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Suppression of inflammation and structural damage in experimental arthritis through molecular targeted therapy with PPI-2458.

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ARTHRITIS & RHEUMATISM
Vol. 56, No. 3, March 2007, pp 850–860
DOI 10.1002/art.22402
© 2007, American College of Rheumatology
Suppression of Inflammation and Structural Damage in
Experimental Arthritis Through Molecular Targeted
Therapy With PPI-2458
Gerhard Hannig, Sylvie G. Bernier, Jennifer G. Hoyt, Beth Doyle, Ed Clark, Russell M. Karp,
Jeanine Lorusso, and William F. Westlin
Objective. To determine the disease-modifying
activity and mechanism of action of the orally available methionine aminopeptidase type 2 inhibitor,
[(1R)-1-carbamoyl-2-methyl-propyl]-carbamic acid(3R,4S,5S,6R)-5-methoxy-4-[(2R,3R)-2-methyl-3-(3methyl-but-2-enyl)-oxiranyl]-1-oxa-spiro [2.5] oct-6-yl
ester (PPI-2458), in a rat model of peptidoglycan–
polysaccharide (PG-PS)–induced arthritis.
Methods. Arthritis was induced in rats by administration of PG-PS, causing tarsal joint swelling and
histopathologic changes characteristic of rheumatoid
arthritis (RA). PPI-2458, a potent irreversible methionine aminopeptidase type 2 inhibitor, was administered
orally every other day at 1, 5, or 10 mg/kg.
Results. In an in vitro osteoclastogenesis model,
PPI-2458 potently inhibited osteoclast differentiation
and bone resorption. In the rat PG-PS arthritis model,
PPI-2458 afforded significant protection against established disease after therapeutic dosing. This in vivo
activity of PPI-2458 was linked to the inhibition of
methionine aminopeptidase type 2. Histopathologic assessment of affected joints showed improvement in
processes of inflammation, bone resorption, and cartilage erosion, associated with significant improvement in
all clinical indices. The protective effects of PPI-2458
against bone destruction in vivo, including the structural preservation of affected hind joints, correlated
with improvements in bone histomorphometric markers, as determined by microfocal computed tomography
and a significant decrease in systemic C-telopeptide of
type I collagen, suggesting decreased osteoclast activity
in vivo. Moreover, PPI-2458 prevented cartilage erosion
as shown by a significant decrease in systemic cartilage
oligomeric matrix protein.
Conclusion. The findings of this study suggest
that PPI-2458 exerts disease-modifying activity in experimental arthritis through its direct inhibition of
several pathophysiologic processes of this disease.
These results provide a rationale for assessing the
potential of PPI-2458 as a novel RA therapy.
Rheumatoid arthritis (RA), which affects ⬃1%
of the US population, is a systemic, chronic inflammatory autoimmune disease with a multifactorial, poorly
defined etiology. The progression of RA is driven by an
interdependent network of closely connected pathogenetic mechanisms, which ultimately lead to chronically
inflamed joints and structural joint damage (1). The
disease is mediated by pathologic neovascularization
and the secretion of proinflammatory mediators, such as
tumor necrosis factor ␣ (TNF␣), interleukin-1 (IL-1),
and IL-17, from activated endothelial cells. Cells of the
synovial intima (macrophage-like synoviocytes and
fibroblast-like synoviocytes) and activated T cells, which
are recruited to the inflamed joint, also contribute to
synovial hyperplasia and the growth of the destructive
pannus (2–4). The major structural damage in RA is
associated with periarticular bone erosions and juxtaarticular osteoporosis (5). Bone erosion is mediated by
osteoclasts, highly specialized multinucleated cells which
are derived from hematopoietic precursors through a
sequence of processes involving proliferation, differentiation, fusion, and activation (6,7). In the inflamed joint,
Gerhard Hannig, PhD, Sylvie G. Bernier, PhD, Jennifer G.
Hoyt, BS (current address: Infinity Pharmaceuticals, Cambridge, Massachusetts), Beth Doyle, MS, Ed Clark, BS, Russell M. Karp, BS,
Jeanine Lorusso, BS, William F. Westlin, PhD: Praecis Pharmaceuticals, Waltham, Massachusetts.
Address correspondence and reprint requests to Gerhard
Hannig, PhD, Praecis Pharmaceuticals Inc., 830 Winter Street,
Waltham, MA 02451. E-mail: Gerhard.Hannig@praecis.com.
Submitted for publication August 1, 2006; accepted in revised
form November 3, 2006.
850
DISEASE-MODIFYING ACTIVITY OF PPI-2458 IN ARTHRITIS
activated synoviocytes and T cells secrete RANKL,
which, in synergy with TNF␣, ⌱L-1, and other osteoclastogenic mediators, stimulates pathologic bone resorption through osteoclast recruitment, differentiation, and
activation (5,7–10).
