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Reduction of pro-inflammatory cytokines in rats following 7-day oral supplementation with a proprietary eggshell membrane-derived product

Authors:
  • Stratum Nutrition, a business of ESM Technologies, LLC

Abstract and Figures

NEM® brand eggshell membrane is a novel dietary supplement that has been clinically shown to alleviate arthritis joint pain and stiffness; however the mechanism of action is not well understood. Preliminary evidence from an in vitro study of NEM® indicated that the mechanism of action may be based on the reduction of pro-inflammatory cytokines. In vivo studies were therefore initiated to evaluate the effects of NEM® on pro-inflammatory and anti-inflammatory cytokines following oral administration in rats. NEM® was administered daily at doses of 6.13 mg/kg bw/day (Study 1), 10.0 mg/kg bw/day (Study 2), or at doses of 0 (control), 26.0 or 52.0 mg/kg bw/day (Study 3) by oral gavage for 7 consecutive days. Inflammation was induced in the Study 3 rats by intraperitoneal injection of lipopolysaccharide. Changes in plasma cytokine levels from baseline following 7 days of oral supplementation with NEM® at 6.13 mg/kg bw/ day (Study 1) were statistically significant at Day 8 for IL-2, TIMP-1 and VEGF, at Day 21 for IL-10, and at Day 35 for MCP-1, MCP-3 and TIMP-1, and at 10.0 mg/kg bw/day (Study 2) were statistically significant at Day 8 for VEGF, at Day 21 for MIP-1β, MIP-2 and VEGF, and at Day 35 for MCP-3, MIP-1β, MIP-2 and VEGF. Changes in serum cytokine levels versus control at 26.0 mg/kg bw/day (Study 3) were statistically significant at all time-points for IL-1β and at 1.5 hours for IL-10, and at 52.0 mg/kg bw/day (Study 3) were statistically significant at 1.5 hours for IFN-γ, IL-1β and IL-10, and at 3 hours for IL-1β, and at 24 hours for IL-10. Taken together, these studies demonstrate that oral supplementation with NEM® can influence both early-phase pro-inflammatory cytokines like IL-1β and TNF-α (Study 3), as well as later-phase cytokines like MCP-1, MIP-1α & β, RANTES and VEGF (Study 1 & 2). These studies provide a possible basis for the mechanism of action of NEM® in vivo.
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Vol.3, No.1, 19-25 (2014) Modern Research in Inflammation
http://dx.doi.org/10.4236/mri.2014.31003
Copyright © 2014 SciRes. OPEN ACCESS
Reduction of pro-inflammatory cytokines in rats
following 7-day oral supplementation with a
proprietary eggshell membrane-derived product
Kevin J. Ruff1*, Dale P. DeVore2
1ESM Technologies, LLC, Carthage, USA; *Corresponding Author: kruff@esmingredients.com
2Membrell, LLC, Carthage, USA
Received 21 January 2014; revised 14 February 2014; accepted 20 February 2014
Copyright © 2014 Kevin J. Ruff, Dale P. D eVore. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
In accordance of the Creative Commons Attribution License all Copyrights © 2014 are reserved for SCIRP and the owner of the
intellectual property Kevin J. Ruff, Dale P. DeVor e. All Copyright © 2014 are guarded by law and by SCIRP as a guardian.
ABSTRACT
NEM® brand eggshell membrane is a novel die-
tary supplement that has been clinically shown
to alleviate arthritis joint pain and stiffness;
however the mechanism of action is not well
understood. Preliminary evidence from an in
vitro study of NEM® indicated that the mechan-
ism of action may be based on the reduction of
pro-inflammatory cytokines. In vivo studies were
therefore initiated to evaluate the effects of
NEM® on pro-inflammatory and anti-inflamma-
tory cytokines following oral administration in
rats. NEM® was administered daily at doses of
6.13 mg/kg bw/day (Study 1), 10.0 mg/kg bw/day
(Study 2), or at doses of 0 (control), 26.0 or 52.0
mg/kg bw/day (Study 3) by oral gavage for 7
consecutive days. Inflammation was induced in
the Study 3 rats by intraperitoneal injection of
lipopolysaccharide. Changes in plasma cytokine
levels from baseline following 7 days of oral
supplementation with NEM® at 6.13 mg/kg bw/
day (Study 1) were statistically significant at Day
8 for IL-2, TIMP-1 and VEGF, at Day 21 for IL-10,
and at Day 35 for MCP-1, MCP-3 and TIMP-1, and
at 10.0 mg/kg bw/day (Study 2) were statistically
significant at Day 8 for VEGF, at Day 21 for
MIP-1β, MIP-2 and VEGF, and at Day 35 for
MCP-3, MIP-1β, MIP-2 and VEGF. Changes in
serum cytokine levels versus control at 26.0
mg/kg bw/day (Study 3) were statistically sig-
nificant at all time-points for IL-1β and at 1.5
hours for IL-10, and at 52.0 mg/kg bw/day (Study
3) were statistically significant at 1.5 hours for
IFN-γ, IL-1β and IL-10, and at 3 hours for IL-1β,
and at 24 hours for IL-10. Taken together, these
studies demonstrate that oral supplementation
with NEM® can influence both early-phase pro-
inflammatory cytokines like IL-1β and TNF-α
(Study 3), as well as later-phase cytokines like
MCP-1, MIP-1α & β, RANTES and VEGF (Study 1
& 2). These studies provide a possible basis for
the mechanism of action of NEM® in vivo.
