Etanercept, a TNF-alpha Binding Agent

Jonathan M Buscaglia
1
, Brian W Simons
2
, Brent J Prosser
1
, Dawn S Ruben
2
,
Samuel A Giday
1
, Priscilla Magno
1
, John O Clarke
1
, Eun Ji Shin
1
, Anthony N Kalloo
1
,
Sergey V Kantsevoy
1
, Kathleen L Gabrielson
2
, Sanjay B Jagannath
1
1Division of Gastroenterology and Hepatology and 2Department of Molecular and Comparative
Pathobiology, Johns Hopkins University School of Medicine, Johns Hopkins Hospital.
Baltimore, MD, USA
ABSTRACT
Context The incidence of post-ERCP
pancreatitis is 1-22%. It continues to be a
difficult problem for endoscopist and patient.
Uncovering an agent that may be used to
prevent its occurrence is critical.
Objective The aim of our study was to
investigate the role of etanercept in the
prevention of post-ERCP pancreatitis.
Design
Endoscopic
retrograde
pancreatography (ERP)-induced injury was
performed in dogs using a previously
established endoscopic model of post-ERCP
pancreatitis.
Animals Eight study dogs underwent ERP: 4
were pre-treated with etanercept one day
before the procedure and 4 were untreated. In
addition, three control dogs not undergoing
ERP were also studied.
Main outcome measures Serum levels of
amylase, lipase, and TNF-alpha, as well as the
ratio of urinary trypsinogen activation peptide
(TAP) and urinary creatinine, were measured
before and after ERP. Necropsy was
performed on post-operative day 5. All
pancreatic specimens were graded by two
blinded pathologists according to a validated
scoring system.
Results Eight study dogs developed mild to
moderate clinical pancreatitis with
hyperamylasemia (11,538±4,065 U/L vs.
701±157 U/L; post-ERP peak levels vs.
baseline values: P<0.001) and hyperlipasemia
(3,637±2,333 U/L vs. 246±125 U/L;
P=0.003). Mean total injury score was
significantly elevated in study dogs compared
to control dogs (6.16±1.85 vs. 1.06±0.49;
P=0.001). There were escalating total injury
scores concordant with more elaborate
methods of endoscopically-induced injury
although the trend did not reach the statistical
significance (P=0.223). When comparing
untreated to etanercept-treated dogs, there
were no significant differences in serum
amylase levels (P=0.903), serum lipase levels
(P=0.771), TAP/creatinine urinary ratio
(P=0.912), and pancreatic injury score
(P=0.324).
Conclusion Etanercept is ineffective in
prevention of mild to moderate post-ERCP
pancreatitis in canines. ERP-induced
pancreatic injury can be used as a reliable
animal model for studies investigating therapy
and prevention of post-ERCP pancreatitis.

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INTRODUCTION
Since the inception of endoscopic retrograde
cholangiopancreatography (ERCP) in the
early 1970’s, physicians have described the
unfortunate complication of pancreatitis
following this procedure [1]. Although it
occurs in the minority of patients, it is
completely iatrogenic, and it may cause
significant morbidity, or even mortality [2].
Post-ERCP pancreatitis is said to occur in
approximately 1-22% of all cases, yet in some
patient populations that are at high-risk, its
incidence may be as high as 20-30% [3].
In acute pancreatitis, the activation of
inflammatory cells with the release of
cytokines plays an important role in the
disease process [4]. Several studies have
demonstrated the unique characteristics of
different mediators such as interleukin-6 (IL-
6), IL-8, and IL-10 [5, 6, 7, 8]. The
significance of tumor necrosis factor (TNF) in
acute pancreatitis, however, is less well-
established. Although some have shown the
concentration of soluble TNF receptors to
correlate with disease severity [9], others have
found significant levels in only the early
stages of severe acute pancreatitis [10]. In a
rat model of acute pancreatitis, the chimeric
TNF antibody, infliximab, was found to
significantly decrease serum amylase activity
and pancreas histopathologic scores [11].
More recently, etanercept (a soluble TNF-
alpha binding agent) has been shown to
attenuate the development of acute
pancreatitis in a murine model of disease [12].