Over the past decade, treatment regimens for RA
patients have shifted, and clinical strategies involving the
“therapeutic pyramid” have been supplanted by a new
paradigm which emphasizes the early use of diseasemodifying antirheumatic drugs (DMARDs) (3,11). A
DMARD is used, either as monotherapy or in combination with other DMARDs, in patients with the potential
for progressive disease (3,11). Intensive translational
research in RA has led to the identification and validation of novel molecular targets, such as TNF␣, RANK,
and RANKL, and the elucidation of their molecular
mechanisms and functions in the pathology of this
disease (7,12). These studies have directly contributed to
the successful clinical development of novel DMARDs,
such as the anti-TNF␣ agents infliximab, adalimumab,
and etanercept (13,14). In a significant number of
patients, however, RA has not responded or has become
refractory to currently available DMARDs, or treatment
has had to be interrupted due to intolerable side effects.
Hence, the development of innovative therapies that
target novel effectors and unique pathways in RA, and at
the same time provide a safe alternative to existing
DMARDs, remains a high priority in the management
of this disease (11).
The fumagillin analog [(1R)-1-carbamoyl-2methyl-propyl]-carbamic acid-(3R,4S,5S,6R)-5-methoxy4-[(2R,3R)-2-methyl-3-(3-methyl-but-2-enyl)-oxiranyl]1-oxa-spiro [2.5] oct-6-yl ester (PPI-2458) is an orally
available, irreversible inhibitor of methionine aminopeptidase type 2, the molecular target of this class of
molecules (15–19). Methionine aminopeptidase type 2 is
a cotranslational regulator of protein synthesis involved
in N-terminal protein processing, and recent functional
studies have demonstrated that this enzyme is an important molecular regulator of mammalian cell growth
(20–23). PPI-2458 is a potent inhibitor of the in vitro
proliferation of activated human umbilical vein endothelial cells (HUVECs) and human fibroblast-like synoviocytes (FLS) from RA patients, both cell types that
directly contribute to the pathogenesis of RA (18,23).
This in vitro growth inhibition was directly proportional
to the amount of methionine aminopeptidase type 2
enzyme inhibited in these cells, which suggested that the
inhibition of methionine aminopeptidase type 2 function
is a critical step in the growth inhibition of PPI-2458–
sensitive HUVECs and human FLS from RA patients
851
(18). Moreover, these pharmacologic properties of PPI2458 observed in vitro appear to translate into significant
protection against disease in several rat models of
arthritis (18,24,25).
The principal objective of this study was to show
that the significant protection against disease afforded
by PPI-2458 in an experimental model of arthritis was
linked in vivo to inhibition of the molecular target,
methionine aminopeptidase type 2. We sought to demonstrate that this disease-modifying activity was further
associated with significant improvements in all pathophysiologic processes of inflammation and joint destruction, including protection against osteoclast-mediated
bone resorption, preservation of joint architecture and
integrity, and prevention of the cartilage erosion that
occurs in this model of RA.
MATERIALS AND METHODS
Reagents. PPI-2458 was synthesized at Praecis Pharmaceuticals. For in vitro studies, a 10-mM stock solution in
ethanol was prepared. For in vivo administration, PPI-2458
was dissolved in 11% 2-hydroxypropyl-␤-cyclodextran (Cargill,
Minneapolis, MN). Peptidoglycan–polysaccharide (PG-PS)
was obtained from Lee Biomolecular Laboratories (Grayson,
GA), dexamethasone (DEX) (4 mg/ml in phosphate buffered
saline [PBS]) from Henry Schein (Melville, NY), RANKL
from R&D Systems (Minneapolis, MN), and E-64 from Sigma
(St. Louis, MO).
Osteoclast differentiation assays. Primary human osteoclast precursors (OCPs; Cambrex, Walkersville, MD) were
seeded at 10,000 cells/well (50,000 cells/ml) in OCP growth
medium (Cambrex). The cells were cultured for 7 days with
either macrophage colony-stimulating factor (M-CSF; 33 ng/
ml) alone, M-CSF (33 ng/ml) and RANKL (33 ng/ml), or
with both cytokines and different concentrations of PPI-2458.
Osteoclast differentiation was determined by staining for the
osteoclast marker tartrate-resistant acid phosphatase (TRAP),
using a leukocyte acid phosphatase kit. Briefly, after 7 days in
culture, the cells were rinsed once with PBS, fixed with 37%
formaldehyde in acetone-citrate buffer for 1 minute, and
stained for development of red color, according to the recommendations of the manufacturer (Sigma).
Rat model of PG-PS–induced arthritis. Female Lewis
rats (7–8 weeks old) were obtained from Charles River (Wilmington, MA). PG-PS (25 mg/kg) was injected intraperitoneally on day 1, and responding animals were randomized into
treatment groups on day 14. Vehicle, DEX (1 mg/kg), or
PPI-2458 (1, 5, or 10 mg/kg) was administered orally, every
other day. Paw swelling was monitored using a plethysmometer
(Stoelting, Woodale, IL), according to instrument specifications. The volumes of the 2 hind paws were measured and
averaged on days 1, 4, 6, 8, 10, 13, 15, 17, 20, 22, 23, 27, 29, and
31. Ten animals were assigned to each group, except the
vehicle group (n ⫽ 4) and a group of animals that received 10
mg/kg PPI-2458 but no PG-PS (n ⫽ 4). All animal studies were
852
approved by the Praecis Pharmaceuticals Institutional Animal
Care and Use Committee.