KEYWORDS
Pro-Inflammatory Cytokines; Eggshell
Membrane
1. INTRODUCTION
Many human diseases are characterized by chronic in-
flammation which ultimately leads to tissue destruction.
Inflammatory arthritides like rheumatoid arthritis (RA)
and osteoarthritis (OA) are classic examples of such dis-
eases and the roles that inflammatory chemokines and
cytokines play in the pathogenesis of these diseases are
fairly well accepted [1-6]. Corticosteroids, non-steroidal
anti-inflammatory drugs (NSAIDs) (e.g. ibuprofen, dic-
lofenac, celecoxib), and inflammatory cytokine-specific
biologics (e.g. etanercept, infliximab, adalimumab) are
commonly prescribed to address the underlying inflam-
mation of these debilitating conditions. While some of
these treatments have demonstrated good efficacy in
randomized controlled clinical trials (RCTs), they are
also known to have significant and sometimes severe
side effects [7-10]. NEM® brand eggshell membrane has
previously demonstrated good efficacy in relieving pain
and stiffness associated with OA of the knee in an RCT
[11] and has shown similar efficacy in limited trials for
K. J. Ruff, D. P. DeVore / Modern Research in Inflammation 3 (2014) 19-25
Copyright © 2014 SciRes. OPEN ACCESS
20
other affected joints [12,13] with no reports of any sig-
nificant side effects during these trials.
Eggshell membrane is primarily composed of fibrous
proteins such as Collagen Type I [14]. However, egg-
shell membranes have also been shown to contain other
bioactive components, namely glycosaminoglycans (e.g.
dermatan sulfate [15], chondroitin sulfate [15], hyalu-
ronic acid [16], etc.). ESM Technologies, LLC (Carthage,
MO USA) has developed methods to efficiently and ef-
fectively separate eggshell membrane from eggshells on
a commercial metric-ton scale. The isolated membrane is
then partially hydrolyzed using a proprietary process and
dry-blended to produce NEM® brand eggshell membrane.
Compositional analysis of NEM® conducted by ESM has
identified a high content of protein and moderate quanti-
ties of glucosamine (up to 1% by dry weight), chondroi-
tin sulfate (up to 1%), hyaluronic acid (up to 2%), and
collagen (Type I, up to 5%).
Although NEM® has been clinically shown to alleviate
joint pain and stiffness from arthritis, the mechanism of
action of this eggshell membrane preparation is not well
understood. Preliminary evidence from an in vitro study
of NEM® indicated that the basis for the mechanism of
action may be via the reduction of pro-inflammatory
cytokines [17]. In vivo studies were therefore initiated to
evaluate the effects of NEM® on pro-inflammatory and
anti-inflammatory cytokines following oral administra-
tion in rats. The results of these preliminary studies are
reported herein.
2. MATERIALS AND METHODS
2.1. Animals, Care and Diet
Studies were conducted utilizing the facilities and staff
of Charles River Laboratories (Spencerville, OH) (Study
1 & 2) and Ricerca Biosciences (Taipei, Taiwan) (Study
3). Animals were housed and cared for in general accor-
dance with the “Guide for the Care and Use of Labora-
tory Animals” (National Academies Press, Washington,
D.C. USA, 1996). Male Sprague Dawley rats were ob-
tained from Harlan Sprague Dawley, Inc., Indianapolis,
IN USA (Study 1 & 2) and BioLASCO Taiwan Co., Ltd.
