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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 7 (October 1), 1999:
pp. 2252-2258
The Effect of a Metalloproteinase Inhibitor (GI5402) on Tumor Necrosis
Factor- (TNF-) and TNF- Receptors During Human Endotoxemia
By
Pascale E.P. Dekkers,
Fanny N. Lauw,
Tessa ten Hove,
Anje A. te
Velde,
Philip Lumley,
David Becherer,
Sander J.H. van Deventer, and
Tom van der Poll
From the Academic Medical Center, University of Amsterdam, Laboratory
of Experimental Internal Medicine, Amsterdam, the Netherlands;
Department of Clinical Pharmacology, Glaxo Wellcome, Greenford, UK; and
the Department of Biochemistry, Glaxo Wellcome, Research Triangle Park,
NC.
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ABSTRACT |
Tumor necrosis factor- (TNF-) is released from the cell
surface by cleavage of pro-TNF- by metalloproteinases (MPs). In cell cultures, inhibition of MPs has been found not only to reduce the
release of TNF-, but also to enhance the surface expression of
TNF- and TNF- receptors, which might lead to a proinflammatory effect. To determine the effect of MP inhibition during inflammation in
humans, 7 healthy subjects were studied after intravenous injection of
lipopolysaccharide (LPS; 4 ng/kg) preceded ( 20
minutes) by an oral dose of the MP inhibitor GI5402 (100 mg) or
matching placebo. GI5402 strongly reduced LPS-induced TNF- release
(P < .001), but did not influence the increase in
monocyte-bound TNF-. In addition, GI5402 attenuated the rise in
plasma-soluble TNF- receptors types I and II after LPS injection
(both P < .001), but did not change the LPS-induced decreases
in granulocyte and monocyte TNF- receptor expression. These data
suggest that MP inhibitors may be useful as a treatment modality in
diseases in which excessive production of TNF- is considered to play
an important role. Furthermore, unlike in vitro, no evidence has been
found in vivo with MP inhibition for a potential proinflammatory effect
due to increases in membrane-bound TNF- and TNF- receptor number.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
TUMOR NECROSIS FACTOR- (TNF-) has
been implicated as an important mediator in the pathogenesis of a
variety of inflammatory diseases, including septic shock, rheumatoid
arthritis, and Crohn's disease.1 Two forms of TNF-
have been identified, a membrane-bound precursor protein of 26 kD, and
a 17-kD mature secreted form. Both forms can assume a homotrimeric
configuration and are biologically active.2,3 The cleavage
of cell-associated TNF- is mediated by metalloproteinases (MPs),
including TNF--converting enzyme (TACE).4,5 MP
inhibitors have been found to prevent TNF- release in mononuclear
cell cultures in vitro and in mice in vivo, and to protect mice from a
lethal dose of lipopolysaccharide (LPS).6-14 MPs are also
involved in the shedding of other surface molecules, including TNF-
receptors types I and II.9,10,13,15,16
In theory, inhibition of MPs can result in the accumulation of
biologically active pro-TNF- at the cell surface. Indeed, TACE-deficient mouse T cells showed an almost complete abrogation of
TNF- release upon activation, with a simultaneous increase in
surface TNF- expression compared with normal wild-type
cells,4 and treatment of mononuclear cells with different
MP inhibitors similarly enhanced cell-associated TNF-
expression.4,6,8 Of considerable interest, a recent report
has suggested that inhibition of MPs may also render cells more
sensitive to TNF- effects by preventing the shedding of TNF-
receptors from the cell surface, thereby increasing the number of
transmembrane TNF- receptors available for signal
transduction.17 In accordance, TACE-deficient cells failed
to shed the type II TNF- receptor upon stimulation with various
stimuli, including LPS.15 Hence, the therapeutic potential
of MP inhibitors in inflammatory diseases may be hampered by increased
expression of membrane-bound TNF- and TNF- receptors and thus a
potential proinflammatory effect.