Histologic assessment of PG-PS arthritis. The histopathologic evaluation was performed on the left and right
hind joints of randomly selected animals from each study
group, by an independent histopathologist who had no knowledge of specific interventions. After completion of the treatment, the left and right hind ankles were removed, fixed in
10% buffered formalin, decalcified in 5% formic acid, embedded in paraffin, sectioned, and stained with hematoxylin and
eosin for histologic evaluation. A joint histology scoring system, which grades the severity of 4 histopathologic processes
(cell infiltration, pannus formation, cartilage erosion, and bone
resorption), was used to quantify hind joint involvement (26).
The total score was the sum of the scores assigned for each
parameter (0 ⫽ normal, 1 ⫽ minimal, 2 ⫽ mild, 3 ⫽ moderate,
4 ⫽ marked); the maximum possible total score was 16 per
ankle and 32 per animal.
Methionine aminopeptidase type 2 pharmacodynamic
assay. The methionine aminopeptidase type 2 assay measures the amount of uninhibited methionine aminopeptidase
type 2 in cells or tissue that has not been derivatized by prior
treatment with PPI-2458 (18,23). Briefly, white blood cells
(WBCs) from animals of each study group were pooled, and
cell lysates were prepared as previously described (18,23).
WBC protein (10–20 ␮g) was incubated with a biotinylated
analog of PPI-2458, and the biotinylated methionine aminopeptidase type 2–inhibitor complex was captured on a plate
with immobilized streptavidin (Pierce, Rockford, IL). The
complex was detected with the methionine aminopeptidase
type 2 antibody CM33 (0.5 ␮g/ml) (Zymed, South San Francisco, CA), followed by horseradish peroxidase–conjugated
goat anti-rabbit IgG secondary antibody (Amersham, Pittsburgh, PA). The amount of uninhibited methionine aminopeptidase type 2 was determined by measuring absorption at
450 nm using a Multiskan plate spectrophotometer (Labsystems, Helsinki, Finland). The detection limit of this assay
was 0.47 ng methionine aminopeptidase type 2 protein/mg
WBC protein.
Enzyme-linked immunosorbent assays (ELISAs) for
cartilage and bone biochemical markers. The amount of
cartilage oligomeric matrix protein (COMP) in serum was
measured with a competitive enzyme immunoassay, according
to the recommendations of the manufacturer (MD BioSciences, St. Paul, MN). The detection limit of this ELISA is
0.2 units/liter. Helical peptide (amino acids 620–633) from the
␣1-chain of bone-specific human C-telopeptide of type I
collagen was measured either in cell culture supernatants of
primary human OCPs cultured on OsteoAssay plates (Cambrex) as described above, or in urine with a competitive
enzyme immunoassay (Quidel, San Diego, CA). The detection
limit of this ELISA is 8 ␮g/liter. All measurements of
C-telopeptide of type I collagen in urine were corrected for
urinary creatinine excretion for each sample to account for
potential differences in renal clearance rates among the different study groups. Urinary creatinine was measured with a
colorimetric assay (Quidel).
Microfocal computed tomography (micro-CT). All
specimens were scanned on an AG ␮CT 40 system (Scanco
Medical,Wayne, PA). Images were obtained with an isotropic
voxel resolution of 20␮. A matrix size of 1,024 ⫻ 1,024 with
HANNIG ET AL
1,000 projections was used for all scans. A total of 1,836 slices
were scanned for each specimen (the number of slices scanned
was determined by the length of the scan needed to cover the
entire ankle including the distal tibia). The scan time per
specimen was ⬃2.6 hours. The images were then volume
rendered using 2 different fixed thresholds, 255 and 140. The
total bone volume and bone mineral density were calculated
over the same regions of all specimens. Micro-CT was performed at Scanco.
RESULTS
PPI-2458–induced inhibition of osteoclast differentiation and bone resorption in vitro. We developed an
in vitro osteoclastogenesis model to examine the effects
of PPI-2458 on osteoclast differentiation and bone resorption, based on the ability of primary human OCPs to
recapitulate essential aspects of osteoclast differentiation and activity in vitro. Primary human OCPs were
cultured for 7 days in the presence of M-CSF and
RANKL, and treated with either vehicle or increasing
concentrations of PPI-2458. Cells cultured with only
M-CSF and RANKL differentiated into large, multinucleated osteoclasts, as demonstrated by the appearance
of numerous TRAP-stained cells. Cells cultured with
M-CSF, RANKL, and PPI-2458 at a concentration of 1
nM yielded almost no TRAP-positive multinucleated
osteoclasts, while PPI-2458 at a concentration of 0.1 nM
had little detectable effect on the ability of cells to
differentiate into TRAP-positive, mature osteoclasts
(Figure 1A). The ability of PPI-2458 to inhibit osteoclast
differentiation was fully reversible (results not shown),
consistent with previously reported findings in human
FLS from RA patients (18), and was dependent on rates
of metabolic turnover of the methionine aminopeptidase
type 2 enzyme and normal turnover of affected cell
types. Furthermore, incubation of these cells with PPI2458 at concentrations of up to 100 nM did not induce
cytotoxicity (data not shown).