(a Charles River licensee), Taipei, Taiwan (Study 3) at
approximately 7 - 10 weeks of age. Upon receipt, tags
with unique identification numbers were used to indivi-
dually identify the animals. Cage cards displaying the
study number, animal number, and sex were affixed to
each cage. The rats were acclimatized for 5 days prior to
study commencement and were observed daily for overt
physical or behavioral abnormalities, general health/ mo-
ribundity, and mortality. Healthy rats weighing 300 ± 20
g were randomized into groups of three and were housed
in cages under standard experimental conditions (22˚C ±
3˚C; 30% - 70% humidity; 12-hour light/dark cycle;
minimum 10 room air changes per hour) and had access
to standard rat chow [PMI Certified Rodent Chow #5002
(PMI Nutrition International, St. Louis, MO USA)
(Study 1 & 2) or Laboratory Rodent Diet MF-18 (Orien-
tal Yeast Co., Ltd., Tokyo, Japan)(Study 3)] and water
ad libitum.
2.2. Test Article Preparation and Dosing
The test article was prepared by suspending NEM®
powder (ESM Technologies, LLC, Carthage, MO USA)
in 0.5% methylcellulose (Sigma-Aldrich, St. Louis, MO
USA) in distilled water at a concentration of 0.613
mg/mL (Study 1), 1.0 mg/mL (Study 2), or 2.6 mg/mL
and 5.2 mg/mL (Study 3), corresponding to a dose vo-
lume of 10 mL/kg. The test article was stored at ap-
proximately 4˚C with constant stirring between daily
uses. The NEM® suspension was administered daily to
groups of 3 rats (Study 1 & 2) or groups of 8 rats (Study
3) at doses of 6.13 mg/kg bw/day (Study 1), 10.0 mg/kg
bw/day (Study 2), or at doses of 0 (control, vehicle only),
26.0 mg/kg bw/day, or 52.0 mg/kg bw/day (Study 3) by
oral gavage for 7 consecutive days. The rats were ob-
served twice daily following administration of the test
article for mortality and clinical abnormalities during the
study period.
2.3. Induction of Inflammation (Study 3)
Inflammation was induced in the Study 3 rats by
intraperitoneal (i.p.) injection (2.5 mg/kg) of a solution
of lipopolysaccharide (LPS) (E. coli serotype 055:B5,
Sigma-Aldrich, St. Louis, MO USA) in pyrogen free
saline (Taiwan Biotech Co., Ltd., Taoyuan, Taiwan).
Control rats received an i.p. injection of saline only.
2.4. Blood Collection and Cytokine
Measurement
Blood samples (~0.5 mL) were collected via jugular
vein (Study 1 & 2) or tail vein (Study 3) pre-dose (Day 0)
(all studies) and on Days 8, 21 and 35 (Study 1 & 2) or
1.5, 3 and 24 hours post LPS injection (Study 3). Blood
samples were processed at the time of collection into
plasma samples (Study 1 & 2) or serum samples (Study 3)
and were stored at 70˚C until cytokine determination
could be performed.
Cytokine determination for Study 1 & 2 was accom-
plished utilizing the facilities and services of Rules
Based Medicine, Inc., Austin, TX USA using their Ro-
dent MAP® multi-analyte profile platform following the
manufacturer’s instructions. In this instance, the levels of
42 different biomarkers, chemokines and cytokines were
evaluated, however only 16 of these which are related to
inflammation [Granulocyte Chemotactic Protein 2 (GCP-
2), Interferon gamma (IFN-γ), Interleukin 1 beta (IL-1β),
K. J. Ruff, D. P. DeVore / Modern Research in Inflammation 3 (2014) 19-25
Copyright © 2014 SciRes. OPEN ACCESS
21
IL-2, IL-4, IL-6, IL-10, Monocyte Chemotactic Protein 1
(MCP-1), MCP-3, Macrophage Inflammatory Protein 1
alpha (MIP-1α), MIP-1β, MIP-2, Regulated upon Acti-
vation Normal T-cell Expressed and Secreted (RANTES),
Tissue Inhibitor of Matalloproteinase Type 1 (TIMP-1),
Tumor Necrosis Factor alpha (TNF-α), and Vascular
Endothelial Growth Factor (VEGF)] are reported herein.
Cytokine determination for Study 3 was performed by
Ricerca Biosciences, LLC, Bothell, WA USA using the
Luminex xMAP® bead-based multiplex platform (Austin,
TX USA) following the manufacturer’s instructions. In
this instance, the levels of 5 chemokines/cytokines re-
lated to inflammation (IFN-γ, IL-1
β
, IL-6, IL-10, TNF-α)
were evaluated and are reported herein.