Knowledge of the effects of MP inhibitors on the processing of TNF-
and TNF- receptors through in vivo models of inflammation is
limited. In the present study, we sought to determine the effect of a
newly developed MP inhibitor on soluble and cell-bound TNF- and
TNF- receptor levels in healthy humans injected with a single dose
of LPS.
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MATERIALS AND METHODS |
Matrix MP and TACE inhibitory activity of GI5402.
Ki (inhibitory constant) values for GI5402
(Glaxo Wellcome, Greenford, UK) were determined for human 19-kD
truncated collagenase (matrix MP [MMP]-1), 20-kD truncated
collagenase-3 (MMP-13), stromelysin-1 (MMP-3), and 50-kD truncated
gelatinase B (MMP-9) using the fluorogenic substrate,
Dnp-Pro-Cha-Gly-Cys(Me)-His-Ala-Lys (N-methylanthranilic acid)-NH2.18 Assays were conducted in a total
volume of 0.180 mL assay buffer (200 mmol/L NaCl, 50 mmol/L Tris, 5 mmol/L CaCl2, 10 µmol/L ZnSO4, 0.005% Brij
35, pH 7.6) in each well of a black 96-well microtiter plate. 19-kD
collagenase-1, 20-kD collagenase-3, stromelysin-1, and 50-kD gelatinase
B concentrations were adjusted to 500 pmol/L, 30 pmol/L, 5 nmol/L, and
100 pmol/L, respectively. A concentration-effect curve was generated
for GI5402 using an 11-point 3-fold serial dilution with initial
starting concentrations of 100, 10, or 1 µmol/L. Inhibitor and enzyme
were incubated for 30 minutes at room temperature and then the reaction
initiated with addition of 10 µmol/L fluorogenic substrate (above).
The product formation was measured at
EX343/EM450 nm
(excitation/emission) after 45 to 180 minutes (time dependent on MP
studied and hence rate of substrate cleavage) using a Fluostar
SLT fluorescence analyzer (TecanUS Inc, Research
Triangle Park, NC). Under the above conditions, the substrate
concentrations are << than Km and the Ki can be determined directly by plotting the
percentage inhibition versus the log of the inhibitor concentration.
Assays to determine inhibition constants for TACE were run in a 96-well
plate using a biotinylated and tritiated peptide substrate based on the
cleavage sequence surrounding precursor TNF-. A microsomal
preparation of TACE5 was diluted 1/25 into 10 mmol/L HEPES,
pH 7.5, and incubated with a biotinylated and tritiated version of the
TACE substrate SPLAQAVRSSSRTPS-NH2. After 2 hours at room temperature,
the substrate turnover was determined in the presence or absence of
inhibitor using avidin-conjugated scintillation proximity beads.
Percentage inhibition versus log (inhibitor) was plotted to determine
the IC50 (inhibitory concentration, 50%), where Ki = IC50/(1 + [S]/Km).
Depletion of substrate was followed using a scintillation proximity
assay (SPA).5
Whole-blood stimulation.
Whole-blood stimulation was performed as described
previously.19 Briefly, on the first study occasion, and
before dosing, blood was collected aseptically from subjects using a
sterile collecting system consisting of a butterfly needle connected to a syringe (Becton Dickinson, Rutherford, NJ). Anticoagulation was
obtained using sterile heparin (LEO Pharmaceutical, Weesp, the
Netherlands) (final concentration, 10 U/mL blood). Whole blood, diluted
1:1 in sterile RPMI-1640 (GIBCO-BRL, Life Technologies, Grand Island,
NY), was stimulated for 24 hours at 37°C with LPS (final
concentration, 10 ng/mL) with or without increasing concentrations of
GI5402 (30 to 10,000 ng/mL) in sterile polypropylene tubes (Becton
Dickinson). A 24-hour incubation period was chosen, since TNF levels
have reached a plateau by this time.20 For these experiments, polypropylene tubes were prefilled with 0.75 mL RPMI containing the appropriate concentrations of LPS and GI5402, after which 0.75 mL heparinized blood was added. Tubes were then gently mixed
and placed in the incubator. After the incubation plasma was prepared
by centrifugation and stored at 20°C until TNF- assays
were performed.
