macrophages and
monocytes.13,17,19,52 Moreover, sargramostim significantly
counteracted dexamethasone-induced inhibition of superoxide anion
release by monocytes, and the fungicidal activity of
dexamethasone-treated monocytes against Aspergillus fumigatus was enhanced (Fig
2).95
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| Fig 2.
(A) Fumigatus hyphal damage induced by elutriated
human monocytes incubated with 500 nmol/L dexamethasone (DEX) alone and
with either 5 ng/mL sargramostim (rHuGM-CSF) or 1.2 ng/mL
interferon- (IFN). Vertical bars denote standard errors of means,
and the number of experiments performed are shown in
parentheses. *P < .05. (Reprinted with
permission.95)
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Substantial evidence exists from in vitro and in vivo studies that
rHuGM-CSF activates and enhances the ability of neutrophils and
macrophages to phagocytize and destroy bacteria and fungi. Enhancement
of the microbicidal activity of neutrophils by rHuGM-CSF was shown in
vitro against Staphylococcus aureus,96,97
Torulopsis glabrata,98 and Candida
albicans.16,88,99 Neutrophils treated with rHuGM-CSF
killed 90% of intracellular C albicans in comparison to 50%
of intracellular yeast cells killed by untreated
neutrophils.99 Similarly, enhancement of the microbicidal
activity of monocytes by rHuGM-CSF was shown in vitro against C
albicans,16 A fumigatus,95,100 Histoplasma capsulatum,101 Cryptococcus
neoformans,102 and Trypanosoma cruzi.103 Functional studies of neutrophils and
monocytes isolated from patients treated with rHuGM-CSF at 250 µg/m2/d indicate that phagocytic and cytotoxic activity
against S aureus is increased.97,104 The percentage
of S aureus phagocytosed or killed after 20 minutes
significantly increased from 62% before rHuGM-CSF treatment to 72%
during treatment (P = .0028).97
Sargramostim also promotes killing of Mycobacterium avium
complex.105-108 Significant growth inhibition of
Mycobacterium avium complex was observed in human macrophages
treated with sargramostim or tumor necrosis factor (TNF;
Table 3).108 A similar effect was observed using a mouse model of disseminated Mycobacterium avium complex infection. Significantly (P = .04) lower
concentrations of Mycobacterium avium complex were present in
the liver and spleen of mice treated with sargramostim for 14 days
compared with control mice.105 These data also suggest that
sargramostim enhances the antimycobacterial effect of clarithromycin,
azithromycin, amikacin, and ofloxacin.105,107
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Table 3.
Growth Inhibition of Mycobacterium Avium
Complex in Human Macrophages Treated With Sargramostim or
TNF108
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In other animal studies, enhancement of microbicidal activity by
rHuGM-CSF has been confirmed. Survival was significantly (P < .05) improved in neonatal rats when sargramostim was administered 6 hours before inoculation of a lethal dose of S
aureus.109 Similarly, in neutropenic mice,
administration of molgramostim 1 to 5 µg/d protected against lethal
infections of S aureus and Pseudomonas aeruginosa;
survival was significantly increased in molgramostim-treated mice
infected with either S aureus (70% v 20%,
P < .05) or P aeruginosa (50% v 0%, P
< .01).110
Recombinant murine GM-CSF protected 62% of neutropenic rats from a
lethal inoculum of C albicans and reduced lung injury. Importantly, there was no effect of murine GM-CSF on the neutrophil count, suggesting that the protective mechanism involved led to enhanced host defense mechanisms.111
Adjunctive treatment of fungal infections.
The effect of sargramostim on the incidence and severity of fungal
infections was observed in randomized, double-blind studies of the drug
in patients undergoing AuBMT and in patients with AML.112,113 Fungal infections developed in 4 AuBMT patients
who received placebo in comparison to 2 AuBMT patients who received sargramostim.112 Two of the infections in placebo-treated
patients were disseminated aspergillosis. In the phase III ECOG trial
of 99 elderly patients undergoing chemotherapy for AML, sargramostim significantly (P = .02) reduced mortality due to fungal
infection.113 One of 8 patients who received sargramostim
died as a result of fungal infection, whereas 9 of 12 placebo-treated
patients developed fatal fungal infections.