2.5. Statistical Analysis
Comparisons of baseline data between groups (Study 3)
were made with a Kruskal-Wallis test for multiple inde-
pendent samples to validate randomization. Statistical
significance was accepted at an α value of < 0.05. Post-
baseline statistical analyses were done as repeated meas-
ures univariate Analysis of Variance (rm-ANOVA) ver-
sus baseline (Study 1 & 2) or versus control (Study 3).
Items found to have statistical significance with
rm-ANOVA were then compared using a post hoc test
for repeated measures. Statistical significance was ac-
cepted at an α value of < 0.05 for both determinations. In
cases where post-baseline cytokine values were below
the Limit of Quantitation (LOQ), a value of ½LOQ (well
above the Limit of Detection for the assays) was incor-
porated for statistical calculations as opposed to incor-
porating zero values (Study 1 & 2). This substitution
approach was developed in consultation with the assay
manufacturer and cases where this approach was used are
denoted in the data tables. Additionally, data points were
excluded in cases where there was >35% variance be-
tween replicates (Study 3). This occurred at a rate of
<4% in the overall dataset and appeared to be randomly
distributed throughout. SYSTAT software (version 13)
was used for all statistical analyses [18].
3. RESULTS
Changes in plasma cytokine levels from baseline fol-
lowing 7 days of oral supplementation with NEM® at
6.13 mg/kg bw/day (Study 1, Table 1) were statistically
significant at Day 8 for IL-2 (153% increase, p = 0.033),
TIMP-1 (11.2% reduction, p = 0.002) and VEGF (27.8%
reduction, p = 0.022), at Day 21 for IL-10 (65.1% reduc-
tion, p = 0.033), and at Day 35 for MCP-1 (30.0% reduc-
tion, p = 0.034), MCP-3 (26.6% reduction, p = 0.007)
and TIMP-1 (14.6% reduction, p = 0.032). There were
non-detectable levels of MIP-1
β
at Day 21 and MIP-2
and TNF-α at Day 35.
Table 1. Change in plasma cytokine levels from baseline in
healthy rats following 7 days of oral supplementation with
NEM® at 6.13 mg/kg bw/day.
Baseline
(Day 0) NEM
(Day 8) NEM
(Day 21) NEM
(Day 35)
n = 3 n = 3 n = 3 n = 3
GCP-2a 0.07 ± 0.02 0.22 ± 0.10 0.24 ± 0.07 0.06 ± 0.02
IFN-γb < LOQ < LOQ < LOQ < LOQ
IL-1
β
a 1.29 ± 0.99 0.72 ± 0.34 0.62 ± 0.22 0.30 ± 0.12c
IL-2b 11.2 ± 6.2 28.3 ± 9.0* 33.9 ± 23.6 26.0 ± 13.0
IL-4b < LOQ < LOQ < LOQ < LOQ
IL-6b < LOQ < LOQ < LOQ < LOQ
IL-10b 616 ± 98 503 ± 85 215 ± 105* 477 ± 62
MCP-1b 457 ± 65 449 ± 91 553 ± 144 320 ± 101*
MCP-3b 222 ± 51 207 ± 39 256 ± 70 163 ± 42*
MIP-1αa 0.26 ± 0.05 0.17 ± 0.08 0.15 ± 0.03 0.24 ± 0.06
MIP-1
β
b 28.7 ± 11.5 44.2 ± 9.1 39.0 ± 0.0c 27.4 ± 17.0
MIP-2b 3.0 ± 0.7 3.3 ± 0.7 3.6 ± 1.2 3.6 ± 0.0c
RANTESb 86.2 ± 36.6 152 ± 92 319 ± 70 62.0 ± 22.5
TIMP-1a 8.9 ± 1.4 7.9 ± 1.3* 8.3 ± 0.6 7.6 ± 1.1*
TNF-αa 0.05 ± 0.03 0.05 ± 0.04c 0.05 ± 0.04c 0.07 ± 0.0c
VEGFb 227 ± 38 164 ± 40* 196 ± 55 208 ± 30
Values represent means ± standard deviation, a = ng/mL, b = pg/mL, <LOQ
= below limit of quantitation, P-values determined by repeated measures
ANOVA versus baseline, *P < 0.05, P < 0.10, c = contained cases where
values measured were below the Limit of Quantitation (LOQ) wherein
values of ½LOQ were incorporated for statistical calculations.