Human endotoxemia.
The study was approved by the Research and Ethics Committees of the
Academic Medical Center, Amsterdam, the Netherlands, and written
informed consent was obtained from all volunteers before study entry.
The study was designed as a double-blind, randomized, cross-over,
placebo-controlled study in which 7 male volunteers (mean age, 22 years; range, 20 to 25 years; weight range, 74 to 90 kg) were treated
with LPS on 2 occasions, with an interval of 6 weeks between. All study
subjects had an unremarkable medical history, physical examination, and
routine laboratory examination before study entry. During the 2 study
periods, they were admitted to the hospital Clinical Research Unit. On
1 study occasion, fasted subjects were given an oral dose of GI5402, as
a 100-mg tablet, which was followed 20 minutes later by an intravenous
injection of LPS. On the other occasion, matching placebo preceded the
LPS injection. Placebo tablets were visually and chemically identical with the exception of the presence of GI5402. Escherichia coli LPS, lot G (UPS, Rockville, MD) was administered over 1 minute into
an antecubital vein at a dose of 4 ng/kg body weight.
Heart rate, blood pressure (by Dinamap, Critikon, Tampa, FL), and oral
temperature were measured before GI5402 administration, 20 minutes
postadministration (ie, just before LPS injection), and at intervals
thereafter up to 24 hours. Clinical symptoms were recorded throughout
the study periods using a scoring list for separate signs and symptoms,
such as headache, shivers, nausea, vomiting, tiredness, and malaise (0 = absent; 1 = weak; 2 = moderate; 3 = severe).
Sampling and assays.
Blood was obtained from a cannulated forearm vein 0.5 hours before LPS
injection (ie, directly before administration of GI5402 or placebo),
directly before LPS administration (time = 0 hours) and at 0.5, 1, 1.5, 2, 3, 4, 5, 6, 8, 12, and 24 hours thereafter. Blood for FACScan
analyses was collected in heparin-containing vacutainer tubes; all
other samples were drawn in K3-EDTA-containing tubes.
Leukocyte counts and differentials were assessed by a Stekker analyzer
(counter STKS; Coulter counter, Bedfordshire, UK). All blood samples
(except samples for flow cytometry) were centri-fuged at 2,000g
for 20 minutes at 4°C and plasma was stored at 20°C until assays were performed. The following enzyme-linked immunosorbent assays (ELISAs) were used according to the instructions of the manufacturer and/or were described in detail previously (with detection
limits): TNF- (Medgenix Diagnostics, Brussels, Belgium; 21 pg/mL)
and soluble TNF- receptors type I and II (Hoffman La Roche, Basel,
Switzerland; both 70 pg/mL).21 The TNF- ELISA measures
total TNF- (free and complexed with its receptors)22; the soluble TNF- receptor assays detect unoccupied TNF-
receptors.21 Blood obtained for FACScan analysis was
immediately put on ice. Erythrocytes were lysed with ice-cold isotonic
NH4Cl solution (155 mmol/L NH4Cl, 10 mmol/L
KHCO3, 0.1 mmol/L EDTA, pH 7.4) for 10 minutes. Cells were
centrifuged at 600g for 5 minutes at 4°C. The remaining
cells were brought to a concentration of 4 × 106
cells/mL in FACS buffer (phosphate-buffered saline [PBS] supplemented with 0.5% bovine serum albumin [BSA], 0.01% NaN3, and
100 mmol/L EDTA). Expression of cell-surface TNF- was determined
using a fluorescein isothiocyanate (FITC)-labeled anti-human TNF-
monoclonal antibody (MoAb) (clone 6401; R&D Systems, Abingdon, UK).
