Pharmacology & Pharmacy, 2011, 2, 17-30
doi:10.4236/pp.2011.21003 Published Online January 2011 (http://www.SciRP.org/journal/pp)
17
Pharmacodynamics of CNTO 530 and
Darbepoetin-α in Human TNF-α Transgenic Mice,
a Murine Model of Anemia of Chronic Disease
Ram Achuthanandam1, Dorie Makropoulos1, Laura Johns1, Amy Volk1, Kerry Brosnan1, Jin Lu1,
Wojciech Krzyzanski2, Peter J. Bugelski1
1Centocor R&D, Radnor, USA; 2Department of Pharmaceutics, State University of New York, Buffalo, USA.
Received October 22nd, 2010; revised November 16th, 2010; accepted December 13th, 2010.
ABSTRACT
CNTO 530 and darbepoetin-  are long lived erythropoietin receptor agonists (ERAs). Clinically, anemia of chronic
disease (ACD) is associated with increased expression of tumor necrosis factor-  (TNF-  ) and mice transgenic for
human TNF-
 develop ACD. The purpose of this investigation was to compare the effects of these agents in a murine
model of ACD. Human TNF-  expressing (Tg197) mice were administered a single subcutaneous dose of CNTO 530 or
darbepoetin-
 and the pharmacodynamic response in bone marrow spleen and peripheral blood evaluated. RBC life
span and reticulocyte age distribution were also evaluated. CNTO 530 induced a dose responsive increase in reticulo-
cytes, RBCs and Hgb in both wild type and Tg197 mice. Although the reticulocyte response was similar to wild types,
the RBC and Hgb response to CNTO 530 in Tg197 mice was blunted. There was no statistically significant difference in
RBC life span with either compound. Darbepoetin-α caused a greater peak in % dead Pro/basophilic erythroblasts,
greater peak EMH in the spleen and a greater increase in reticulocyte maturation time. In contrast, despite a similar
peak increase, CNTO 530 caused a more sustained response of reticulocyte, EMH, RBC and Hgb, consistent with in-
creased exposure. In conclusion, CNTO 530 and darbepoetin-
 increased RBC and hemoglobin in a murine model of
ACD. Compared to darbepoetin-  , CNTO 530 had a more sustained effect, consistent with increased exposure.
Keywords: Fusion Protein, Recombinant, Flow Cytometry, Erythropoiesis
1. Introduction
Anemia of chronic disease (ACD) is an important cause
of co-morbidity in rheumatoid arthritis (RA), a systemic
autoimmune disease afflicting 1-2% of the population
[1,2]. Tumor necrosis factor- (TNF-) has been shown
to play a central role in disease progression in RA (see
review by Sfikakis and Kollias [3]) and has been impli-
cated in the pathogenesis of ACD. In ACD patients there
is increased local production of TNF- in the bone mar-
row [4] and TNF- is thought to contribute to ACD by
decreasing red blood cell (RBC) survival, causing dys-
functional erythropoiesis and impairing mobilization of
iron stores [5,6].
Darbepoetin- and CNTO 530 are long-lived erythro-
poietin receptor agonists. CNTO 530 is a 58 kD glyco-
protein MIMETIBODY™ construct that has been shown
to cause a long-lived stimulation of erythropoiesis in
mice and rats [7,8]. CNTO 530 has no sequence homol-
ogy with erythropoietin (EPO). Rather, CNTO 530 in-
cludes two EMP1 sequences, a 20-amino acid peptide
that binds to EPO receptors (EPO-R), on a human IgG4
Fc framework and expresses EPO-like bioactivity [9,10]
The purpose of this study was to determine if darbepo-
etin- and CNTO 530 had beneficial effects in a murine
model of ACD.
2. Materials and Methods
2.1. Reagents and Monoclonal Antibodies
Darbepoetin- (Aranesp®, Amgen, Inc. Thousand Oaks,
CA) was purchased commercially and CNTO 530 was
supplied by Centocor R & D (Radnor, PA). Phosphate
buffered saline (PBS) without Ca++ and Mg++, fetal calf
serum (FCS), PE-anti-Ter-119, APC-streptavidin; PE-
streptavidin; FITC-annexin V, isotype-matched IgG con-