A pilot study of molgramostim as adjuvant therapy for fungal infections
was conducted in cancer patients with proven major-organ or
disseminated fungal infection.114 Of 8 evaluable patients, 6 had a neutrophil response to molgramostim; 4 of these patients were
completed cured of the fungal infection and the other 2 had a partial
response. Several case series have reported a response to adjunctive
treatment with sargramostim for fungal infections. Three human
immunodeficiency virus (HIV)-infected patients with oropharyngeal
candidiasis refractory to fluconazole at doses of 200 mg daily or
greater for at least 14 days were treated with sargramostim at 125 µg/m2/d.115 Fluconazole treatment was
maintained at the same dose. All patients experienced improvement in
signs and symptoms of oropharyngeal candidiasis by week 2. No
significant adverse events occurred, including no upregulation of HIV-1
replication, and therapy was well tolerated. When sargramostim was
discontinued, 2 of 3 patients relapsed.115
Sargramostim also has been administered to patients with rhinocerebral
and disseminated mucormycosis, a rare opportunistic infection
associated with a mortality rate exceeding 50%.116 Three
of four patients with mucormycosis have been successfully treated with
sargramostim (doses ranging from 250 to 500 µg/d for 14 days to 6 months) in combination with traditional surgical and medical treatment.
The three patients were disease-free at periods of 6 months, 18 months,
and 3 years after surgery.
HIV infection.
Initial use of sargramostim in patients with HIV infection focused on
its ability to ameliorate drug-induced
myelosuppression.117-119 In a phase I/II study in patients
with Kaposi's sarcoma who became neutropenic while receiving
zidovudine and interferon , administration of sargramostim resulted
in a prompt increase in absolute neutrophil count in all patients and
an absolute neutrophil count greater than 1,000 cells/µL within 7 days; there was no increase in p24 antigen levels.118
Sargramostim (250 or 500 µg/m2/d administered by
subcutaneous injection) also has been used to ameliorate
chemotherapy-induced neutropenia in patients with acquired
immunodeficiency syndrome (AIDS)-related Kaposi's sarcoma receiving a
regimen of doxorubicin, bleomycin, and vincristine (ABV).117 Although both sargramostim doses allowed the
chemotherapy regimen to be continued without a dose reduction, the
lower sargramostim dose was better tolerated. In a recent study in 12 patients with advanced HIV infection (CD4+ cell count
 200/µL) who were receiving zidovudine (300 to 1,200 mg/d),
administration of sargramostim in a dosage of 50, 125, or 250 µg/m2/d by subcutaneous injection resulted in significant
increases in absolute neutrophil count and monocyte counts at all three dosage levels.119
Potential uses for the drug in HIV patients were expanded when it
became evident that rHuGM-CSF activates and enhances the ability of
neutrophils and macrophages to phagocytize bacteria, fungi, and
intracellular parasites, which has important implications for the
prophylaxis and treatment of opportunistic infections in this patient
population.
Although there were initial concerns that use of rHuGM-CSF in
HIV-infected patients might stimulate HIV replication and increase viral load, these concerns have not been substantiated. In vitro studies are somewhat conflicting regarding the effect of
rHuGM-CSF on HIV replication. Numerous studies have
demonstrated enhancement of viral replication when HIV-infected
monocytes or macrophages are exposed to rHuGM-CSF120-124;
however, three studies have reported suppression of HIV expression by
rHuGM-CSF.125-127 An additional finding from in vitro
studies is that rHuGM-CSF enhances the antiretroviral activity of some
dideoxynucleoside antiretroviral agents, such as zidovudine and
stavudine, possibly by increasing intracellular phosphorylation of
these agents to their active metabolite.124,128,129
Early clinical trials evaluating the effect of rHuGM-CSF on viral
replication in HIV patients failed to show an increase in viral load as
determined by serum p24 antigen levels as long as patients received
concurrent zidovudine117,118,130-132; however, in studies
in which molgramostim was administered without an antiretroviral, some
patients did experience an increase in serum p24 antigen levels.133,134 Subsequent trials using a more sensitive
polymerase chain reaction assay for viral load determination confirmed
that rHuGM-CSF does not result in an increase in viral load during or
after CSF therapy in HIV patients receiving concurrent
zidovudine.119,135 More recently, administration of
sargramostim to patients on stable, highly active antiretroviral
therapy including a protease inhibitor has been shown to result in no
upregulation of viral load by polymerase chain
reaction.136,137 These HIV-positive patients receiving sargramostim have experienced a significant (P = .0372)
increase in CD4 count and decrease in viral load  0.5 log.