This MoAb recognizes both the 26-kD membrane-associated form of TNF and
TNF bound to its cell surface receptor.23,24 Saturation binding of TNF- by white blood cells was determined using
phycoerythrin (PE)-labeled human TNF- (Fluorokine; R&D Systems).
Expression of type I and type II TNF- receptors was determined using
biotinylated MoAbs directed against either the type I (p55; clone
MR1-2) or type II (p75; clone MR2-1) TNF-
receptor (Monosan, Uden, the Netherlands). These MoAbs are
nonneutralizing, ie, they bind to receptor epitopes not involved in
TNF- binding (information supplied by the manufacturer). All FACS
reagents were used in concentrations as recommended by the
manufacturers, and all analyses were also conducted with the
appropriate control antibodies (murine IgG1 or
IgG2 [Becton Dickinson]). For each test, 105
cells were counted. Mean cell fluorescence (MCF) at greater than 570 nm
of forward and side angle scatter-gated granulocytes, monocytes, and
lymphocytes was assessed. Data are presented as the difference (linear
units) between MCF intensities of specifically and nonspecifically stained cells.
Blood samples (1 mL) for parent drug assay (times relative to LPS
administration), were taken pre-GI5402, 10, 0, 10, 25, 40 minutes, and 1, 1.5, 2, 3, 4, 5, 6, 8, 12, and 24 hours post-LPS dose.
Samples were drawn into clotting tubes, allowed to clot at room
temperature for approximately 30 minutes, centrifuged within 15 minutes
at 1,500g for 10 minutes at 4°C, and serum separated and
stored at 20°C until assay. GI5402 was measured by liquid chromatography-mass spectrometry following solid-phase
extraction with a lower limit of quantification of 1 ng/mL. The maximum
observed serum concentration (Cmax) and the time at which
the maximum serum concentration was observed (tmax) were
calculated using PEARS, an in-house PC-based pharmacokinetic software package.
Statistical analysis.
Values are given either as arithmetic mean ± SEM or geometric mean
with 95% confidence intervals (CIs). Differences between GI5402 and
placebo treatment periods were tested by analysis of variance (ANOVA)
for repeated measures. Changes of variables over time were analyzed
using 1-way ANOVA. Differences in maximum symptom scores between the 2 study periods were analyzed with the paired t-test. The
correlation between in vitro IC50 values and percentage inhibition of TNF- in vivo was calculated using the Pearson
correlation test. The area under the curve for plasma concentration of
TNF- was calculated from time point 0.5 hours pre-LPS up to
the point at which the second derivative of the curve changes sign (5 hours post-LPS in all subjects). All tests were done using SPSS for Windows (Microsoft, Redmond, WA). A 2-tailed P value less than .05 was considered significant.
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RESULTS |
Inhibitory activity of GI5402 on isolated MMPs and TACE.
GI5402 is a potent inhibitor in vitro of a range of human isolated
MMPs, including collagenase-1 (MMP-1), collagenase-3 (MMP-13), stromelysin-1 (MMP-3), and gelatinase B (MMP-9) with IC50
values (Ki) in the low nanomolar range (Table
1). The compound was relatively nonselective across this range of MMPs. It was also a potent inhibitor of isolated TACE, with an IC50 of 6.6 nmol/L (equivalent to
2.6 ng/mL).
Effect of GI5402 in whole blood in vitro.
In 6 of 7 healthy volunteers, the effect of GI5402 on TNF- release
in LPS-stimulated whole blood was investigated before their
participation in the in vivo phase of the study. GI5402 caused a
concentration-dependent inhibition of LPS-induced TNF- release in
vitro in all subjects with, on average, greater than 95% inhibition
achieved at 10,000 ng/mL GI5402. The inhibitory potency was similar in
all subjects, with a mean (n = 6) IC50 of 515 ng/mL (95%
CI, 405 to 654 ng/mL) (Table 2). This
potency is substantially lower ( 200-fold) than that observed on the
isolated TACE enzyme. Apart from differences in methodologies in
determining these IC50 values, the difference in potency
between whole cell and isolated enzyme may suggest a reduced ability of
GI5402 to access the cellularly located enzyme.