Pharmacodynamics of CNTO 530 and Darbepoetin-α in Human TNF-α Transgenic Mice,
a Murine Model of Anemia of Chronic Disease
18
trols and the viability/DNA probe 7-amino-actinomycin
D (7-AAD) were purchased from Invitrogen (Carlsbad,
CA). EZ-Link NHS biotin was purchased from Pierce
(Rockford, IL).
2.2. Mice
Heterozygous, 9 wk old Tg197 mice and CBA/C57Bl/6
F1 (CBA F1) control mice were supplied by Ace Labo-
ratories (Boyertown, PA). Tg197 mice carry a human
TNF-/β globin transgene on a C57Bl/6 background [11].
For these experiments, homozygous TNF- transgenic
males were bred to CBA females to provide heterozy-
gous mice. All mice were maintained in the pathogen-
free animal facility at Centocor, Radnor PA. The Cento-
cor Institutional Animal Care and Use Committee ap-
proved all procedures.
2.3. Hematology and Histology
Blood was collected from mice anesthetized with a CO2
mixture via open chest cardiac puncture into EDTA
coated microtubes. Analyses were performed on whole
blood using an Advia 120 blood analyzer (Siemens,
Tarrytown, NY). Spleens were collected, fixed in 10%
neutral buffered formalin and processed for paraffin sec-
tions, sectioned and stained with Giemsa using routine
methods. Extramedullary hematopoiesis (EMH) was
evaluated morphometrically. Three 4x microscope fields
from each mouse at each time point were digitally im-
aged. Polychromic/orthochromic erythroblasts (P/O EB)
were identified by their size and staining characteristics
and the percent area measured using ImagePro Plus V
5.1 (Media Cybernetics, Inc. Bethesda, MD). Mean val-
ues were calculated for each mouse. Data are shown as
group mean ± standard deviation.
2.4. Bone Marrow Cell Preparation and Analysis
Flow cytometric analysis was performed as described
previously [12,13]. Briefly, femoral bone marrow was
suspended in PBS without Ca++ or Mg++ supplemented
with 2% heat-inactivated fetal calf serum (FCS) and
0.1% NaN3. Following Fc block with anti-murine CD16/
CD32, cells were incubated with anti-Ter-119 and anti-
CD71 to discriminate erythroblasts from non-erythroid
cells and pro/basophilic erythroblasts (Pro/BasoEB) from
poly/orthochromic erythroblasts (Poly/OrthoEB). Apop-
totic and dead cells were enumerated with annexin V and
7-aminoactinomycin-D (7AAD) as per manufacturer’s
instructions kit (BD Biosciences, San Jose, CA).
Samples were analyzed on a FACSCalibur flow cy-
tometer (BD Biosciences, San Jose, CA). Data analysis
was performed with CellQuest ProTM (BD Biosciences,
San Jose, CA) and FlowJo (Treestar, Seattle, WA). Data
for at least 10,000 cells/sample were collected. A nested
analysis strategy was used to enumerate cell populations
of interest: Debris and aggregates were excluded using
FSC and SSC; apoptotoic (7AAD negative/annexin V+)
and dead cells (7AAD+) and Pro/Basophilic (Pro/Ba-
soEB, Ter119+/CD71 bright) and Poly/Orthochomic
Erythroblasts (Poly/OrthoEB, Ter119+/CD71 dim) de-
limited using a Boolean “AND” operator.
2.5. Pharmacokinetics
Normal female C57Bl/6 mice received a single sc dose of
0.3 mg/kg of CNTO 530 or darbepoetin-. Mice were
euthanized (3-6 mice per time point) and blood samples
collected by cardiac puncture into EDTA-coated micro-
tubes. A total of 63 mice were used in this analysis. The
plasma concentration of CNTO 530 was measured as
described previously [12]. The plasma concentration of
darbepoetin- was measured in mouse plasma using a
double-antibody sandwich ELISA (R&D Systems, Inc.,
Minneapolis, MN) adapted for use with darbepoetin-
and murine plasma as described previously for recombi-
nant human EPO [7].
2.6. RBC Lifespan
An in vivo biotinylation technique was used to determine
RBC survival [14,15]. On Day 1, groups of Tg197 mice
(5-6/group) received a single subcutaneous (sc) dose of
saline, CNTO 530 or darbepoetin- (0.3 mg/kg). On
Days 2, 3 and 4, the mice received EZ-Link Sulfo-NHS
biotin (1 mg/mouse, intravenous in 200 L PBS). Blood
samples (25 L) were collected from the retro-orbital
sinus (EDTA anticoagulant) on 1, 3, 6, 10, and 17 days
after the last injection of biotin reagent. Blood (5 L)
was mixed with 1 mL of 5 g/mL of streptavidin-PE in
PBS, and incubated for 30 min. After a wash, the cells
were resuspended in 200 uL of PBS and analyzed by
flow cytometry. Data for mean % biotinylated RBC was
plotted as survival fraction vs. time. The initial slope 0 S
of such a survival curve determines the mean RBC life-
span TRBC according to the equation [16]:
0
1
RBC
TS 
Although derived under homeostatic conditions, this
relationship has been shown to be accurate for a non-
stationary applications [17]. To determine the initial
slope the RBC survival curve for each animal was fitted
with the liner-exponential model [18,19]:
 