The primary aim of our study was to
investigate the role of etanercept in the
prevention post-ERCP pancreatitis using a
previously established endoscopic model of
disease in canines [13]. The secondary aim
was to ascertain levels of TNF-alpha in mild
to moderate acute pancreatitis resulting from
ERCP.
MATERIALS AND METHODS
Pre-Procedure
We performed endoscopic retrograde
pancreatography (ERP) procedures on eight
consecutive male 25-35 kg hound dogs (Canis
familiaris). Animals undergoing ERP were
divided into one of two categories: untreated
group and treatment group. Those assigned to
the treatment group received etanercept
(Amgen Co., Thousand Oaks, CA, USA) at a
dose of 1 mg/kg administered subcutaneously
dorsal to the scapula one day prior to the
procedure. Those dogs in the untreated group
did not receive any injection before ERP.
Twenty-four hours prior to the procedure, all
dogs were fed a standard canine lab diet and
allowed free access to water. Twelve hours
prior to the procedure, all solid food was held.
On the morning of the ERP, pre-anesthesia
medication was administered consisting of an
intramuscular injection of 0.03 mg/kg
acepromazine (10 mg/mL; Fort Dodge Inc.,
Ames, IA, USA). Thirty minutes later an
intravenous catheter was placed in the foreleg
cephalic vein of each dog, and the following
was administered: 100 mg/mL Telazol
(tiletamine HCl plus zolazepam HCl; Leerle
Parenterals Inc., Carolina, Puerto Rico)
reconstituted with 2.5 mL of 100 mg/mL
ketamine HCl and 2.5 mL of 100 mg/mL
xylazine at a total dose of 0.02 mL/kg. All
procedures were performed under 1.5% to
2.0% isoflurane general anesthesia with
endotracheal intubation.
Once on the operating table, all animals had
continuous monitoring of end tidal carbon
dioxide, peripheral pulse and oxygen
saturation by pulse oximetry. A standard 8-F
Foley catheter was inserted through the
urethra and into the bladder. After the internal
balloon was inflated with 3 mL of water, the
external end and stop-cock portions of the
catheter were sutured in place to the dog’s
skin surface. Baseline urine was collected,
and blood samples were drawn from the
internal jugular vein. All animals were given
0.9% NaCl saline solution throughout the
procedure.
Procedure
A standard overtube (Olympus Optical Co.
Ltd., Tokyo, Japan) was advanced into the
dog’s stomach with a standard upper
endoscope (GIF-160, Olympus Optical Co.
Ltd., Tokyo, Japan) inside the tube. All

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remaining gastric contents were adequately
suctioned out through the endoscope. Once
the tip of the overtube was positioned in the
distal esophagus, the endoscope was
withdrawn. A regular, adult size, side-viewing
duodenoscope (TJF-160, Olympus Optical
Co. Ltd., Tokyo, Japan) was then advanced
down through the pylorus and into the
proximal intestine. The intestinal mucosa was
scanned in order to locate the minor duodenal
papilla. The minor papilla serves as the main
duct of drainage in the canine pancreas. The
more proximal major papilla acts as an
accessory drainage duct, and it joins the bile
duct prior to emptying into the duodenal
lumen [14].
After locating the minor papilla, a standard 7-
F endoscopic sphincterotome (Tri-tome®,
Cook Endoscopy, Winston-Salem, NC, USA)
loaded with a 0.035-inch guidewire
(Jagwire®, Boston Scientific Corp., Natick,
MA, USA) was used to cannulate the
papillary orifice. Following cannulation, 1-2
mL of contrast (Omnipaque®, Amersham
Health, Princeton, NJ, USA) was injected into
the pancreatic duct in order to confirm proper
position. Once successful access to the
pancreatic duct was obtained, ERP was
performed in one of four manners based on
our previously established endoscopic model
of disease [13]. Method 1: pancreatic duct
acinarization with 20 mL of contrast; method
2: pancreatic duct acinarization with 30 mL of
contrast; method 3: pancreatic duct
acinarization with 30 mL of contrast, plus 5-
minute balloon occlusion of the papillary
orifice, plus endoscopic pancreatic
sphincterotomy; method 4: pancreatic duct
acinarization with 30 mL of contrast, plus
injection of 3 g of ursodeoxycholic acid
(powder) mixed in 10 mL of sterile water,
plus 5-minute balloon occlusion of the
papillary orifice, plus endoscopic pancreatic
sphincterotomy. Balloon occlusion was
performed with a 6 mm biliary dilating
balloon (Bard Inc., Covington, GA, USA) in
all cases. Sphincterotomy was performed
using 40 W blended current electrocautery
(Valley Lab-Electrosurgery Unit, Tyco Corp.,
Boulder, CO, USA).