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Table 2.
Comparison of the In Vitro IC50 to Inhibit
TNF Release in LPS-Stimulated Whole Blood With the Magnitude
of Inhibition of TNF In Vivo for Each Subject
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Clinical signs and circulating leukocyte counts.
Injection of LPS induced a febrile response, peaking after 3.5 hours
(38.8 ± 0.3°C), together with transient flu-like
symptoms, including headache, nausea, retching, malaise, and chills. In addition, LPS administration resulted in a biphasic change in neutrophil counts, characterized by an initial neutropenia (from 3.4±0.4 to 1.1 ± 0.2 × 109/L after 1 hour)
followed by neutrophilia (12 hours: 14.8 ± 1.0 × 109/L), monocytopenia (from 0.57 ± 0.04 to 0.02 ± 0.003 × 109/L after 3 hours), and lymphocytopenia
(from 1.7 ± 0.2 to 0.3 ± 0.02 × 109/L after 6 hours). None of these changes were influenced by GI5402 (data not shown).
Soluble and cell-bound TNF-.
Following placebo, LPS elicited a monophasic increase in plasma soluble
TNF- concentrations peaking after 2 hours (2,839 ± 1,041 pg/mL)
(Fig 1). This LPS-induced rise in soluble
TNF- levels was associated with an increase in monocyte-bound
TNF- immunoreactivity from 6.2 ± 2.0 to 20.0 ± 6.5 U (Fig
1). GI5402 produced a 74% reduction in the peak LPS-induced increase
in plasma-soluble TNF- levels (2 hours: 750 ± 226 pg/mL;
P < .001 v placebo), while it did not significantly
alter the enhanced expression of monocyte TNF- (Fig 1). Granulocyte-
and lymphocyte-associated TNF- immunoreactivities were low and
remained unchanged in both study periods (data not shown).
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| Fig 1.
Mean (±SE) plasma concentrations of soluble TNF-
(pg/mL) and monocyte membrane-anchored TNF- after intravenous
injection of LPS in subjects either receiving GI5402 ( ) or placebo
( ) given 20 minutes before LPS challenge (lot G, 4 ng/kg). P
value indicates the difference between treatment groups. NS,
nonsignificant.
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The GI5402 IC50 against LPS-induced TNF- release in
vitro obtained in 6 of 7 volunteers did not correlate significantly
with the relative potency of GI5402 to inhibit LPS-induced TNF-
release in vivo in these same subjects (r2 = .36, P = .2) (Table 2). GI5402 Cmax values in individual
subjects and tmax are also shown in Table 2.
Cmax values ranged from 360 to 1,065 ng/mL and were greater
than the IC50 values determined in vitro for 5 of 6 subjects. Time to peak serum concentration of GI5402 was similar in
most subjects (0.75 to 1.0 hours), which was approximately 1 hour
before the peak in soluble TNF-; mean plasma half-life was 4.8 hours
(range, 2.8 to 7.2). An examination of the relationship between percent
inhibition of soluble TNF- in vivo and serum concentration of drug
was not possible due to the relative lack of pharmacokinetic data for
each subject around the time of the peak in soluble TNF-.
Soluble and cell-bound TNF- receptors.
Because MP inhibitors have been found to influence the cell-surface
expression of TNF- receptors,10,11,14,16 we determined the effect of GI5402 on the shedding and surface expression of type I
and type II TNF- receptors. LPS injection induced increases in the
plasma concentrations of soluble TNF- receptor type I and type II,
peaking after 2 hours (7.08 ± 0.38 ng/mL and 15.32 ± 0.85 ng/mL, respectively). GI5402 inhibited the release of both soluble
TNF- receptor species (Fig 2); peak
concentrations of the type I receptor were 3.75 ± 0.38 ng/mL, and
of the type II receptor 8.52 ± 0.89 ng/mL (both P < .001 v placebo). LPS also induced a transient decrease in the
binding of PE-labeled TNF- by circulating monocytes and
granulocytes, reaching its nadir at 2 to 4 hours (Figs
3 and 4).