00
0
1, ,
1, .
t
t
StTS eiftT
SF tSe iftT








 





Pharmacodynamics of CNTO 530 and Darbepoetin-α in Human TNF-α Transgenic Mice,
a Murine Model of Anemia of Chronic Disease
19
where  is the first-order rate constant of RBC random
destruction, T denotes the RBC lifespan due to senes-
cence, and 0 
  1 is the weight factor between these
two processes of the RBC removal. The parameters T, ,
and S0 were estimated, and S0 was calculated as follows:

0
1
1
ST




The nonlinear regression was performed by WinNonlin
5.0 (Pharsight, Mountain View, CA).
2.7. Reticulocyte Age Distribution
Oxazine 750 has been used as a marker of reticulocyte
age. The fluorescent intensity signal s is correlated to the
levels of RNA present in the reticulocyte. If the reticulo-
cyte RNA degrades at a first-order rate, one can establish
a relationship between the reticulocyte age and s, know-
ing that s is proportional to the RNA content [20]. To
determine the absolute reticulocyte age, the signal from
the bone marrow reticulocytes is necessary. Thus, the
signal s0 at which a reticulocyte becomes a mature RBC
has been used as a reference, and an absolute reticulocyte
age/maturity has been replaced by a relative time to be-
come RBC (RT) according to the following equation:
1/2
0
ln
ln 2
RNA
t
s
RT
s



where t1/2RNA is the half-life of the RNA degradation in
reticulocytes. RT densities were calculated at times t = 1,
2, 4, 10, and 21 days after test article administration. The
RT density r(RT) function is defined as [20]:
 
#,
,a
ofreticulocytes ofRTaaa
rta Unit Volume ofblood



where, Δa is an infinitesimally small age increment. The
RT densities were approximated by the product of abso-
lute reticulocyte count at time t and the frequency histo-
gram for RT at time t:
  
*,
,RETthistt RT rtRTRT 
where, ΔRT is the length of the histogram bin. The pro-
duction rate of mature RBC at time t was calculated as:
 
0
max ,
RET RT
kt rtRT


and the maximal value of the RT density was used to
approximate the reticulocyte release rate from the bone
marrow to blood:
 
0
max ,
RET RT
kt rtRT


The mean RT was calculated from the histogram hist (t,
RT). The median of r(RT) was interpreted as the median
cell age (mid age of reticulocytes). Since the half-life of
the RNA degradation in Tg197 transgenic mice has not
been determined, RT was expressed in t1/2RNA units. The
ADVIA 120 FCS files were converted to ASCII files
containing reticulocyte oxazine 750 absorption intensity
data using FCS Express v. 3 (De Novo Software). The
RT densities, and other reticulocyte related parameters
were obtained using MATLAB 7.6 (MathWorks, Natick,
MA).
2.8. Statistical Analysis
Data were expressed as means +/– standard deviation.
Significant differences between experimental groups
were determined by ANOVA (Bonferroni’s correction).
P values less than 0.05 were considered significant.
3. Results
3.1. Dose Response of CNTO 530 in Tg197 Mice
As shown in Table 1, Tg197 mice have a mild compen-
sated, hypochromic aqnemia. To establish the dose re-
sponse for CNTO 530, female Tg197 mice (4/group)
received a single subcutaneous (sc) dose of CNTO 530 at
0.03, 0.1 or 0.3 mg/kg. Control Tg197 mice received a
single sc dose of PBS. As shown in Figures 1(a)-(c),
CNTO 530 caused a dose responsive increase in reticu-
locytes, RBC and Hgb in Tg197 mice. Based on these
results, a dose of 0.3 mg/kg was chosen for the remaining
experiments.
3.2. Comparative Effects of CNTO 530 in
Normal and Tg197 Mice
To compare the response of normal (CBF1) and Tg197
mice to CNTO 530, 9 week-old female mice (3/group)
received a single sc dose of 0.3 mg/kg. Control mice re-
ceived a single sc dose of PBS. As shown in Figure 2(a),
the reticulocyte response to CNTO 530 in CBF1 and
Table 1. Combined control values for normal and Tg197
mice.
Parameter CBF1 Tg197
Reticulocytes 210 ± 27 256 ± 65*
Total RBC 9.7 ± 0.3 9.6 ± 0.7
MCV 54.2 ± 0.5 53.1 ± 1.8
MCH 16.4 ± 0.7 15.5 ± 0.9*
Hgb 15.8 ± 0.4 14.8 ± 0.9*
*Significantly different from CBF1 (t-test).