Interestingly, the studies by Massari et al126 and Matsuda
et al127 that reported suppression of HIV expression by
rHuGM-CSF were conducted before the role of coreceptors for HIV
infection was appreciated. Indeed, the investigators examined the
effects of rHuGM-CSF on CD4 expression as a potential mechanism for the reduction in HIV expression, but found CD4 expression to be unchanged. A recent study provides a possible mechanism for their findings. Exposure to sargramostim has recently been shown to downregulate expression of the C-C chemokine receptor CCR5, a -chemokine receptor on macrophages, and to reduce the susceptibility of macrophages to
infection by a macrophage-tropic strain of HIV.125,138 CCR5 has been shown to be the major coreceptor required for infection of
macrophages by macrophage-tropic strains of HIV.
Sargramostim-stimulated monocytes produced high levels of
-chemokines, macrophage inflammatory protein-1 (MIP-1), and
MIP-1 in the medium. This medium was able to protect bystander cells
from entry by JRFL, a macrophage-tropic strain of HIV.
Another possible role for sargramostim in HIV-infected patients is in
the prevention or treatment of opportunistic infections. Based on
results of in vitro studies demonstrating that rHuGM-CSF promotes
killing of Mycobacterium
avium-intracellulare105,107 and in vitro and murine
studies indicating that rHuGM-CSF can enhance the antimycobacterial
effects of some antimicrobial agents, including azithromycin,
ofloxacin, and clarithromycin,105,107 investigations of
sargramostim for the adjunctive treatment of Mycobacterium
avium-intracellulare were initiated. In a small study, AIDS
patients with disseminated Mycobacterium avium-intracellulare were randomized to receive azithromycin with or without sargramostim for 6 weeks.139 Mycobacteremia and monocyte function were
assessed biweekly. Mean superoxide anion production was significantly
increased in monocytes obtained from all 4 patients receiving
sargramostim (53% to 199% relative to controls) and these patients
had a 60% reduction in the number of viable intracellular
Mycobacterium avium-intracellulare per milliliter at the end of
treatment. Patients receiving azithromycin alone had no increase in
superoxide anion production and only a 28% reduction in viable
Mycobacterium avium-intracellulare per milliliter. These data
indicate that sargramostim activates monocytes in AIDS patients with
Mycobacterium avium-intracellulare bacteremia and deserves
further study as adjunctive therapy in these patients.
A multicenter phase III randomized, double-blind, placebo-controlled
trial of sargramostim in patients with advanced HIV disease is ongoing
to compare the incidence and time to first opportunistic infection or
death. Patients with CD4 count 50 cells/µL and receiving a stable
antiretroviral regimen before and during the study are eligible.
Patients are randomized to receive either sargramostim 250 µg/d 3 days per week for a minimum of 24 weeks or placebo. Secondary
objectives include incidence of AIDS-related opportunistic malignancies
and esophageal candidiasis; survival; pharmacoeconomic and
quality-of-life parameters; changes in HIV viral load or
CD4+ lymphocyte counts; incidence, degree, and duration of
neutropenia; and concurrent use of open-label cytokines.