Following ERP all dogs were extubated and
allowed to recover from anesthesia in their
own cages. They were examined at regular
intervals by a member of the veterinary staff
and assessed for pain or evidence of
procedure-related complications. Analgesic
medicine for animal discomfort was
administered at the discretion of the
examining staff member.
All dogs were sacrificed for necropsy on post-
operative day 5, or sooner if clinical
symptoms of pancreatitis were felt to be
severe or inhumane.
Controls
Three identical species dogs that did not
undergo ERP, abdominal intervention, or
etanercept injection were euthanized, and
their pancreas was obtained at necropsy to
serve as controls. Control dogs were used in
other animal institutional review board-
approved trials involving non-GI protocols.
Serum and urine were not collected in control
dogs.
Sample Collection
Urine samples were aspirated from the
urinary catheter of each dog in order to
measure trypsinogen activation peptide (TAP;
nM) (TAP Assays, Biotrin Inc., Dublin,
Ireland) levels and urine creatinine (mg/dL)
levels (Antech Diagnostics, Lake Success,
NY, USA). Samples were collected at
baseline, immediately following ERP, and
then 30 min, 3 h, 6 h, and 12 h post-
procedure. The dog’s bladder was completely
emptied after each sample was obtained.
Individual urinary TAP levels (nM) were
divided by the corresponding urine creatinine
level (mg/dL) in order to obtain the urinary
TAP/creatinine ratio. The peak urinary
TAP/creatinine values of the five samples
collected after ERP were recorded in each
dog.
Serum levels of amylase and lipase were also
measured (Antech Diagnostics, Lake Success,
NY, USA). Samples were obtained at baseline
and 2 h post-procedure. Additional samples
were collected on days 1, 2, and 5 following
ERP. The peak amylase and lipase values of

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the four samples collected after ERP were
recorded in each dog.
Cytokine Analysis
Serum samples for TNF-alpha measurement
were obtained at the identical time intervals as
amylase and lipase. Blood was collected,
centrifuged at 3,000 rpm for 15 minutes,
aliquoted and stored in a -70°C freezer until
analyzed. Serum levels of canine TNF-alpha
were determined using a commercially
available EIA kit (R&D Systems,
Minneapolis, MN, USA). Serum samples
were tested in duplicate according to the
manufacture's instructions. The plates were
read immediately after application of the stop
solution. The optical density of each sample
was determined using VERSAmax® tunable
microplate reader (Molecular Devices,
Sunnyvale, CA, USA). Results were
calculated from a standard curve and reported
accordingly in pg/mL. The minimum
detectable dose for TNF-alpha is 2.4 pg/mL.
Histology
Following euthanasia, the pancreas was
resected and fixed in 10% buffered formalin.
The pancreas was systematically divided into
six transverse sections representing the
following areas: distal left lobe, proximal left
lobe, body of the pancreas at the major
papilla, body of the pancreas at the minor
papilla, proximal right lobe, and distal right
lobe. The sections underwent routine
histological processing, stained with
hematoxylin and eosin, and evaluated with
light microscopy by two pathologists in
tandem who were blinded to the treatment
arm for each dog. Each section was scored for
eight different categories of pancreatic injury:
neutrophilic inflammation, mononuclear
inflammation, acinar cell necrosis, fibrosis,
acinar cell atrophy, fat necrosis, edema, and
hemorrhage. A previously described and
validated pathologic scoring system [15] was
used with the following modifications: acinar
cell necrosis was defined as necrosis of
individual acinar cells, and mononuclear
inflammation was defined as the presence of
lymphocytes, plasma cells, or macrophages
within the pancreatic parenchyma or peri-
pancreatic tissue. The severity of the lesions
was graded on a scale of 0 to 4 using a
modification of the previous scoring system
[15]. Severity scores were defined as: grade 0
(lesion absent); grade 1 (less than 10% of the
section affected); grade 2 (10-33% of the
section affected); grade 3 (33-66% of the
section affected); grade 4 (more than 66% of
the section affected). In all dogs, the severity
scores (0-4) in each of the six transverse slide
sections were used to calculate an average
value for the eight different categories of
pancreatic injury. These eight values were
then summed to calculate the total pancreatic
injury score for each dog.