To determine whether the observed inhibition of
osteoclast differentiation by PPI-2458 resulted in inhibition of bone resorption in vitro, we cultured primary
human osteoclasts on a thin layer of human bone particles in the presence of M-CSF and RANKL, with
vehicle or increasing concentrations of PPI-2458. The
nonspecific cysteine proteinase inhibitor E-64 (100 nM),
a known inhibitor of bone resorption in vitro, was used
as a positive control. After 7 days, the culture supernatant was collected and the amount of bone-specific
C-telopeptide of type I collagen was measured by
ELISA. PPI-2458 potently inhibited the bone-resorbing
activity of human osteoclasts in a dose-dependent man-
DISEASE-MODIFYING ACTIVITY OF PPI-2458 IN ARTHRITIS
853
Figure 1. Inhibition, by PPI-2458, of osteoclast differentiation and bone resorption in vitro. A, Morphology of primary human osteoclast precursors
(OCPs; 10,000 cells/well) cultured for 7 days with 33 ng/ml macrophage colony-stimulating factor (M-CSF) and 33 ng/ml RANKL, and vehicle or
PPI-2458 at concentrations of 0.1 nM or 1 nM. Numerous multinucleated tartrate-resistant acid phosphatase (TRAP)–staining cells were seen with
M-CSF/RANKL treatment. TRAP staining was inhibited by treatment with PPI-2458 at 1 nM, but not at 0.1 nM. Results are representative of 3
independent experiments (original magnification ⫻ 4). B, Amount of C-telopeptide of type I collagen (CTX-I) in cell culture supernatants. Primary
human OCPs (10,000 cells/well) were seeded into wells coated with a thin layer of human bone particles (duplicate wells per experimental condition)
and cultured with vehicle alone (first unlabeled bar), 33 ng/ml M-CSF and 33 ng/ml RANKL plus vehicle (second unlabeled bar), or 33 ng/ml M-CSF
and 33 ng/ml RANKL plus 100 nM E-64 or PPI-2458 at concentrations of 0.01, 0.1, 1, or 10 nM. After 7 days, cell culture supernatants were collected
and the amount of CTX-I was measured by enzyme-linked immunosorbent assay. The detection limit in this assay was 8 ␮g/liter. Values are the mean
and SEM and are representative of 2 experiments with similar results.
ner (50% inhibition concentration ⱕ0.1 nM), and the
degree of inhibition at 1 nM and 10 nM was comparable with the inhibitory activity of E-64 at 100 nM
(Figure 1B).
Association of potent antiinflammatory activity
of PPI-2458 with inhibition of methionine aminopeptidase type 2 function in the rat arthritis model. The
potent in vitro inhibition of PPI-2458—sensitive
HUVECs and human FLS from RA patients has previously been shown to be directly proportional to the
amount of methionine aminopeptidase type 2 enzyme
inhibited in these cells (18). We investigated whether
the therapeutic effects of PPI-2458 on established disease in the rat PG-PS arthritis model were linked in vivo
to inhibition of the molecular target, methionine aminopeptidase type 2. The progression of disease in this
model follows a biphasic mode, with an early acute,
predominantly neutrophil-driven phase which persists to
day 6 or 7, followed by a chronic, T cell–dependent
phase (evident at approximately day 12) (27).
Therapeutic dosing of animals administered
vehicle or PPI-2458 (1, 5, or 10 mg/kg orally, every
other day) started on day 15, after the chronic destructive phase of the disease was established, and
terminated on day 31. PPI-2458 at all 3 doses resulted
in significant amelioration of joint swelling and inflammation, as measured by swelling of the hind paws,
when compared with vehicle-treated animals (Figure 2A).
We measured the amount of uninhibited methionine aminopeptidase type 2 in WBCs of animals from
all study groups at the conclusion of the treatment
protocol, using the methionine aminopeptidase type 2
pharmacodynamic assay (18,23). In animals administered PPI-2458 at a dose of 1 mg/kg, ⱖ60% of the
methionine aminopeptidase type 2 in WBCs was inhib-
854
HANNIG ET AL
Figure 2. In vivo antiinflammatory activity of PPI-2458 and methionine aminopeptidase type 2 inhibition. A,
Paw swelling in rats administered vehicle, dexamethasone (DEX; 1 mg/kg), or PPI-2458 (1, 5, or 10 mg/kg).