Pharmacodynamics of CNTO 530 and Darbepoetin-α in Human TNF-α Transgenic Mice,
a Murine Model of Anemia of Chronic Disease
20
(a)
(b)
(c)
Figure 1. Dose response for CNTO 530 in Tg197 mice. (a), (b) and (c) show reticulocyte, RBC and HgB responses to CNTO
530 as a function of time. *statistically significant difference from control.

Pharmacodynamics of CNTO 530 and Darbepoetin-α in Human TNF-α Transgenic Mice,
a Murine Model of Anemia of Chronic Disease
21
(a)
(b)
(c)
Figure 2. Comparative pharmacodynamics of CNTO 530 in normal CBF1 and Tg197 mice. (a) reticulocyte response. (b) RBC
response. (c) Hgb response. *statistically significant difference from untreated control. **statistically significant difference
from Tg197 mice.

Pharmacodynamics of CNTO 530 and Darbepoetin-α in Human TNF-α Transgenic Mice,
a Murine Model of Anemia of Chronic Disease
22
Tg197 mice was similar. However, as shown in Figures
2(b) and 2(c), the RBC and Hgb response was attenuated,
suggesting that Tg197 mice are resistant to CNTO 530.
3.3. Comparative Pharmacokinetics of CNTO
530 and Darbepoetin-
Female C57Bl/6 mice (5-6/group) received a single sc
dose of CNTO 530 or darbepoetin- (0.3 mg/kg) and
blood samples collected from 2 hrs to 10 days after dos-
ing. Plasma concentrations are shown in Figure 3 and
the pharmacokinetic parameters in Table 2. Compared to
darbepoetin-, there was a statistically significant in-
crease in the area under the curve (AUC) and terminal
half-life for CNTO 530.
3.4. Pharmacodynamics of CNTO 530 and
Darbepoetin- in Tg197 Mice
Female Tg197 mice (4/group) received a single sc dose
of CNTO 530 or darbepoetin- (0.3 mg/kg) and blood,
bone marrow and spleens were collected 1, 2, 4, 10 and
Figure 3. Comparative pharmacokinetics of CNTO 530 and
darbepoetin- in normal mice.
Table 2. Pharmacokinetics of CNTO 530 and darbepoetin-
following a single subcutaneous dose in mice.
Parameter CNTO 530
(mean ± SD)
Darbepoetin-
(mean ± SD)
Cmax (ug/mL) 2.2 ± 0.5 2.9 ± 0.5
Tmax (hr) 20 ± 8 19 ± 5
AUCt (ughr/mL) 152 ± 28* 80 ± 10
AUC (ughr/mL) 160 ± 20* 95 ± 11
t1/2 (hr) 41 ± 9* 14 ± 4
*Statistically greater than darbepoetin-.
21 days after dosing. Controls received a single sc dose
of PBS. As shown in Figure 4(a), CNTO 530 and dar-
bepoetin- caused a similar peak reticulocyte response.
However, the reticulocyte response to CNTO 530 was
longer-lived. CNTO 530 and darbepoetin- also caused
an increase in RBC (Figure 4(b)) and Hgb (Figure 4(c)).
Despite a similar peak effect on RBC and Hgb, the ef-
fects on CNTO 530 were significantly longer-lived than
the effects of darbepoetin-.
Representative scatter plots showing CD71 vs. Ter119
and annexin-V vs. 7-AAD for bone marrow from Tg197
mice are shown in Figures 5(a) and (b). As shown in
Figure 5(c), with both test articles the relative percent-
ages of Pro/BasoEB initially fell and then recovered to
control values by Day 10 and then again fell below con-
trol values on Day 21. Similarly, Poly/OrthoEB percent-
ages also initially fell below control values and then re-
covered for darbepoetin- or rose above control values
for CNTO 530 by Day 10 (Figure 5(d)). Poly/OrthoEB
were elevated for both test articles on Day 21. The results
of the analysis of apoptosis and cell death in the bone
marrow are shown in Figures 5(e) and (f). Both test arti-
cles transiently increased cell death in Pro/BasoEB. In
contrast, neither test article had a significant effect on
apoptosis of Pro/BasoEB until Day 21 when apoptosis
was decreased. Neither test article had an effect on apop-
tosis or cell death in Poly/OrthoEB at any time point
evaluated (Data not shown).
In the spleen, animals treated with CNTO 530 or dar-
bepoetin- showed an expansion of extramedullary he-
matopoiesis (EMH) (Figure 6). On Day 1, erythroid pre-
cursors were the predominant cell types and were found
in clusters in the red pulp. In these clusters, some cells
could be identified as Pro/BasoEB, however many cells
appeared to be more primitive, with large pale nuclei that
contained multiple nucleoli. This early progenitor popu-
lation was the predominant cell type in roughly half of
the animals. By Day 2, the primitive population and the
Pro/BasoEB had expanded to a high degree and mitotic
figures could be observed in the early progenitors. By
Day 4, EMH peaked in both groups of treated animals.
At this time, there was a shift from the primitive cells
toward Pro/BasoEB. Additionally, the number of Poly/
OrthoEB and mature RBC had expanded to nearly the
degree of the earlier precursors. There also was also an
increase in apoptotic bodies in both groups of treated
animals compared with control animals. Although the
effects of CNTO 530 and darbepoetin- were similar on
Days 1 and 2, by Day 4 the degree of EMH was greater
in animals treated with darbepoetin- relative to those
treated with CNTO 530. By Day 10, there was a striking
difference between CNTO 530 and darbepoetin--treated