This decreased monocyte and granulocyte PE-labeled TNF- binding was
associated with a reduced expression of both type I and type II TNF-
receptors at the surface of these cell types, also reaching minimum
values at 2 to 4 hours. In contrast to its effect on soluble TNF-
receptors, GI5402 did not influence LPS-induced changes in monocyte and
granulocyte TNF- receptor expression (Figs 3 and 4). Lymphocyte
TNF- receptor expression was low throughout both study periods (data
not shown).
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| Fig 2.
Mean (±SE) plasma concentrations of soluble TNF-
receptor I and II after intravenous injection of LPS in subjects
receiving either GI5402 () or placebo () 20 minutes before LPS
challenge (lot G, 4 ng/kg). P value indicates the difference
between treatment groups.
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| Fig 3.
Monocyte surface TNF- receptor and monocyte PE-labeled
TNF- binding after intravenous injection of LPS in subjects
receiving either GI5402 () or placebo () given 20 minutes before
LPS challenge (lot G, 4 ng/kg). Results are expressed as the difference
between specific mean channel fluorescence (MCF) and nonspecific MCF
(mean ± SE).
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| Fig 4.
Granulocyte surface TNF- receptor and granulocyte
PE-labeled TNF- binding after intravenous injection of LPS in
subjects receiving either GI5402 () or placebo () given 20 minutes before LPS challenge (lot G, 4 ng/kg). Results are expressed as
the difference between specific MCF and nonspecific MCF (mean ± SE).
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DISCUSSION |
Although MP inhibitors have been advocated as therapeutic agents for a
number of inflammatory diseases, knowledge of their effects in humans
in vivo is limited. Here, we have shown that the orally administered MP
inhibitor GI5402 markedly reduced the appearance of soluble TNF- in
the circulation after a bolus intravenous injection of LPS into healthy
humans. GI5402 did not influence the LPS-induced increase
in monocyte-bound TNF- immunoreactivity or the decreases in
monocyte and granulocyte TNF- receptor expression, but did attenuate
the rise in the plasma concentrations of soluble TNF- receptors.
These results demonstrate for the first time the in vivo effects of a
MP inhibitor on TNF- and TNF- receptor processing in humans.
Soluble mature TNF- is released from cells by proteolytic cleavage
of a 26-kD biologically active transmembrane precursor between Ala-76
and Val-77. Several years ago, it was recognized that MP inhibitors
were able to reduce TNF- release by stimulated cells, indicating
that one or more MP-like enzymes were involved in this
process.6-8 Furthermore, it was reported that MP
inhibitors could inhibit TNF- release in rodents in vivo
and prevent LPS-induced lethality in
D-galactosamine sensitized
mice.6-8,11,12,14 however, recent investigations have
raised concerns about possible unwanted proinflammatory actions of MP
inhibitors secondary to their inhibiting effects on not only TNF-
release, but also on TNF- receptor processing. Indeed, inhibition of
MPs has been associated with enhanced expression of cell-surface
TNF- and TNF- receptors.4,6,8,17 Therefore, we sought
to determine the effects of GI5402, a broad spectrum inhibitor of MPs,
on changes in cell-associated and soluble forms of both TNF- and its
receptors in a well-characterized human model of inflammation in which
rises in the plasma concentrations of soluble TNF- and soluble
TNF- receptors have been found to be accompanied by decreased
expression of monocyte and granulocyte TNF-
receptors.25-27
In previous studies in healthy male volunteers, single oral doses of
GI5402 (10 to 200 mg) were rapidly absorbed, and peak concentrations
increased proportionally with dose. In one study in 12 healthy fasted
subjects, pharmacokinetic parameters following a 100-mg tablet of
GI5402 were Cmax 1,091 (984 to 1,210) ng/mL (geometric mean
with 95% CI), tmax 45 (30 to 90) minutes, and half-life
3.9 (3.7 to 4.2) hours (both median and range) (unpublished data, May,
1997). These values compare closely with those obtained in the present study. The concentrations of GI5402 required to inhibit
soluble TNF- production in vivo and in whole blood in vitro also
appeared to be similar. The peak plasma concentrations achieved in vivo
for the majority of subjects were slightly greater than the
IC50 values determined in vitro for inhibition of soluble TNF- release. The resultant mean inhibition of soluble TNF- in
vivo of approximately 70% is therefore consistent with these slightly
greater plasma concentrations of GI5402. Given the Cmax and
IC50 concentration data obtained in the present study, this would suggest that measurable inhibition of TNF- in the LPS model with 100 mg oral GI5402 might be expected to persist for between 4 to 8 hours (ie, 1 to 2 half-lives).