Changes in plasma cytokine levels versus baseline
following 7 days of oral supplementation with NEM® at
10.0 mg/kg bw/day (Study 2, Table 2) were statistically
significant at Day 8 for VEGF (50.1% reduction, p =
0.038), at Day 21 for MIP-1
β
(84.8% reduction, p =
0.022), MIP-2 (77.1% reduction, p = 0.005) and VEGF
(61.5% reduction, p = 0.014), and at Day 35 for MCP-3
(67.2% reduction, p = 0.047), MIP-1
β
(88.4% reduction,
p = 0.002), MIP-2 (76.5% reduction, p = 0.006) and
VEGF (66.4% reduction, p = 0.002). There were trends
toward significance at Day 8 for MIP-2 (64.0% reduction,
p = 0.063), at Day 21 for MCP-3 (61.9% reduction, p =
0.081) and TNF-α (70.0% reduction, p = 0.097), and at
Day 35 for GCP-2 (53.8% reduction, p = 0.075), IL-2
(69.0% reduction, p = 0.098) and MCP-1 (67.3% reduc-
tion, p = 0.079). There were non-detectable levels of
IL-2, MIP-2 and TIMP-1 at Day 35.
There were no differences in serum cytokine levels
between groups (control, 26.0 mg/kg bw/day or 52.0
mg/kg bw/day) at baseline for any of the five cytokines
evaluated (IFN-γ, IL-1
β
, IL-6, IL-10, TNF-α)(Study 3,
Table 3). Changes in serum cytokine levels versus con-
trol following 7 days of oral supplementation with
NEM® at 26.0 mg/kg bw/day (Study 3, Table 4) with
K. J. Ruff, D. P. DeVore / Modern Research in Inflammation 3 (2014) 19-25
Copyright © 2014 SciRes. OPEN ACCESS
22
Table 2. Change in plasma cytokine levels from baseline in
healthy rats following 7 days of oral supplementation with
NEM® at 10.0 mg/kg bw/day.
Baseline
(Day 0) NEM
(Day 8) NEM
(Day 21) NEM
(Day 35)
n = 3 n = 3 n = 3 n = 3
GCP-2a 0.13 ± 0.05 0.06 ± 0.04 0.02 ± 0.03 0.06 ± 0.02
IFN-γb < LOQ < LOQ < LOQ <LOQ
IL-1
β
a < LOQ < LOQ < LOQ <LOQ
IL-2b 108 ± 44 31.3 ± 21.3 25.3 ± 14.1c 33.5 ± 0.0c†
IL-4b < LOQ < LOQ < LOQ <LOQ
IL-6b < LOQ < LOQ < LOQ <LOQ
IL-10b 460 ± 265 305 ± 15 314 ± 25 294 ± 83
MCP-1b 721 ± 233 439 ± 113 250 ± 122 236 ± 28
MCP-3b 354 ± 93 226 ± 72 135 ± 44 116 ± 6*
MIP-1αa < LOQ < LOQ < LOQ <LOQ
MIP-1
β
b 250 ± 31 81 ± 103 38 ± 46* 29 ± 16*
MIP-2b 15.3 ± 1.5 5.5 ± 3.4c† 3.5 ± 0.2c* 3.6 ± 0.0c*
RANTESb 195 ± 118 93 ± 100 23 ± 20 20 ± 4
TIMP-1a 0.28 ± 0.16 0.09 ± 0.06c
0.08 ± 0.03c 0.09 ± 0.00c
TNF-αa 0.20 ± 0.09 0.06 ± 0.06 0.06 ± 0.03c† 0.04 ± 0.03c
VEGFb 429 ± 42 214 ± 75* 165 ± 25* 144 ± 33*
Values represent means ± standard deviation, a = ng/mL, b = pg/mL, < LOQ
= below limit of quantitation, P-values determined by repeated measures
ANOVA versus baseline, *P < 0.05, P < 0.10, c = contained cases where
values measured were below the Limit of Quantitation (LOQ) wherein
values of ½LOQ were incorporated for statistical calculations.
Table 3. Mean serum cytokine concentrations (pg/mL) in
NEM-supplemented and control groups at baseline.
Control
(0 mg/kg) NEM
(26 mg/kg) NEM
(52 mg/kg)
n = 8 n = 8 n = 8
IFN-γ 2.49 ± 0.16 2.40 ± 0.00 2.49 ± 0.27
IL-1
β
15.8 ± 10.0 13.0 ± 5.5 18.6 ± 13.2
TNF-α 11.1 ± 2.0 10.2 ± 0.7 11.2 ± 2.9
IL-6 9.80 ± 0.00 9.96 ± 1.14 10.2 ± 1.1
IL-10 10.2 ± 1.0 9.80 ± 0.00 10.0 ± 0.6
Values represent means ± standard deviation, P-values determined by
Kruskal-Wallis test for multiple independent samples, *P < 0.05, P < 0.10.
subsequent inflammatory challenge (LPS, i.p.) were sta-
tistically significant at 1.5 hours (43.7% reduction, p =
0.013), 3 hours (28.8% reduction, p = 0.034) and 24
hours (20.8% reduction, p = 0.006) for IL-1
β
and at 1.5
hours (27.6% reduction, p = 0.028) for IL-10. There was
a trend toward significance at 24 hours for IL-10 (74.6%
increase, p = 0.097). No other changes in serum cytokine
levels were statistically significant at this dose level.