ETHICS
The study was approved by the Johns
Hopkins University Animal Care and Use
Committee and Institutional Review Board. It
met all federal guidelines for the humane use
and treatment of animals according to the
“Guide for the Care and Use of Laboratory
Animals (1996)”, prepared by the National
Academy of Sciences. A review committee
was implemented to monitor data acquisition
and animal safety. There were no violations in
safety guidelines or the approved protocol
plan.
STATISTICS
Data are shown as mean and standard
deviation (SD). Peak serum amylase and
lipase activities, as well as peak
TAP/creatinine urinary ratio, were compared
to baseline values by using the paired t-test.
The differences in these peak values between
the untreated and the treated group were
analyzed using the unpaired t-test.
The eight different scores of pancreatic injury,
as well as the total injury score, in the eight
dogs that underwent ERP were compared to
the control dogs using the unpaired t-test.
Similar comparisons were made between the
four untreated and the four etanercept-treated
dogs.

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The one-way linear term ANOVA was
applied to test the relationship between the
total injury score and the severity of
endoscopic approach (ERP method). Data
were analyzed by using the VassarStats web
site
(http://faculty.vassar.edu/lowry/VassarStats.ht
ml).In all analyses, a two-tailed P value of
less than 0.05 was considered to be
statistically significant.
RESULTS
A total of eight survival experiments were
performed. Selective cannulation of the minor
papilla was successful in all animals.
Pancreatic duct acinarization was achieved in
each dog (Figure 1). There were no
significant alterations in the animals’ vital
signs during the procedures (data not shown).
There were no peri-procedural complications
such as bleeding, perforation, infection, etc.
Each animal was recovered uneventfully from
anesthesia following ERP. Seven animals
were sacrificed for necropsy on post-operative
day 5, as planned, while the dog of the
untreated group operated with ERP method 4
had severe clinical pancreatitis with
associated fever, tachycardia, vomiting,
hypotension, and renal dysfunction. This
animal was euthanized on post-operative day
2.
Figure 2 demonstrates the changes in amylase
and lipase levels before and after ERP in all
eight study dogs. Peak amylase levels were
significantly increased compared to baseline
values registered before the procedure
(11,538±4,065 U/L vs. 701±157 U/L;
P<0.001). Peak lipase levels were also
elevated following ERP, and the difference
reached statistical significance (3,637±2,333
U/L vs. 246±125 U/L; P=0.003). When
comparing peak pancreatic enzyme levels
amongst untreated and etanercept-treated
dogs, however, there were no significant
differences in those dogs that received
etanercept prior to ERP (Figure 3). For
amylase levels, the mean value in the
untreated group was 11,735±4,596 U/L vs.
11,341±4,162 U/L in the treatment group
(P=0.903). For lipase levels, the value in the
Figure 1. Fluoroscopic image demonstrating
acinarization of the pancreatic duct by endoscopic
retrograde pancreatography.
Figure 2. Mean (±SD) peak serum amylase and lipase
levels compared to baseline in all eight study dogs.
Figure 3. Mean (±SD) peak serum amylase and lipase
levels in untreated and etanercept-treated dogs.

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untreated group was 3,906±3,253 U/L vs.
3,368±1,389 U/L in the treatment group
(P=0.771).
Mean peak TAP/creatinine urinary ratio was
compared to the corresponding baseline value
in all eight animals. Although peak values
were greater than baseline values (0.71±0.64
vs.0.23±0.17), this difference was not
statistically significant (P=0.097). There was
no significant difference in peak
TAP/creatinine urinary ratio between the four
untreated dogs and the four etanercept-treated
ones (0.74±0.71 vs. 0.68±0.76, respectively;
P=0.910).