| |
USE AS A VACCINE ADJUVANT |
rHuGM-CSF is the principal mediator of proliferation, maturation, and
migration of dendritic cells, important antigen-presenting cells that
play a major role in the induction of primary and secondary T-cell
immune responses.14,20,140 Dendritic cells display antigens on their surface in conjunction with class II major histocompatibility complex (MHC). rHuGM-CSF also increases class II MHC
expression.12 Once presented, the antigen can be recognized
by helper CD4+ T cells,141 which provide
support for the development of B cells and cytotoxic CD8+ T
cells. By augmenting antigen presentation to lymphocytes by dendritic
cells, rHuGM-CSF stimulates T-cell immune responses.12,14 rHuGM-CSF has been demonstrated to augment the primary in vitro immune
response to sheep red blood cells by murine spleen
cells.142 rHuGM-CSF also is important to the immune
response to vaccination, because it enhances expression of
costimulatory molecules such as B7 and adhesion molecules (eg,
intercellular adhesion molecule [ICAM]) that are
necessary for the interaction of antigen-presenting cells with T cells;
it also enhances production of other cytokines such as IL-1, TNF, and
IL-6, which promote expansion and differentiation of B and T
lymphocytes. In addition, rHuGM-CSF primes T cells for IL-2-induced
proliferation143 and augments lymphokine-activated killer
(LAK) cell generation in conjunction with IL-2.15,144 The
important role of rHuGM-CSF in the maturation and function of
antigen-presenting cells, such as dendritic cells and macrophages, as
well as its ability to affect T-cell immunity, provides the basis for
its potential evaluation as a vaccine adjuvant in new immunotherapy
strategies for infectious diseases and cancer.
Local injection of rHuGM-CSF would be expected to enhance vaccine
immunogenicity and would likely be well tolerated based on clinical
experience in other uses. Disis et al145 evaluated the use
of sargramostim as an adjuvant for protein- and peptide-based vaccines
in rats. Tetanus toxoid was used as the foreign antigen system, and
peptides derived from a self antigen, rat neu protein, were
used as the tumor antigen system. A series of initial experiments demonstrated that intradermal injections of sargramostim every 24 hours
for a total of five inoculations increased the number of class II
MHC+ cells in regional lymph nodes that peaked at the
fourth inoculation, whereas subcutaneous injections of sargramostim on
the same schedule increased these cells with a peak after the second
inoculation. This conditioning schema was then used, with tetanus
toxoid administered at the beginning or end of the immunization cycle.
Intradermal immunization was more effective than subcutaneous
immunization in eliciting specific immunity to the tetanus toxoid
antigen. In addition, intradermal injection of sargramostim as a single dose with antigen was similarly effective in eliciting specific antibody and cellular immunity as the use of Freund's adjuvant or alum
(Fig 3). Inoculation with rat neu
peptides and sargramostim elicited a strong delayed-type
hypersensitivity response, whereas the peptides alone were
nonimmunogenic. Sargramostim was as effective as Freund's adjuvant in
generating rat neu-specific delayed-type hypersensitivity
responses after immunization with the peptide-based vaccine. These
studies demonstrated that sargramostim was an effective adjuvant for
elicitation of immunity to both antigen systems, comparing favorably
with other standard adjuvants.
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| Fig 3.
rHuGM-CSF, as an adjuvant, elicits delayed-type
hypersensitivity (DTH) responses to tetanus toxoid (tt) similar to
those seen in animals immunized with a standard adjuvant. Rats were
injected with Freund's adjuvant (CFA) subcutaneously (sq), alum sq,
rHuGM-CSF intradermally (id) or sq (5 µg), and
phosphate-buffered saline (PBS) sq with tt at a concentration of 3 limit flocculation (Lf ) units. Immunizations were administered on 1 day only with no repeated administration of rHuGM-CSF. Six rats were
included in each experimental group. Figure represents data collected
from two separate experiments. Twenty days after immunization, a DTH
response was measured in the immunized animals. Antigen was
applied to rat ear and responses measured at 48 hours. Ear swelling of
experimental compared with control ear was measured. Results are shown
as the mean and standard deviation of measurements taken
from each experimental group. (Reprinted with
permission.145)
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Results of several preliminary studies using molgramostim in
conjunction with hepatitis B141,146 and tetravalent
influenzae virus vaccine147 suggest that rHuGM-CSF may have
a potential role as an antiviral vaccine adjuvant; however, further
evaluation is needed in this setting. Its evaluation as an adjuvant to
vaccines and other immunotherapies for tumors is promising and is
discussed in the subsequent section.
| |
USE IN ANTITUMOR THERAPY |
Antitumor effects.