Our finding that GI5402 not only reduced the release of soluble
TNF-, but also that of soluble TNF- receptors is in accordance with earlier in vitro studies,9,10,13,17 and indicates that the shedding of TNF- receptors is regulated at least in part by MPs
in humans in vivo. The fact that GI5402 did not alter LPS-induced changes in cell-surface TNF- receptor expression contrasts with in
vitro findings with the broad-spectrum MP inhibitor BB2275. This agent
reversed the downmodulation of TNF- receptors on several cell lines
stimulated with phorbol myristate acetate, and enhanced the sensitivity
of rhabdomyosarcoma cells to TNF--induced
cytotoxicity.17 Possible explanations for this discrepancy
include differences in experimental setting (cell lines in vitro
v humans in vivo) and/or differences in the modes or spectrum
of action of the different MP inhibitors used. Our finding that during
human endotoxemia the release of soluble TNF- receptors and the
downregulation of blood cell-associated TNF- receptors are not
directly linked phenomena is supported by previous observations in this
model of human endotoxemia. Indeed, in earlier studies, no correlation existed between the extent of down-modulation of cellular TNF- receptors and peak plasma levels of soluble TNF-
receptors,25 and neutralization of endogenous TNF-
activity strongly reduced soluble TNF- receptor release with no or
only a modest effect on cell-surface TNF- receptor
expression.27 Possible explanations for this lack of
correlation are that (1) soluble TNF- receptors in plasma are, at
least in part, derived from surface TNF- receptors of cells that are
not present in blood (eg, endothelial cells, tissue macrophages); (2)
the soluble receptors are derived from an intracellular pool, not from
the cell surface; (3) the MP inhibitor may not only reduce the shedding
of the receptors, but also their expression; and (4) the downmodulation
of peripheral blood monocyte and granulocyte TNF- receptors is at
least partly caused by internalization of their surface receptors,
rather than by shedding. In accordance with the latter possibility is
the in vitro finding that exposure of macrophages or monocytes to
endotoxin results in a rapid and complete loss of cell-surface TNF-
binding sites secondary to internalization of TNF-
receptors.28
Our study also demonstrated a transient rise in monocyte TNF-
immunoreactivity after injection of LPS into normal humans. Although
the anti-TNF- MoAb used cross-reacts with both the 26-kD membrane
form of TNF- and TNF- bound to its cell-surface
receptor,23,24 we consider it likely that TNF-
immunoreactivity detected in the present study predominantly
represented the membrane-anchored cytokine. Indeed, at the time point
monocyte-bound TNF- peaked, after 2 hours, the expression of
(occupied and unoccupied) TNF- receptors was strongly reduced, as
determined by TNF- receptor MoAbs binding to epitopes of TNF-
receptors that are not involved in TNF- binding. These data
therefore suggest that monocytes within the circulation contribute to
soluble TNF- release during endotoxemia. Membrane-anchored TNF-
remained low on circulating granulocytes and lymphocytes during
endotoxemia, suggesting that these cell types are not important sources
for the appearance of soluble TNF- in the circulation. GI5402 did
not cause an accumulation of pro-TNF- on the surface of circulating
cells, indicating that the previously reported rise in cell-associated
TNF- after treatment of cells with MP inhibitors in vitro may not be
of great importance when using an MP inhibitor in
vivo.4,6,8 The fact that membrane-anchored TNF- (ie,
pro-TNF-) on monocytes from subjects treated with LPS plus GI5402