Histology
The mean total injury score for the study dogs
was 6.16±1.85 vs. 1.06±0.49 for the control
dogs (P=0.001). When compared to the three
control dogs, the eight study dogs had an
elevated injury score in all histological
categories except for fibrosis (Figure 4).
Mononuclear inflammation (P=0.040), acinar
cell necrosis (P<0.001), and atrophy
(P=0.020) were all significantly worse in the
study dogs. Figure 5 highlights some of the
Figure 4. Mean (±SD) pancreatic injury scores for the
three control dogs and all eight study dogs. (Neu-Infl:
neutrophilic inflammation; Mn-Infl: mononuclear
inflammation; Fat-Nec: fat necrosis).
Figure 5. Histological analysis of the pathology in the pancreas specimens of control dogs and study (ERP) dogs. a-f.
Hematoxylin-eosin staining of pancreatic sections. a,d. Control dog pancreas showing normal lobular architecture with
tightly associated acinar cells. b,e. Representative sections of the pancreas (dog of the untreated group, method 1)
showing mild leukocytic infiltration and acinar cell atrophy. c,f. Representative sections of the pancreas (dog of the
untreated group, method 4) showing inter- and intra-lobular edema, and marked acute inflammation. (Scale bar in a,b,c:
100 μm; scale bar in d,e,f: 25 μm).

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microscopic differences between control dogs
and study dogs.
When the dogs were paired according to the
method of ERP-induced injury, there were
escalating total injury scores concordant with
the severity of endoscopic approach (Figure
6). method 1 had the lowest total mean injury
score (4.38), while method 4 registered the
highest summed score (7.17) although the
trend did not reach the statistical significance
(P=0.223).
The total score for the untreated group was
6.86±1.95 vs. 5.47±1.70 in the treatment
group (P=0.324). Individual pancreatic injury
scores for the two groups of dogs are shown
in Figure 7. There were no significant
differences between the two groups of dogs in
each of the eight different categories of
histological injury.
Cytokine Analysis
TNF-alpha measurements were ascertained in
all eight study dogs at each of the five
collection intervals described above, except
for the dog of the untreated group who was
euthanized prior to the last serum sample
collection due to severe clinical symptoms. In
this dog, as well as, in six of the other dogs,
all collections yielded TNF-alpha concentrat-
ions below the minimal detectable level of 2.4
pg/mL. Only in the remaining dog
(etanercept-treated; ERP method 4) there was
a slight elevation of TNF-alpha in the
duplicate samples recorded at post-procedure
times 2 h (3.09±1.55 pg/mL) and day 2
(3.37±0.39 pg/mL), while all the other
samples (baseline, day 1, and day 5) of this
dog too had undetectable values.
DISCUSSION
Post-ERCP pancreatitis has been a serious
and frustrating problem for endoscopists since
the inception of ERCP in the early 1970’s. It
is relatively common complication that bears
an incidence of roughly 1-22% [3]. In some
patients who are considered high-risk for
post-ERCP pancreatitis (e.g. female gender,
suspected sphincter of Oddi dysfunction, or
previous episode of post-ERCP pancreatitis),
the incidence may be as high as 20-30% [3].
As a result, numerous pharmacological agents
have been studied in an attempt to prevent or
ameliorate this disease. Drugs such as
gabexate, allopurinol, diclofenac, nifedipine,
corticosteroids, somatostatin analogues,
interleukin-10, and glyceryl trinitrate have all
been previously evaluated with varying
results [16, 17, 18, 19, 20, 21, 22, 23, 24, 25,
26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36].
Figure 6. Total pancreatic injury scores in the eight
study dogs according to the four different methods of
endoscopic injury. (Method 1: pancreatic duct
acinarization with 20 mL of contrast; method 2:
pancreatic duct acinarization with 30 mL of contrast;
method 3: pancreatic duct acinarization with 30 mL of
contrast, plus 5-minute balloon occlusion of the
papillary orifice, plus endoscopic pancreatic
sphincterotomy; method 4: pancreatic duct
acinarization with 30 mL of contrast, plus injection of
3 g of ursodeoxycholic acid (powder) mixed in 10 mL
of sterile water, plus 5-minute balloon occlusion of the
papillary orifice, plus endoscopic pancreatic
sphincterotomy).