Treatment started on day 15, after the onset of chronic disease. Values are the mean ⫾ SEM paw volume (n ⫽
10 rats per group, except for naive rats treated with vehicle [n ⫽ 4]) and are representative of at least 6
independent experiments. ⴱ ⫽ P ⬍ 0.0001 versus rats treated with peptidoglycan–polysaccharide (PG-PS) and
vehicle, by one-way analysis of variance followed by Dunnett’s multiple comparison test. B, Amount of
methionine aminopeptidase type 2 inhibited in white blood cell (WBC) lysates, determined by methionine
aminopeptidase type 2 pharmacodynamic assay at the completion of the treatment. Values are the mean
percent amount of methionine aminopeptidase type 2 compared with that in vehicle-treated rats with
PG-PS–induced arthritis (which corresponded to 41 ng methionine aminopeptidase type 2/mg of WBC protein,
arbitrarily set at 100% for comparison across groups) (n ⫽ 10 rats per group, except for naive rats treated with
vehicle [n ⫽ 4]).
ited relative to the vehicle-treated PG-PS arthritis
group, while ⱖ95% of methionine aminopeptidase type
2 was inhibited with PPI-2458 at doses of 5 and 10 mg/kg
(Figure 2B). Notably, ⱖ90% methionine aminopeptidase type 2 inhibition was also observed after the
administration of DEX. No methionine aminopeptidase
type 2 inhibition was observed in naive animals treated
with DEX for 12 days.
Decrease in the severity of indices of inflammation and joint destruction in rats with experimental
arthritis treated with PPI-2458. The PG-PS animal
model of arthritis is characterized by aggressive synovitis, extensive pannus formation, cartilage degradation,
and focal bone erosion. We investigated whether the
protective activity of PPI-2458 was mediated through a
decrease in the severity of all of these clinical indices, or
whether the activity of PPI-2458 affected only specific
pathogenetic processes. Therapeutic dosing with PPI2458 at doses of 1, 5, and 10 mg/kg resulted in significant
improvements in the total arthritis score, compared with
vehicle-treated animals (mean ⫾ SEM 7.00 ⫾ 0.58,
7.60 ⫾ 0.67, and 5.83 ⫾ 0.71, respectively, versus 13.85 ⫾
0.75) (Table 1). Moreover, this significant improvement
in the total arthritis score was reflected in significantly
improved scores for all clinical indices, with the highest
level of protection (⬃80%) observed for cartilage erosion with PPI-2458 at a dose of 10 mg/kg (Table 1).
These results demonstrate that protection against arthritis in this model was mediated through a significant
decrease in all indices of inflammatory and destructive
processes.
Preservation of the structural integrity of affected joints by PPI-2458 treatment. We further investigated the effect of PPI-2458 treatment on the structural preservation of hind joints in rats with established
disease. Three-dimensional–rendered micro-CT imaging, which allows for the noninvasive visualization of
pathologic joint changes, demonstrated major structural
damage and degenerative changes in the joint architecture of vehicle-treated animals (Figure 3). Therapeutic
dosing with PPI-2458 (10 mg/kg) resulted in significant
protection against bone destruction and preserved the
architecture of affected hind joints. Moreover, quantitative micro-CT analysis demonstrated that treatment with
PPI-2458 led to preservation of bone volume and protection against loss of bone mineral density, 2 impor-
DISEASE-MODIFYING ACTIVITY OF PPI-2458 IN ARTHRITIS
855
Table 1. Joint histology scores by treatment group*
Treatment group
Cell
infiltration
Pannus
formation
Cartilage
erosion
Bone
resorption
Arthritis
(total)
Vehicle alone
PG-PS plus vehicle
PG-PS plus DEX (1 mg/kg)
PG-PS plus PPI-2458 (1 mg/kg)
PG-PS plus PPI-2458 (5 mg/kg)
PG-PS plus PPI-2458 (10 mg/kg)
0
3.45 ⫾ 0.26
0.25 ⫾ 0.08†
1.85 ⫾ 0.15†
2.15 ⫾ 0.18†
1.38 ⫾ 0.31†
0
3.65 ⫾ 0.16
0.55 ⫾ 0.08†
2.00 ⫾ 0.14†
2.40 ⫾ 0.20†
1.72 ⫾ 0.20†
0
3.15 ⫾ 0.19
0.60 ⫾ 0.14‡
1.10 ⫾ 0.12‡
1.25 ⫾ 0.18‡
0.72 ⫾ 0.14‡
0
3.60 ⫾ 0.17
0.95 ⫾ 0.13‡
2.05 ⫾ 0.24‡
1.80 ⫾ 0.20‡
2.00 ⫾ 0.16‡
0
13.85 ⫾ 0.75
2.35 ⫾ 0.23‡
7.00 ⫾ 0.58‡
7.60 ⫾ 0.67‡
5.83 ⫾ 0.71‡
* Values are the mean ⫾ SEM joint histology score on a 5-point scale, where 0 ⫽ normal, 1 ⫽ minimal, 2 ⫽ mild, 3 ⫽ moderate, and 4 ⫽ marked.
For each group, the left and right hind ankles of 5 rats (n ⫽ 10 joints) were scored, except for the group of naive rats treated with vehicle (n ⫽ 4
joints). Results are representative of at least 3 independent experiments. DEX ⫽ dexamethasone.
† P ⬍ 0.01 versus rats treated with peptidoglycan–polysaccharide (PG-PS) plus vehicle, by one-way analysis of variance followed by Dunnett’s
multiple comparison test.
‡ P ⬍ 0.0001 versus rats treated with PG-PS plus vehicle, by one-way analysis of variance followed by Dunnett’s multiple comparison test.
tant indices of bone homeostasis, although the observed improvements were not statistically significant
(Table 2).