Pharmacodynamics of CNTO 530 and Darbepoetin-α in Human TNF-α Transgenic Mice,
a Murine Model of Anemia of Chronic Disease
23
(a)
(b)
(c)
Figure 4. Comparative pharmacodynamics of CNTO 530 and darbepoetin in Tg197 mice. (a), (b) and (c) show reticulocyte,
RBC and HgB responses to CNTO 530 as a function of time. *statistically significant difference from untreated control.
**significantly different from control and darbepoetin-.

Pharmacodynamics of CNTO 530 and Darbepoetin-α in Human TNF-α Transgenic Mice,
a Murine Model of Anemia of Chronic Disease
24
(a) (b)
(c) (d)
(e) (f)
Figure 5. Comparative effects of CNTO 530 and darbepoetin- in the bone marrow of Tg197 mice. (a) representative scatter
plot showing flow cytometric discrimination of erythroid precursors. Proerythroblasts and basophilic erythroblasts
(Pro/BasoEB) are identified as Ter119 high/CD71+. Polychromic and orthochromic erythroblasts (Poly/OrthoEB) are identi-
fied as Ter199 hi/CD71-. (b) representative scatter plot showing flow cytometric discrimination of live, apoptotic and dead
erythroid precursors. Live cells are identified as AnnexinV-/7AAD-, apoptotic cells as AnnexinV+/7AAD- and dead cells as
7AAD+. (c) comparative pharmacodynamic effect of CNTO 530 and darbepoetin- on Pro/BasoEB. (d) comparative phar-
macodynamic effect of CNTO 530 and darbepoetin- on Poly/OrthoEB. (e) and (f) comparative pharmacodynamic effects on
% apoptotic and % dead Pro/BasoEB. *statistically significant difference from untreated control. **significantly different
from control and darbepoetin-.