Changes in serum cytokine levels versus control fol-
lowing 7 days of oral supplementation with NEM® at
52.0 mg/kg bw/day (Study 3, Table 5) with subsequent
inflammatory challenge (LPS, i.p.) were statistically sig-
nificant at 1.5 hours for IFN-γ (33.5% reduction, p =
0.047), IL-1
β
(39.4% reduction, p = 0.003) and IL-10
(29.8% reduction, p = 0.015), and at 3 hours for IL-1
β
(23.9% reduction, p = 0.044), and at 24 hours for IL-10
(57.5% increase, p = 0.021). There was a trend toward
significance at 24 hours for IL-1
β
(9.3% reduction, p =
0.093). No other changes in serum cytokine levels were
statistically significant at this dose level.
4. DISCUSSION
Although OA has not traditionally been considered an
inflammatory arthropathy, the scientific understanding of
the pathophysiological progression of the disease has
been gradually trending towards that of a disease involv-
ing the whole jointwith significant localized inflam-
mation [19]. Evidence of an inflammatory process in OA
is reflected in many of the clinical symptoms of the pro-
gressive disease, including swelling of affected joints,
Table 4. Change in mean serum cytokine concentrations
(pg/mL) in 7-day NEM-supplemented (26 mg/kg bw/day) and
control groups from baseline at 1.5, 3, and 24 hours post LPS
treatment.
Hours post-
treatment
Control NEM % Difference
(NEM-vs-ctrl)
(0 mg/kg)
n = 8 (26 mg/kg)
n = 8
IFN-γ Baseline 2.49 ± 0.16 2.40 ± 0.00 3.5
1.5 6.35 ± 2.48 4.88 ± 2.60 23.2
3 135 ± 43 144 ± 85 6.2
24 3.55 ± 2.18 2.49 ± 0.14 29.8
IL-1
β
Baseline 15.8 ± 10.0 13.0 ± 5.5 17.9
1.5 62.4 ± 17.1 35.1 ± 16.7* 43.7*
3 87.9 ± 23.5 62.6 ± 15.9* 28.8*
24 12.6 ± 2.1 9.96 ± 0.42* 20.8*
TNF-α Baseline 11.1 ± 2.0 10.2 ± 0.7 7.6
1.5 1157 ± 828 934 ± 332 19.3
3 84.5 ± 46.7 75.5 ± 28.7 10.7
24 9.80 ± 0.00 9.80 ± 0.00 0.0
IL-6 Baseline 9.80 ± 0.00 9.96 ± 1.14 1.7
1.5 321 ± 175 385 ± 182 20.0
3 386 ± 172 329 ± 133 14.8
24 10.0 ± 0.6 10.7 ± 2.3 6.6
IL-10 Baseline 10.2 ± 1.0 9.80 ± 0.00 3.8
1.5 42.7 ± 12.3 30.9 ± 8.3* 27.6*
3 18.7 ± 5.6 19.3 ± 7.4 3.1
24 14.2 ± 3.2 24.9 ± 11.5 74.6
Values represent means ± standard deviation. P-values determined by re-
peated measures ANOVA, *p < 0.05, p < 0.10. ctrl = control.
K. J. Ruff, D. P. DeVore / Modern Research in Inflammation 3 (2014) 19-25
Copyright © 2014 SciRes. OPEN ACCESS
23
Table 5. Mean serum cytokine concentrations (pg/mL) in 7-day
NEM-supplemented (52 mg/kg bw/day) and control groups at
baseline and 1.5, 3, and 24 hours post LPS treatment.