Figure 3. Three-dimensional (3-D)–rendered microfocal computed
tomography (micro-CT) images of hind paws from representative rats
from each of 4 study groups, showing preservation of joint architecture
by PPI-2458. After completion of the treatment protocol, hind paws
from rats were excised, and joints were visualized using 3-D–rendered
micro-CT to detect pathologic bone changes. All images were obtained
using a Scanco Medical AG ␮CT 40 system with an isotropic voxel
resolution of 20␮. See Figure 2 for other definitions.
PPI-2458–induced inhibition of bone resorption
and cartilage erosion in vivo in the PG-PS arthritis
model. Bone resorption represents the best surrogate
marker of joint destruction. To assess the activity of
PPI-2458 in protection against ongoing in vivo bone
resorption during the RA process in this disease model,
we measured the amount of C-telopeptide of type I
collagen in urine, normalized for urinary creatinine
excretion, at the conclusion of the treatment. Therapeutic administration of PPI-2458 resulted in significantly decreased systemic urinary levels of C-telopeptide
of type I collagen compared with vehicle-treated animals
(P ⬍ 0.0001 at doses of 1 and 5 mg/kg, P ⬍ 0.00001 at
10 mg/kg) (Figure 4A). These results confirmed that
PPI-2458 inhibited bone resorption in this model, consistent with the clinical assessment of this parameter
(Table 1).
COMP is a major component of the extracellular
matrix of the musculoskeletal system that mediates
chondrocyte attachment through interactions with integrins (28). The amount of COMP present in the serum
of animals treated with vehicle or PPI-2458 was measured at the conclusion of the treatment. A significant
decrease in systemic levels of this marker, even below
the level of serum COMP measured in naive animals
treated with vehicle, was detected after treatment with
PPI-2458 at all doses (all P ⬍ 0.00001) (Figure 4B),
consistent with the clinical assessment of protection
against cartilage erosion (Table 1).
DISCUSSION
In the chronically inflamed joints in RA, synovial
hyperplasia, along with local neovascularization, ultimately results in the destruction of articular bone and
856
HANNIG ET AL
Table 2.
Total bone volume and bone mineral density (BMD), by treatment group*
Group
Bone volume, voxels
BMD, mg hydroxyapatite/cm3
Vehicle alone (n ⫽ 2)
PG-PS plus vehicle (n ⫽ 3)
PG-PS plus DEX (1 mg/kg) (n ⫽ 3)
PG-PS plus PPI-2458 (10 mg/kg) (n ⫽ 5)
206.7 ⫾ 8.93
162.6 ⫾ 9.91
240.7 ⫾ 12.68
213.8 ⫾ 5.23
1,161 ⫾ 1.15
1,044 ⫾ 17.88
1,125 ⫾ 2.59
1,087 ⫾ 17.44
* Values are the mean ⫾ SEM. The n values are the number of joints examined. See Table 1 for other
definitions.
cartilage (2–4). Angiogenesis has been recognized as a
major contributing factor in the pathologic process in
RA, and the concept of antiangiogenesis has recently
emerged as a new strategy for the treatment of disease
states involving proliferative synovitis, particularly RA
(29). Earlier studies of TNP-470, a member of the
fumagillin class of irreversible methionine aminopeptidase type 2 inhibitors like PPI-2458, have shown that it
provides protection against experimental arthritis, thus
validating angiogenesis as a therapeutic target in RA
(30–32). TNP-470 was originally developed as an anticancer agent, but its clinical development was discontinued, primarily due to dose-limiting toxicities to the
central nervous system and an unfavorable pharmacokinetic profile (33–35).
PPI-2458 was designed to overcome the clinical
deficiencies of TNP-470, while at the same time maintaining the potent antiangiogenic and antiproliferative
activity of this class of molecules. A previous study of
PPI-2458 demonstrated a significant reduction in adverse effects on the central nervous system, as compared
with TNP-470, and showed that it was effective when
administered orally (18). Moreover, PPI-2458 potently
inhibited the in vitro proliferation of activated endothelial cells and synoviocytes, both cell types which
directly contribute to the pathogenesis of RA, through a
Figure 4. Inhibition, by PPI-2458, of bone resorption and cartilage erosion in the rat model of PG-PS–
induced arthritis. A, Levels of C-telopeptide of type I collagen (CTX-I) in urine, measured by enzyme-linked
immunosorbent assay (ELISA) and corrected for urinary creatinine excretion. On the day rats were killed,
urine was collected from naive rats treated with vehicle or with PPI-2458 (10 mg/kg), and from rats with
PG-PS–induced arthritis treated with either vehicle, DEX (1 mg/kg), or PPI-2458 (1, 5, or 10 mg/kg). B,
Amount of cartilage oligomeric matrix protein (COMP) in serum, measured by ELISA. At completion of the
treatment, serum was prepared from peripheral blood of naive rats treated with vehicle or with PPI-2458
(10 mg/kg), and from rats with PG-PS–induced arthritis treated with vehicle, DEX (1 mg/kg), or PPI-2458
(1, 5, or 10 mg/kg). Values are the mean and SEM and are representative of 2 experiments with similar results
(in both assays, n ⫽ 10 rats per group, except for naive rats treated with vehicle or PPI-2458 [n ⫽ 4 rats per
group]). ⴱ ⫽ P ⬍ 0.0001; ⴱⴱ ⫽ P ⬍ 0.00001, versus rats treated with PG-PS and vehicle, by one-way analysis
of variance followed by Dunnett’s multiple comparison test. See Figure 2 for other definitions.