Pharmacodynamics of CNTO 530 and Darbepoetin-α in Human TNF-α Transgenic Mice,
a Murine Model of Anemia of Chronic Disease
25
(a) (b) (c)
(d) (e) (f)
(g)
Figure 6. Comparative pharmacodynamic effects of CNTO 530 and darbepoetin- on extramedullary erythropoiesis (EMH).
Photomicrographs of the red pulp of formalin fixed, paraffin embedded, Giemsa stained sections of the spleen from Tg197
mice. (a) control Day 4, (b) CNTO 530 Day 4, (c) darbepoetin- Day 4, (d) CNTO 530 Day 10, (e) darbepoetin- Day 10 and
(f) CNTO 530 Day 21. Basophilic erythroblasts are recognized as the large deeply blue stained nuclei. Bar = 100 m. (g)
morphometric analysis of EMH. CNTO 530 caused a longer-lived increase in expansion of P/OEB in the spleen.
animals. While there remained significant EMH in ani-
mals treated with CNTO 530, EMH was reduced in the
darbepoetin- group. In the CNTO 530 treated animals,
early stage progenitors were still present, however the
overall EMH had shifted to later stage Poly/OrthoEB and
there were widespread areas of mature RBCs. Scattered
apoptotic bodies could be observed in this group. In the
darbepoetin--treated animals, the progenitor population
was mixed, with sporadic progenitors at varying stages
present at low frequency and there were abundant mature

Pharmacodynamics of CNTO 530 and Darbepoetin-α in Human TNF-α Transgenic Mice,
a Murine Model of Anemia of Chronic Disease
26
RBC. By Day 21, the control animals were also showing
moderate EMH, probably reflecting a response to disease
progression. At this time, there were no notable differ-
ences between the CNTO 530 and darbepoetin- treated
animals.
In peripheral blood, increases in reticulocytes are ob-
served by Day 1for both compounds, and by Day 4 there
is a ~6 fold increase in circulating reticulocytes. After
Day 4, the levels of reticulocytes reflect the pharma-
cokinetic properties of each of the molecules as shown in
Figure 4. The increase in reticulocytes is followed by a
delayed increase in RBCs observed at Day 4. Again, the
RBC profiles reflect the pharmacokinetic properties with
increased RBC levels observed up till Day 21 in CNTO
530 treated group.
3.5. Effects of CNTO 530 and Darbepoetin- on
RBC Life Span
As shown in Figure 7(g), the surviving fraction of RBC
was significantly lower than control for both treatment
groups on Day 1 and significantly higher than control for
the CNTO 530 group on Day 17. There was no statisti-
cally significant effect of either test article on mean RBC
lifespan: control = 22 ± 6 days; CNTO 530 = 24 ± 2 days
and darbepoetin- = 19 ± 1 days.
3.6. Effects of CNTO 530 and Darbepoetin- on
Reticulocyte and RBC Production Rates and
Reticulocyte Age Distribution
The reticulocyte rates of release to the blood are pre-
sented in Figure 7(d). For treated mice kRET gradually
increased up to Day 2, to rapidly increase within the next
two days, and reached a peak on Day 4. Following the
peak, the production rates declined to reach the values
below the control group on Day 21. The kRET for mice
that received PBS was at the same level of about 0.34 ±
0.08 1012cells/L/t1/2RNA. Darbepoetin- increased kRET
higher than CNTO 530 (2.07 ± 0.16 vs. 1.71 ± 0.36
1012cells/L/t1/2RNA) but the difference was not statistically
significant. This effect of CNTO 530 on kRET lasted
longer than darbepoetin-. Both darbepoetin- and
CNTO 530 resulted in kRET values that were significantly
less than the control group (0.21 ± 0.04 and 0.16 ± 0.05
1012cells/L/t1/2RNA, respectively) at Day 21.
The time courses of the mean time for reticulocyte to
become mature RBC (RT) for the three groups of mice
are shown in Figure 7(e). For control mice, the mean
RT(mRT) values remained at the same level of 4 t1/2RNA.
The mRT values for both CNTO 530 and darbepoetin-
groups reached the peaks of 7.4 ± 0.6 t1/2RNA and 9.3 ± 0.9
t1/2RNA, respectively, on Day 2 which were significantly
different. The time courses of the mRT following the
peak were different for two groups. The RT values for
mice treated with darbepoetin- declined more rapidly
and reach a nadir of 2.9 ± 0.6 t1/2RNA on Day 10 and
reached a plateau at this value, whereas the mean for the
CNTO 530 group gradually declined to reach 3.5 ± 0.4
t1/2RNA on Day 21. The mRT on Day 21 for both treatment
groups was significantly smaller than the mRT value for
the control group.
The mature RBC production rate vs. time plot is
shown in Figure 7(f). The kRBC values for the PBS group
were relatively constant 0.16 ± 0.03 1012cells/L/t1/2RNA.
The time courses of kRBC for treated animals exhibited a 2
day lag prior to a rapid increase followed by a gradual
decline. The peak of 0.46 ± 0.08 1012cells/L/t1/2RNA for the
darbepoetin- group occurred on Day 4 whereas the peak
of 0.46 ± 0.04 1012cells/L/t1/2RNA for the CNTO 530 group
was on Day 10. The peak values were not significantly
different. This effect of CNTO 530 on kRBC lasted longer
than darbepoetin-. The kRBC values on Day 21 for the
treated animals were significantly lower than for the PBS
group.
4. Discussion
In our studies in huTNF- transgenic mice, it was ob-
served that CNTO 530 and darbepoetin- caused a dose
responsive stimulus of erythropoiesis. Moreover, CNTO
530 had a superior pharmacokinetic and pharmacody-
namic profile compared to darbepoetin- in this model
where the mice were relatively resistant to CNTO 530
(blunted response) compared to normal mice at the same
dose level.
The model of ACD used in these experiments, Tg197
mice, are transgenic for human TNF- and develop a
progressive polyarthritis starting at 4-5 weeks of age [1].
Arthritis is well established by 10 weeks of age and ane-
mia is correlated with disease progression and huTNF-
concentrations in the serum, despite the presence of ade-
quate iron stores in the spleen and normal levels of en-
dogenous erythropoietin [2]. That TNF- can induce
anemia in mice has been reported previously. Single in-
traperitoneal doses of murine TNF- have been reported
to cause a decrease in hematocrit [3] and transplantation
of CHO cells transfected with a human TNF- gene
caused anemia within 3 weeks [4]. Similarly, mice trans-
genic for human TNF- developed a microcytic anemia
at 16 weeks of age [5].
When compared to normal mice, Tg197 mice were
relatively resistant to CNTO 530. In contrast to our pre-
vious results in normal mice where a single sc dose of
epoetin- or CNTO 530 caused an expansion of Pro/
BasoEB in bone marrow [6,7], in Tg197 mice CNTO 530
and darbepoetin- caused a decrease in Pro/BasoEB. As