Figure 7. Mean (±SD) pancreatic injury scores for the
four untreated dogs and the four etanercept-treated
dogs.
Neu-Infl: neutrophilic inflammation
Mn-Infl: mononuclear inflammation
Fat-Nec: fat necrosis).

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Theories as to how these agents may reduce
the risk of post-ERCP pancreatitis include
inhibition of premature intrapancreatic
enzyme activation, reduction in sphincter of
Oddi hypertension, and control of the
inflammatory cascade [37].
In acute pancreatitis, the activation of
inflammatory cells with the release of
cytokines plays an important role in the
disease process [4]. Studies have
demonstrated a substantial rise in both
interleukin-6 (IL-6) and IL-8 immediately
following an episode of acute pancreatitis [5,
6]. Serum levels of these cytokines correlate
with disease severity, and higher levels are
seen in those patients with severe acute
pancreatitis [4]. Conversely, IL-10 has been
studied and shown to reduce the inflammatory
response in acute pancreatitis. Deviere et al.
demonstrated that a single intravenous dose of
IL-10 given 30 minutes before an ERCP
procedure independently reduces the risk of
post-ERCP pancreatitis [7]. Furthermore,
higher levels of IL-10 have been
demonstrated in the sera of those patients with
milder forms of the disease [4, 8].
Tumor necrosis factor-alpha (TNF-alpha) also
plays a role in the pathogenesis of acute
pancreatitis, but its part may be less well-
defined compared to other cytokines. In a rat
or mouse model of disease, TNF-alpha has
been shown to be elevated in the sera of
animals with experimentally-induced acute
pancreatitis [38, 39, 40, 41]. Furthermore,
TNF-alpha blocking agents, such as
etanercept, have been shown to attenuate the
disease process and even improve survival in
some studies [12, 42]. In human studies,
however, significantly elevated levels of
TNF-alpha are seen only in the very early
stages of severe acute pancreatitis [10].
Large animal models that study acute
pancreatitis exist, but one that specifically
mimics post-ERCP pancreatitis has not been
fully developed. Prior studies that have
attempted to create pancreatitis in large
animals have done so by performing an open
laparotomy with surgical duodenostomy and
the injection of a caustic agent directly into
the pancreatic duct. Sodium taurocholate,
trypsin, or autologous bile are usually the
agents of choice in these studies [2, 43, 44,
45]. The problem with these models is that
they do not simulate what occurs inside the
boundaries of a human ERCP. We have
previously demonstrated that post-ERCP
pancreatitis is effectively induced in dogs
with increasing severity by acinarization of
the pancreatic duct, with or without balloon-
occlusion and sphincterotomy [13]. A similar
model was utilized in the present study to
provide the platform for investigating the role
of etanercept in the prevention of ERCP-
induced pancreatitis.
In the current study, we have effectively
induced acute pancreatitis by means of ERP
in eight consecutive animals, using four
escalating endoscopic methods of inducing
pancreatic injury. Post-ERCP pancreatitis was
documented by a significant elevation in
pancreatic enzyme levels, as well as a
significant increase in the total histological
pancreatic injury scores of all eight study
dogs compared to three control dogs. The
trend in severity of pancreatic injury
correlated with the severity of endoscopic
technique during ERP. That is, the necropsy
specimens with the least amount of injury
were seen in the dogs of method 1 (pancreatic
duct acinarization with 20 mL of contrast),
and the specimens with the greatest amount of
injury were seen in the dogs of method 4
(acinarization with 30 mL of contrast, plus
injection of 3 g of ursodeoxycholic acid, plus
balloon occlusion of the papillary orifice, plus
endoscopic pancreatic sphincterotomy).
Four of the eight dogs in this study were pre-
treated with etanercept one day prior to their
ERP procedure. This soluble TNF-alpha
binding agent appeared to have no effect on
the development of post-ERCP pancreatitis.
Peak levels of serum pancreatic enzymes and
urinary TAP/creatinine urinary ratio were
similar among the two groups of dogs. In
addition, when comparing necropsy
specimens between the two groups, total
pancreatic injury scores did not significantly
differ from one another. Individual pancreatic
injury scores were also similar in all
categories of pathology.