DISEASE-MODIFYING ACTIVITY OF PPI-2458 IN ARTHRITIS
mechanism that was proportional to the amount of
methionine aminopeptidase type 2 enzyme inhibited in
these cells (18,23). These pharmacologic properties of
PPI-2458 suggest that therapeutic benefit in RA could
be derived by targeting several inflammatory processes,
including synovial hyperplasia and angiogenesis; such
treatment could lead to a decrease in the production
of proinflammatory mediators, an inhibition of leukocyte adhesion and migration through decreased endothelial cell surface area, and a diminished supply of
nutrients to the rapidly proliferating and erosive synovium.
While most current treatment strategies in RA
have targeted the inflammatory cascade, the discovery
of the osteoprotegerin (OPG)/RANKL/RANK axis and
its critical function in bone resorption has not only led to
an improved understanding of the molecular mechanisms involved in bone resorption, but has also guided
the clinical development of novel therapies, such as the
anti-RANKL antibody AMG-162 (denosumab) and
bisphosphonate drugs, which specifically target the
pathologic bone loss in RA (36,37). The destruction of
articular bone in diseased joints, a hallmark of RA, is
caused by excessive activity of bone-resorbing osteoclasts. Due to this critical role of osteoclasts in the
pathologic process in RA, we investigated the activity of
PPI-2458 on the differentiation of human OCPs and the
ability of this agent to prevent bone resorption in an in
vitro model of osteoclastogenesis. We demonstrated a
dose-dependent inhibition of both processes. This further suggested that PPI-2458 may have direct beneficial
effects on bone homeostasis in RA.
The potent in vitro growth inhibition of activated
endothelial cells and synoviocytes through methionine
aminopeptidase type 2 inhibition prompted us to further
investigate whether the therapeutic effects of PPI-2458
on established disease in rat experimental arthritis were
linked to methionine aminopeptidase type 2 inhibition
in vivo. Therapeutic dosing with PPI-2458 administered
orally at 1, 5, or 10 mg/kg significantly reversed joint
swelling and inflammation in PG-PS—induced arthritis,
and this activity of PPI-2458 was associated with the
inhibition of methionine aminopeptidase type 2 in circulating WBCs. These results demonstrate the effectiveness of this orally administered agent and further suggest that the amount of methionine aminopeptidase type
2 enzyme inhibited in WBCs could serve as a pharmacodynamic marker of the biologic activity of PPI-2458 in
vivo, which could potentially be useful in the clinical
setting.
Notably, the amount of free methionine amino-
857
peptidase type 2 in WBCs of PG-PS–treated rats was
significantly elevated over that in naive animals, suggesting a mechanism of inducible methionine aminopeptidase type 2 expression in this compartment in
inflammatory disease. Another unexpected finding was
that the treatment of rats with PG-PS arthritis with
DEX also decreased the amount of uninhibited methionine aminopeptidase type 2 by ⱖ90%, a reduction that
was not observed in naive animals after treatment with
DEX for 12 days with different treatment schedules.
These results suggest a novel mechanism of glucocorticoid activity in protection against experimental arthritis,
possibly linked to a decrease in free methionine aminopeptidase type 2 enzyme, similar to the activity of
PPI-2458.
The development and progression of disease in
the PG-PS arthritis model closely resemble the pathogenetic processes observed in humans with RA. We used
an established scoring system to measure whether the
observed protective activity of PPI-2458 decreased the
severity of all indices of joint inflammation and joint
destruction, or whether this agent selectively targeted
specific pathogenetic processes. Therapeutic administration of PPI-2458, even at the lowest dose (1 mg/kg), not
only significantly reduced the total arthritis score, but
this broad protection also translated into significant
decreases in the severity of all indices tested, suggesting
that PPI-2458 exhibits disease-modifying activity in this
animal model of arthritis.
The most critical issue in the treatment of RA is
the prevention of disability due to joint destruction
caused by excessive bone resorption and cartilage erosion. The recent elucidation of the OPG/RANKL/
RANK axis has broadened our understanding of the
molecular mechanisms of osteoclastogenesis and bone
resorption (5–7). Since histologic assessment of arthritic
joints from rats with PG-PS–induced arthritis treated
with PPI-2458 demonstrated significant protection
against bone resorption in vivo, we investigated further
the potential mechanism by which PPI-2458 afforded
this protection.