Pharmacodynamics of CNTO 530 and Darbepoetin-α in Human TNF-α Transgenic Mice,
a Murine Model of Anemia of Chronic Disease
27
(a) (b) (c)
(d) (e)
(f) (g)
Figure 7. Comparative effects of CNTO 530 and darbepoetin- on reticulocyte and RBC dynamics. (a) representative scatter
plot showing flow cytometric discrimination of recticulocytes, and its age distribution. Data are taken from the Advia 120
from Day 10 and show oxizine staining (absorption) plotted against forward scatter. Gate R1 + R2 represents total RBCs +
Reticulocytes with R2 showing the age of reticulocytes. (b)-(c) representative scatter plot showing flow cytometric discrimina-
tion of biotinylated RBC. 7(b) shows the delineation of RBCs (R3) and (R4) showing the percentage of RBCs that are bioti-
nylated. On Day 1, mice received a single sc dose of saline, CNTO 530 or darbepoetin- (0.3 mg/kg). On Days 2, 3 and 4, the
mice received EZ-Link Sulfo-NHS biotin. (d) production rate of reticulocytes (RT). The rate of production was calculated as
described in Materials and Methods (Units = 1012 Cells/L/t1/2RNA). (e) reticulocyte maturation (kRET). The time to for a
reticulocyte to mature into an RBC was calculated as described in Materials and Methods (Units = t1/2RNA). (f) production
rate of RBC (RT). The rate of production was calculated as describe in Materials and Methods (Units = 1012 Cells/L/t1/2RNA).
(g) surviving fraction of RBC (RBC SF) was calculated as describe in Materials and Methods (Units = % biotinylated RBC).
*statistically significant difference from untreated control. **significantly different from control and darbepoetin-.