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Regarding the lack of efficacy of etanercept in
this study, the near complete absence of
detectable TNF-alpha in the sera of all eight
study dogs must be weighed heavily. Ruaux
et al. previously examined the plasma
samples for TNF-alpha in 60 dogs with
varying degrees of spontaneous acute
pancreatitis [46]. TNF-alpha was detectable in
only 6.7% (4/60) of dogs, and all four of these
animals had severe acute pancreatitis with
multi-organ system failure. Based on these
results, and those of the present study, it
appears as though additional factors may be
involved in the release of TNF-alpha in acute
pancreatitis. It may be that this cytokine
asserts its effect only in the extreme end of
disease severity. If so, this might explain the
lack of detectable TNF-alpha in the eight dogs
of this study; as all but one had mild to
moderate ERCP-induced acute pancreatitis
(one dog had severe disease). Without
significant elevations in TNF-alpha levels
(suggesting its importance in the pathogenesis
of post-ERCP pancreatitis) it is difficult to
imagine how a TNF-alpha binding agent such
as etanercept could be used to limit disease
severity.
Despite the lack of efficacy of etanercept in
the prevention of canine post-ERCP
pancreatitis, this study is unique and uncovers
some important principles. First, it confirms
the utilization of an endoscopically-based
large animal model of post-ERCP
pancreatitis. As procedure-related factors are
known to play a significant role in post-ERCP
pancreatitis, this model replicates the working
conditions of a human ERCP, thus providing
a platform that allows for safe, reliable
investigation of maneuvers, devices, and
future pharmacological agents that may
reduce the occurrence of post-ERCP
pancreatitis. Small-scale studies addressing
different prophylactic agents of choice, or
certain preventative endoscopic techniques,
could be performed using this animal model
before undertaking larger, human clinical
trials. Secondly, this study yields further
information regarding the role of TNF-alpha
in acute pancreatitis. Based on our results, it
seems as though TNF-alpha plays a small
role, if any, in the inflammatory cascade of
mild to moderate acute disease. Therefore,
agents blocking or inhibiting this cytokine
will likely have little effect in this form of
disease severity. On the other hand, TNF-
alpha may play a significant role in severe
acute pancreatitis as suggested in other
studies [10], and future investigations of this
sort should likely focus on the use of anti-
TNF-alpha agents for the prevention of post-
ERCP pancreatitis in cases of severe disease
only.
In conclusion, etanercept, a TNF-alpha
binding agent, is ineffective at preventing
post-ERCP pancreatitis using a canine model
of disease. TNF-alpha appears to have little
role in the inflammatory cascade and
pathogenesis of mild to moderate post-ERCP
pancreatitis in canines. Nonetheless, this
study confirms an endoscopically-based, large
animal model of post-ERCP pancreatitis, and
it serves as a platform for future studies
evaluating this disease.
Received February 5th, 2008 - Accepted April
7th, 2008
Keywords Cholangiopancreatography, Endoscopic
Retrograde; Dogs; Pancreatitis
Abbreviations ERCP: endoscopic retrograde
cholangiopancreatography; ERP: endoscopic
retrograde pancreatography; Fat-Nec: fat
necrosis; Mn-Infl: mononuclear inflam-
mation; Neu-Infl: neutrophilic inflammation;
TAP: trypsinogen activation peptide; TNF:
tumor necrosis factor
Acknowledgements We would like to thank
Diana Scorpio, DVM, and Laurie Pipitone,
BS, for assistance in the preparation of the
study protocol and guidance in the care of
animals used for this study. We also thank
Barbara Detrick, PhD, for her work with
cytokine analysis in this study
Financial support There were no grants or
outside financial support used for this study
Conflict of interest The authors have no
potential conflicts of interest

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JOP. Journal of the Pancreas - http://www.joplink.net - Vol. 9, No. 4 - July 2008. [ISSN 1590-8577]
465
Correspondence
Jonathan M Buscaglia
Johns Hopkins Hospital
1830 E. Monument Street
Room 7100-A
Baltimore, MD 21205
USA
 
 
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