The systemic levels of biochemical markers generated during the destruction of bone in RA, such as
C-telopeptide of type I collagen breakdown products
released into the peripheral blood, have been established as useful markers for monitoring tissue involvement in the development and progression of this disease,
and to assess therapeutic interventions (38,39). Our
results show that PPI-2458 significantly decreased the
level of urinary C-telopeptide of type I collagen, consistent with our findings that PPI-2458 inhibited osteoclast-
858
mediated bone resorption in vitro, and suggest that this
marked effect on bone resorption in vivo is mediated
through a direct effect on osteoclasts. Alternatively, the
potent inhibition of inflammatory processes in vivo by
PPI-2458 could be mediated through a mechanism involving the decrease of mediators of inflammation and
osteoclastogenic mediators, such as TNF␣ and RANKL.
Since progressive destruction of articular joints is
the radiographic hallmark of RA, we examined whether
the observed in vivo protection against bone resorption
afforded after treatment with PPI-2458 would correspond to changes in the architectural structure of arthritic joints. Micro-CT analysis of arthritic hind paws
demonstrated a dramatic deterioration of joint integrity
in vehicle-treated rats with PG-PS–induced arthritis,
while therapeutic dosing with PPI-2458 prevented structural joint damage, as evidenced by the suppression of
focal bone erosions at the joints and the maintenance of
bone integrity. Moreover, the protection of joint integrity was further evidenced by improvements in bone
morphometric indices, including the preservation of
local bone volume in affected joints and a decreased loss
of bone mineral density, a process commonly observed
in rats with experimental arthritis (40,41). These results suggest that the marked protection against bone
destruction afforded by PPI-2458 in this model of arthritis was mediated through a direct effect on osteoclasts,
or that these protective effects were primarily regulated
through the modulation of inflammatory processes in
arthritic joints, a pathogenetic environment known to
promote the recruitment, differentiation, and activation
of osteoclasts.
Novel antiresorptive agents that specifically target the activity of osteoclasts, such as the anti-RANKL
antibody AMG-162 (denosumab) and drugs of the
bisphosphonate class such as zoledronic acid, are currently the subjects of clinical trials for the treatment of
RA (42,43). Results from preclinical studies and clinical
trials, however, have shown that these drugs provided
minimal protection against chronic synovial inflammation ongoing in arthritic joints. Hence, it has been
suggested that the clinical use of antiresorptive drugs in
RA will require combination therapy with potent antiinflammatory DMARDs to target the chronic inflammatory state in this disease (42,43). In contrast, the demonstrated disease-modifying activity of PPI-2458 on
inflammatory and destructive processes in RA suggests
the potential use of this orally available agent as a
monotherapy.
Cartilage erosion is another important pathophysiologic process involved in the destruction of ar-
HANNIG ET AL
thritic joints. Levels of circulating COMP have been
validated for use as a biomarker of cartilage erosion in
RA, and to assess the efficacy of therapeutic interventions (44,45). Therapeutic administration of PPI-2458 at
all doses significantly reduced detectable levels of serum
COMP to below the baseline level of COMP measured
in serum of naive animals treated with vehicle. These
results demonstrated that PPI-2458 potently inhibited
accelerated cartilage turnover and cartilage surface erosion in articular joints, consistent with the histopathologic assessment of this parameter.
The protective activity of PPI-2458 against cartilage erosion could potentially result from direct effects
on chondrocytes. However, the above-suggested alternative mechanism to account for the marked protection
against bone destruction in vivo, involving the potent
inhibition of inflammatory processes, could also play an
important role in the observed cartilage protection, by
preventing the expression and activation of cartilagedegrading enzymes, such as matrix metalloproteinases.
In contrast to PPI-2458, antiresorptive therapies that
specifically target osteoclasts have shown only minor
benefits for cartilage preservation, most likely through
protection of the integrity of the subchondral bone, thus
preventing cartilage erosion from below the bone
(43,46).
In summary, PPI-2458 exhibited diseasemodifying activity in a rat model of arthritis, as demonstrated by the marked suppression of the chronic inflammatory and destructive processes observed in this model.
This potent in vivo activity was linked to the inhibition of
the molecular target of PPI-2458, methionine aminopeptidase type 2, in peripheral WBCs. Moreover, the
marked improvements in the structural preservation of
arthritic joints, through protection against bone resorption and cartilage erosion, observed after treatment with
PPI-2458 suggest a novel mechanism of diseasemodifying activity not previously associated with methionine aminopeptidase type 2 inhibition. These findings
provide a rationale for assessing the therapeutic potential of PPI-2458 in RA in clinical trials.
ACKNOWLEDGMENT
The authors thank Dr. Rasesh Kapadia (Scanco USA,
Wayne, PA) for micro-CT analysis and excellent advice.
AUTHOR CONTRIBUTIONS
Dr. Hannig had full access to all of the data in the study and
takes responsibility for the integrity of the data and the accuracy of the
data analysis.
DISEASE-MODIFYING ACTIVITY OF PPI-2458 IN ARTHRITIS
Study design. Hannig, Bernier, Hoyt, Doyle, Westlin.
Acquisition of data. Hannig, Bernier, Hoyt, Doyle, Clark, Karp,
Lorusso, Westlin.
Analysis and interpretation of data. Hannig, Bernier, Hoyt, Doyle,
Clark, Karp, Lorusso, Westlin.
Manuscript preparation. Hannig, Hoyt, Westlin.
Statistical analysis. Doyle.
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