never exceeded the levels on monocytes from the same subjects treated
with LPS only, suggests that unprocessed TNF- is transient and has a
relatively short half-life on the cell surface. This agrees with
previous pulse-chase studies showing that unprocessed pro-TNF- is
rapidly degraded.8,29 Furthermore, our data are in
accordance with the finding that MP inhibition did not influence the
concentration of membrane-associated TNF- in livers of mice with
Concanavalin A-induced hepatitis.11
Treatment with GI5402 did not reduce clinical signs and symptoms
induced by LPS. In this respect, it should be noted that in previous
studies neither complete neutralization of endogenous TNF by infusion
of a recombinant TNF receptor fusion protein,27,30 nor
treatment with the antiinflammatory cytokine
interleukin-10,31 influenced LPS-induced clinical symptoms,
despite the fact that cytokine release and other inflammatory responses
were strongly inhibited. Therefore, it seems likely that LPS at least
in part induces symptoms directly. In spite of the lack of an effect on LPS-induced clinical signs, we consider it likely that the overall effect of MP inhibition during endotoxemia is antiinflammatory. GI5402,
at oral doses in the range of 9 to 27 mg/kg, produced 90% to 100%
inhibition of plasma soluble TNF- generation in an LPS challenge
model in mice (unpublished data, May 1997). By
administering galactosamine in conjunction with LPS in this model, LPS
induces lethality in the mouse. While GI5402 itself was not tested in the lethality model, a close analog, GW9471 with similar TACE inhibitory potency, did prevent lethality when greater than 90% inhibition of plasma soluble TNF- generation was
achieved.32 Other groups have also demonstrated a
protective effect of a TACE inhibitor against LPS
lethality.6 Thus, it would appear that there is good
evidence for the mechanism of MP inhibition preventing the
pathophysiologic effects of shedding of soluble
TNF-.
MP inhibitors have gained much attention in the past few years as
potentially new antiinflammatory agents, due to their inhibiting effect
on TNF- processing in vitro and animals in vivo, and the fact that
they can be administered orally. Considering that MP inhibitors are
likely to be used in the treatment of patients with a number of
inflammatory disorders in the near future, it is important to obtain
more insight in the effects of these compounds on inflammation in
humans in vivo. We found that the MP inhibitor GI5402 strongly reduced
the release of soluble TNF- in the circulation after a bolus
intravenous injection of LPS in healthy humans. Undesired
proinflammatory effects such as enhanced expression of surface TNF-
or surface TNF- receptors were not observed. Our data therefore
indicate that MP inhibitors may be of use as a treatment modality in
patients with diseases in which excessive production of TNF- is
considered to play a central role.
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ACKNOWLEDGMENT |
We thank Mieke Sala for excellent technical assistance; Jeff Stafford,
Mark Bickett, and Tony Leesnitzer for measurement of the isolated MP
IC50 values; Alison Mackie and Bob Biddlecombe for
pharmacokinetic analysis of GI5402; Emma Seaber for preparation of the
protocol; and Dr Richard P. Koopmans for calculation of the
IC50 values.
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FOOTNOTES |
Submitted December 8, 1998; accepted June 4, 1999.
Supported by Glaxo Wellcome, Middlesex, United Kingdom and a
grant from the Royal Netherlands Academy of Arts and Science to
T.v.d.P.
The publication costs of this
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ABSTRACT
INTRODUCTION