The functional effects of granulocytes, lymphocytes, and macrophages
are important in patients with malignancies because of the ability of
these cells to exhibit antitumor activity. In vitro, rHuGM-CSF has been
shown to slightly enhance the cytotoxic activity of peripheral blood
monocytes and lymphocytes and markedly increase antibody-dependent
cellular cytotoxicity148 and to enhance monocyte cytotoxicity against a malignant melanoma cell line.149
rHuGM-CSF has also been shown to augment the cytotoxic
activity of peripheral blood monocytes in antibody-dependent cellular
cytotoxicity against numerous human tumor cells in the presence of
various monoclonal antibodies150 and to enhance
IL-2-mediated LAK cell function.151,152 In
tumor-infiltrating macrophages, it also increases secretion of matrix
metalloelastase with subsequent production of angiostatin, which
inhibits angiogenesis and suppresses the growth of lung metastases.153 rHuGM-CSF may also enhance the
immunogenicity of tumor cells through facilitation of tumor antigen
presentation.56 In a comparative study in mice, the most
potent stimulator of specific antitumor immunity was tumor cells
engineered to secrete GM-CSF.154 Also, as previously noted,
sargramostim has been shown in rats to be an excellent adjuvant for
generation of immune responses to tumor antigen-derived
peptides.145 Thus, rHuGM-CSF might enhance functions of
cells critical for immune activation against tumor cells, alone or with
other cytokines or monoclonal antibodies, making it potentially useful
in the biotherapy of malignant diseases.
In a phase I study in patients with cancer, administration of
sargramostim enhanced monocyte antibody-dependent cellular cytotoxicity (Fig 4) and increased secretion of both
TNF and interferon.19 In another study in patients with
metastatic solid tumors, sargramostim was administered once daily for
14 days every 28 days; monocyte cytotoxicity against HT29 tumor cells
was enhanced by the cytokine treatment.46 No clinical
effects on tumor regression were apparent in either study. Sargramostim
is under evaluation in an open-label, phase II trial as surgical
adjuvant therapy in patients with advanced melanoma at very high risk
of recurrence.155 Sargramostim at 125 µg/m2/d
was administered subcutaneously for 14 days every 28 days beginning within 60 days of the last evidence of tumor. Treatment is continued until recurrence or a tumor-free interval of 1 year. An interim analysis of 25 patients demonstrated a significant prolongation of
disease-free survival (P = .04) and survival (P = .02) compared with 50 matched historical control
patients.155 These initial results are encouraging;
long-term follow-up is needed.
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| Fig 4.
Antibody-dependent cellular cytotoxicity (ADCC) of
monocytes after treatment with sargramostim. Monocytes were collected
from patients 2 days after a bolus infection (A) and 3 (B) and 10 (C)
days after the start of a continuous infusion of sargramostim.
Antibody-dependent cellular cytotoxicity activity was measured against
antibody-coated chicken erythrocytes by a
Cr51-release assay. Experimental results were compared
statistically with the average of two baseline assays. (Reprinted with
permission.19)
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A phase Ib trial was conducted in 20 patients with metastatic melanoma
to evaluate the use of sargramostim as an adjuvant to R24, a murine
monoclonal antibody that mediates complement-dependent and
antibody-dependent cellular cytotoxicity of melanoma tumor targets.156 The rationale for this combination was the
hypothesis that upregulation of monocyte and granulocyte
antibody-dependent cellular cytotoxicity induced by sargramostim might
enhance antitumor activity. Sargramostim (150 µg/m2/d
administered by subcutaneous injection for 21 days) was administered alone or in conjunction with R24 (10 or 50 mg/m2
administered by continuous intravenous infusion on days 8 through 15).