Hours post-
treatment
Control NEM % Difference
(NEM-vs-ctrl)
(0 mg/kg)
n = 8 (52 mg/kg)
n = 8
IFN-γ Baseline 2.49 ± 0.16 2.49 ± 0.27 0.3
1.5 6.35 ± 2.48 4.22 ± 0.76* 33.5*
3 135 ± 43 130 ± 34 4.3
24 3.55 ± 2.18 2.82 ± 0.75 20.7
IL-1
β
Baseline 15.8 ± 10.0 18.6 ± 13.2 17.3
1.5 62.4 ± 17.1 37.8 ± 9.7* 39.4*
3 87.9 ± 23.5 66.9 ± 15.8* 23.9*
24 12.6 ± 2.1 11.4 ± 1.2 9.3
TNF-α Baseline 11.1 ± 2.0 11.2 ± 2.9 1.5
1.5 1157 ± 828 786 ± 161 32.1
3 84.5 ± 46.7 70.0 ± 15.0 17.2
24 9.80 ± 0.00 9.80 ± 0.00 0.0
IL-6 Baseline 9.80 ± 0.00 10.2 ± 1.1 4.0
1.5 321 ± 175 256 ± 83 20.1
3 386 ± 172 306 ± 64 20.6
24 10.0 ± 0.6 10.3 ± 1.4 3.0
IL-10 Baseline 10.2 ± 1.0 10.0 ± 0.6 1.8
1.5 42.7 ± 12.3 30.0 ± 8.1* 29.8*
3 18.7 ± 5.6 18.1 ± 3.4 3.6
24 14.2 ± 3.2 22.4 ± 6.1* 57.5*
Values represent means ± standard deviation. P-values determined by re-
peated measures ANOVA, *p < 0.05, p < 0.10. ctrl = control.
synovial effusion, and joint stiffness [20]. This clinical
evidence is supported by immunochemical and histolog-
ical data from numerous studies showing infiltration of
the joint synovium by immune cells, primarily macro-
phages and mononuclear lymphocytes such as T-cells
[20-22] accompanied by subsequent inflammatory cyto-
kine expression [23] and synovial fibroblast activation
[24].
The two primary mediators of arthritis inflammation
are IL-1β and TNF-α. These cytokines have been identi-
fied as targets for OA treatment [25,26] and there are
multiple FDA-approved biologic drugs (etanercept, in-
fliximab, adalimumab, etc.) for this indication (mostly
RA). These cytokines, in an autocrine/paracrine manner,
auto-amplify their own expression and induce chondro-
cytes to produce matrix metalloproteinases (MMPs),
chemokines (IL-8, MCP-1, MIP-1α, MIP-1β, RANTES,
etc.), nitric oxide, and prostaglandins [19,26]. This leads
to localized tissue destruction, immune cell infiltration,
inhibition of cartilage matrix synthesis, and increased
pain sensitivity, among others.
The eggshell membrane derived product NEM® has
previously been shown in vitro to reduce a number of
pro-inflammatory cytokines in human immune cells fol-
lowing inflammatory challenge (with phyto-mitogens),
with this effect being most pronounced for IFN-γ and
TNF-α [17]. In this paper, we reported in vivo support for
the reduction of circulating pro-inflammatory cytokines
following oral supplementation with NEM® in both
healthy rats (Study 1 & 2) and inflammatory-challenged
rats (Study 3).
While not statistically significant, NEM® appeared to
demonstrate trends toward reduction in healthy rats for
both IL-1β (6.13 mg/kg bw/day)(Study 1) and TNF-α
(10.0 mg/kg bw/day)(Study 2). Interestingly, there were
statistically significant effects at both dose levels for
nearly all of the chemokines (MCP-1, MIP-1α, MIP-1β,
RANTES, VEGF) currently understood to be key players
in OA/RA inflammation and pathogenesis. MCP-1 and
RANTES have been shown to induce expression of
MMP-3 in both normal and OA chondrocytes [27] and
RANTES has been reported to stimulate MMP-1 release
in chondrocytes as effectively as did IL-1β [28]. These
enzymes are known to degrade chondrocyte extracellular
matrix (ECM) which leads to cartilage destruction.
MCP-1, RANTES, MIP-1α and MIP-1β have all been
shown to inhibit proteoglycan synthesis in chondrocytes
[27,29], a key component of cartilage needed for repair.
VEGF expression is absent in adult healthy cartilage but
is significantly expressed in OA chondrocytes and may
play a role in osteophyte formation [30].
Also interesting is the overall lack of effect from oral
supplementation with NEM® on anti-inflammatory cyto-
kines and chemokines (IL-4, IL-6, IL-10, and TIMP-1) in
healthy rats. While IL-4 and IL-6 were below LOQ at
baseline, there was only a mild downward trend in IL-10
and TIMP-1 levelsconsistent with restoring immune
homeostasis following the reductions seen in pro-in-
flammatory cytokines and chemokines. IL-10 is known
to inhibit the production of IL-1β and TNF-α and is
overexpressed in OA chondrocytes compared to normal,
which is likely the body’s attempt to counteract the de-
trimental effects from these pro-inflammatory cytokines
[31]. MMPs are strictly controlled by TIMPs under nor-
mal conditions and an imbalance toward MMPs is be-
lieved to be the basis for cartilage destruction via ECM
degradation in arthritis [32].