Pharmacodynamics of CNTO 530 and Darbepoetin-α in Human TNF-α Transgenic Mice,
a Murine Model of Anemia of Chronic Disease
28
reported previously, a single intraperitoneal dose of
murine TNF- caused a decrease in the number of col-
ony forming units in the spleen and bone marrow in mice
[3]. Similarly, in human ACD there is a decrease in the
number of erythroblasts and increased apoptosis in the
bone marrow [8] and a relative resistance to EPO [9]. As
the murine p75 TNF-receptor is selective for murine
TNF- and fails to mediate signaling from human
TNF- [10], resistance to CNTO 530 may be mediated
through the p55 TNF-receptor.
Previously, darbepoetin- has been shown to be active
in a model of ACD in rats [11]. In our study, we have
found that compared to darbepoetin-, CNTO 530 has a
superior pharmacodynamic effect on RBC and Hgb in a
murine model of ACD. In an attempt to elucidate the
mechanism of the pharmacodynamics we have evaluated
the effects of CNTO 530 and darbepoetin- on the bone
marrow, EMH, reticulocyte production and maturation
and RBC life span. The superior pharmacodynamic ef-
fects of CNTO 530 are unlikely to be due to differential
effects on the bone marrow. Neither test article caused an
expansion of medullary hematopoiesis. Similarly, both
test articles caused an increase in cell death in Pro/Ba-
soEB that followed a roughly similar time course. More-
over, neither test article had an effect on apoptosis in
Pro/BasoEB nor an effect on apoptosis/cell death in Poly/
OrthoEB. Apoptosis and cell death have been shown
previously to play a critical role in controlling erythro-
poiesis and it is well established that deprivation of EPO-R
stimulation leads to apoptosis of erythroid precursors [12,
13] However, as we have reported previously in normal
mice [6,7], a single sc dose of epoetin- or CNTO 530
caused an increase in cell death in erythroblasts. Simi-
larly, Chang et al. [14] reported increased apoptosis/cell
death in erythroblasts in mice receiving three doses of
recombinant murine EPO. Increased apoptosis/cell death
was also reported by Robinson et al. [15] in erythroblasts
in mice following hemorrhagic shock, despite a strong
endogenous EPO response. Taken together, these find-
ings suggest that apoptosis and cell death play a role in
erythroid homeostasis when EPO-R mediated stimulation
of erythropoiesis is either high or low.
Our data also suggest that the superior pharmacody-
namics of CNTO 530 cannot be attributed to a differen-
tial effect on EMH. Both test articles caused an expan-
sion of EMH, albeit with somewhat different kinetics.
Repeated dosing with epoetin- has been shown to in-
crease EMH in mice and rats [16,17] Similarly, Kapa et
al. [18] found that the response to exogenous EPO after
hypoxia was more pronounced in the spleen than in the
bone marrow. This is consistent with the expansion of
EMH in mice receiving either CNTO 530 or darbepo-
etin- and suggests that in Tg197 mice as has been de-
scribed previously in normal mice [17], the spleen pro-
vides a better microenvironment for EPO mediated ex-
pansion of erythropoiesis than the bone marrow.
Both darbepoetin- and CNTO 530 affected reticulo-
cyte maturation and RBC production profiles. Though,
there was not a statistically significant difference in the
number of reticulocytes in the periphery at Day 1 and 2,
a difference in reticulocyte maturation was observed at
these time points. Both test articles induced release of
immature reticulocytes into the periphery, without
changing the reticulocyte production. Moreover, a de-
crease in RBC production rate is concomitantly observed
with the increase in immature reticulocytes. This sug-
gests that not only are immature reticulocytes released
into the periphery, but both compounds delay the matu-
ration of the reticulocytes to RBCs. However, the com-
pounds did not affect the RBC life span with treatment,
making it unclear how the delayed maturation process of
reticulocytes aided in production of better RBCs.
When we compare the two erythropoietin receptor
agonists, they have similar mode of action in this model
of ACD. Having ruled out fundamentally different ef-
fects on most aspects of erythropoiesis, we are left with
differences in the duration of the stimulation of erythro-
poiesis between the two compounds. Pharmacokinetic
analysis showed that the terminal t1/2 and AUC for
CNTO 530 was significantly greater than darbepoetin-,
resulting in a longer duration of exposure to CNTO 530.
This longer duration of exposure was reflected in the
prolonged increase in RT, kRET, kRBC and reticulocytosis
seen with CNTO 530 and suggests that the differential
effects of CNTO 530 and darbepoetin- on RBC and
Hgb may simply reflect longer lasting effects on the final
stages of erythropoiesis.
5. Acknowledgements
The authors thank Renold Capocasale for generating the
flow cytometry data. All authors (except WK) are em-
ployees of Centocor R&D. CNTO 530 was discovered by
Centocor R&D.
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