Measurement of direct cytotoxicity and antibody-dependent cellular
cytotoxicity indicated that sargramostim enhanced monocyte and
granulocyte cytotoxicity by week 3 in all evaluable
patients.156 Of the 6 patients who received sargramostim
alone, 3 had no response (2 had stable disease) and 3 had disease
progression; in the 14 patients who received sargramostim plus R24, 2 had a partial response, 6 patients had no response (3 had stable
disease), and 6 developed progressive disease.156
The Pediatric Oncology Group performed a phase II study to evaluate the
use of sargramostim to enhance antibody-dependent cellular cytotoxicity
of a chimeric anti-GD2 monoclonal antibody (ch14.18) in the treatment
of recurrent or refractory neuroblastoma.157 Sargramostim
was administered in a dosage of 10 µg/kg daily for 14 days with
5-hour infusions of ch14.18 at 50 mg/m2 daily for 4 days.
Thirty-two patients who had failed to respond to 1 to 4 therapeutic
regimens, including BMT in 18 patients, received 70 courses of
treatment. In 27 patients evaluable for response, there were 1 complete
response, 3 partial responses, 1 mixed response, and 2 stable disease.
When analyzed by site of disease, in 18 patients with marrow disease,
there were 4 complete responses and 1 partial response; in 21 patients
with bone involvement, there were 1 complete response and 2 partial
responses. Two patients with large tumor masses had greater than 60%
reduction in tumor size. Among the responding patients, 4 were alive at
follow-up ranging from 9 to 20 months, whereas those with progressive
disease had a median survival of 3 months. All responding patients had an increase in neutrophil-mediated antibody-dependent cellular cytotoxicity to greater than 20 lytic units, whereas 9 of 12 patients with progressive disease had peak antibody-dependent cellular cytotoxicity activity less than 20 lytic units. These findings were the
basis for a recommendation that a phase III trial in the setting of
minimal residual disease is warranted.157
Adjuvant to tumor vaccines.
Based on enhancement of functional effects on monocytes, macrophages,
and antigen-presenting cells (dendritic cells,
macrophages),15,142 sargramostim has been studied for its
potential to enhance the immune response to antitumor immunotherapies,
including autologous tumor cell vaccines, recombinant peptide tumor
vaccines, and autologous Id-KLH tumor vaccines.
Leong et al158 administered sargramostim at 125 to 250 µg
as an adjuvant to a melanoma vaccine that consisted of irradiated autologous melanoma cells with Bacillus Calmette-Guérin vaccine (BCG vaccine) in 20 stage IV melanoma patients. Patients received multiple cycles that consisted of vaccine plus sargramostim on day 1, with local injection of sargramostim alone in the vaccine site on days
2 to 5; 48 hours before cycles 1, 3, and 4, cyclophosphamide at 300 mg/m2 was administered. Four patients showed partial to
complete responses (20%), 4 had stable disease (20%), and the
remaining 12 patients had disease progression (60%). In the responding
patients, regression of visceral metastases was observed. The results
demonstrated the ability of patients bearing a significant tumor burden
to respond specifically to their autologous melanoma.
Based on the fact that autologous tumor-derived Ig idiotype proteins
(Id) have been shown to induce effective antitumor activity in
experimental models and B-cell lymphoma, a vaccine containing autologous Id-KLH (keyhole limpet hemocyanin, a foreign protein used as
a vaccine adjuvant) conjugates was administered to patients with
multiple myeloma with either rHuGM-CSF or IL-2 as an
adjuvant.159 Results of skin testing with autologous and
unrelated Id were used to assess the specificity of the immune
response; rHuGM-CSF appeared to be a better adjuvant than IL-2 in these
patients.
Immunotherapy for AML.
Future directions in the treatment of AML may include immunotherapy
based on the effect of rHuGM-CSF on T-lymphocyte cytotoxic functions
and surface adhesion proteins. Several preliminary investigations have
been conducted using molgramostim in patients with AML. The effect of
molgramostim at 5 µg/kg/d on activated killer cell activity was
studied in 20 patients with AML undergoing AuBMT.160
Activated killer cell function was investigated before AuBMT, during
rHuGM-CSF therapy, and after withdrawal. The actuarial risk of relapse
was also analyzed and compared with a historical control group of 20 patients transplanted before initiation of this study. Activated killer
cell function was significantly enhanced with rHuGM-CSF (P < .001); during rHuGM-CSF treatment, median activated killer cell
function increased from 1.8% before AutoBMT to 35% and remained increased after withdrawal of rHuGM-CSF (median, 20%). After a median
follow-up of 24 months, the actuarial risk of relapse was 37.4% in
rHuGM-CSF-treated patients compared with 49.5% in controls (P = .05). Additionally, none of the 7 patients with activated killer cell
activity 20% in the first 2 to 5 weeks after AutoBMT have relapsed,
compared with 6 of 9 patients with activated killer cell
activity less than 20% (P < .02).
Exposure of AML cells to rHuGM-CSF upregulates expression of ICAM-1
(CD54) and lymphocyte function associated molecule-3 (LFA-3; CD58), but
does not increase their sensitivity to lysis by IL-2-activated natural
killer cells.161 rHuGM-CSF induces a significantly greater upregulation of ICAM-1 on leukemic CD34+ cells than their
CD34 counterparts. When AML cells are exposed to
rHuGM-CSF before incubation with killer cells, their subsequent
clonogenic activity is significantly reduced. These data suggest that
administration of effector cell activators, such as IL-2, and target
cell modulators, such as rHuGM-CSF, may have therapeutic benefit in
patients with minimal residual myeloid leukemia.
| |
OTHER POTENTIAL USES |
Mucositis, stomatitis, and diarrhea.
Mucositis, stomatitis, and diarrhea are frequent complications of
high-dose chemoradiotherapy. Mucosal epithelial cells in the
gastrointestinal tract are susceptible to direct damage from these
therapies, resulting in dysphagia and decreased oral intake and
potentially leading to airway compromise. Mucosal damage may be further
aggravated by infections or hemorrhage related to myelosuppression. rHuGM-CSF has been shown to stimulate the migration and proliferation of endothelial cells and promote keratinocyte growth, suggesting that
the growth factor has a direct effect on mucosal
cells.162,163 In addition, by decreasing the severity and
duration of neutropenia, rHuGM-CSF may reduce the severity and duration
of mucositis.
Several clinical trials evaluating sargramostim for hematopoietic
support have shown a coincidental benefit of the drug on mucositis. In
addition to enhancing myeloprotection and permitting dose-intensification of chemotherapy, the incidence of mucositis was
reduced in sarcoma patients who received sargramostim after chemotherapy.164 In a phase III placebo-controlled trial of
sargramostim in patients undergoing allogeneic BMT, 8% of
sargramostim-treated patients compared with 29% of placebo-treated
patients developed grade 3 or 4 mucositis (P = .005).165
Based on these results, several prospective trials have been conducted
to evaluate the effect of rHuGM-CSF on mucositis. In a nonrandomized
trial, the effect of sargramostim on oral mucositis was assessed in
pediatric patients undergoing stem cell transplant.166 Children who received thiotepa, etoposide, and total body irradiation followed by sargramostim experienced a significantly shorter duration of mucositis than those children who did not receive sargramostim (12.2 v 20.3 days, P = .02). However, the severity
of mucositis was similar between the groups. In this same study,
patients treated with thiotepa, etoposide, and cyclophosphamide as the
preparative regimen experienced a similar duration and severity of
mucositis regardless of whether they received sargramostim. Although
recovery of neutrophils was faster in sargramostim-treated patients,
there was no correlation between recovery of neutrophils and resolution of mucositis.
A phase I trial of sargramostim as a mucoprotectant was conducted in 10 patients with advanced head and neck cancer who received adjuvant
radiation after surgery or chemotherapy.167 Four patients developed grade 3 or worse mucositis, and the remaining 6 patients had
grade 1 or 2 mucositis. In comparison to 13 historical control patients, grade 3 or worse mucositis was reduced by half from 85% to
40% with sargramostim administration. In a phase I trial of colorectal
cancer patients receiving escalating doses of 5-FU, sargramostim used
in conjunction with leucovorin resulted in decreased rates of diarrhea
relative to historical patients.168
Topical application of molgramostim in the treatment or prevention of
mucositis has also been investigated.169,170 Of 10 </ |
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Introduction
Conclusions