NEM® has been shown in clinical trials to have an ef-
fective dose of 500 mg per day. We initially chose to
evaluate doses of 6.13 mg/kg bw/day (Study 1) and 10.0
mg/kg bw/day (Study 2) which, following allometric
conversion [33], equate to a human equivalent dose
(HED) of 59 mg/day and 97 mg/day, respectively, for a
60 kg person. The number of animals (n = 3) was also
small in the preliminary evaluations of inflammatory
K. J. Ruff, D. P. DeVore / Modern Research in Inflammation 3 (2014) 19-25
Copyright © 2014 SciRes. OPEN ACCESS
24
cytokines. These facts, combined with low basal cyto-
kine levels in healthy rats, made it challenging to obtain
statistically significant changes following oral supple-
mentation with NEM®. In a number of instances (IL-1β
in Study 1 and IL-10 & RANTES in Study 2), there ap-
peared to be substantial percent reductions in mean cyto-
kine levels that nevertheless failed to reach statistical
significance. We therefore set out to employ a rat model
in which inflammation was induced (Study 3) to increase
the likelihood of observing clearer effects from NEM®
supplementation. We also increased the number of ani-
mals (n = 8), narrowed the number of cytokines eva-
luated to five, and increased the doses evaluated to 26.0
mg/kg bw/day and 52.0 mg/kg bw/day (HED: 252 mg/
day & 503 mg/day, respectively).
There was a substantial (39% - 44%) and lasting
(through 24 hours) reduction in IL-1β in this inflamma-
tory-challenge model (Study 3) at both doses evaluated.
And, although not statistically significant, there also ap-
peared to be a substantial (19% - 32%) downward trend
in TNF-α levels for both doses, as well. These effects on
the key mediators of arthritis inflammation provide fur-
ther supportive evidence to the observed clinical efficacy
of NEM®. The ability to influence IL-1β and TNF-α in
vivo likely also explains at least some of the effects ob-
served in the downstream chemokines (MCP-1, MIP-1α,
MIP-1β, RANTES, VEGF) in the initial studies. Interes-
tingly, there was a sinusoidal response for the anti-in-
flammatory cytokine IL-10 over the time-course of this
study, in which there was an initial substantial reduction
(28% - 30%) at 1.5 hours leading to a substantial in-
crease (58% - 75%) by 24 hours when compared to con-
trols. This is particularly interesting in the context that
nearly all of the cytokines evaluated had returned to near
baseline levels by the 24-hour study endpoint in the con-
trol animals. The reason for this divergence isn’t com-
pletely clear, but it may be a result of the delayed time-
course of anti-inflammatory cytokines compared to the
rapid time-course of pro-inflammatory cytokines, espe-
cially in this particular animal model.
Taken together, these studies demonstrate that oral
supplementation with NEM® can influence both early-
phase pro-inflammatory cytokines like IL-1β and TNF-α
(Study 3), as well as later-phase pro-inflammatory cyto-
kines like MCP-1, MIP-1α & β, RANTES and VEGF
(Study 1 & 2). There was also a mild effect on the an-
ti-inflammatory cytokine IL-10 in all three studies. A
natural treatment, such as NEM®, which is suitable for
chronic inflammatory diseases like arthritis that could
potentially avoid the unfortunate side effects of currently
available pharmaceutical treatments is of obvious benefit.
These studies provide a possible basis for the mechanism
of action of NEM® in vivo and serve as an important step
in explaining its observed clinical efficacy seen in mul-
tiple human studies.
ACKNOWLEDGEMENTS
KJR is currently employed by the sponsor of the studies. DPD has
served as a paid consultant to the sponsor of the studies. All three stu-
dies were sponsored by ESM Technologies, LLC.
REFERENCES
[1] Harringman, J.J., Ludikhuize, J. and Tak, P.P. (2004)
Chemokines in joint disease: The key to inflammation?
Annals of the Rheumatic Diseases, 63, 1186-1194.
http://dx.doi.org/10.1136/ard.2004.020529
[2] Martel-Pelletier, J., Alaaeddine, N. and Pelletier, J.P.
(1999) Cytokines and their role in the pathophysiology of
osteoarthritis. Frontiers in Bioscience, 4, d694-d703.
http://dx.doi.org/10.2741/Martel
[3] Choy, E.H.S. and Panayi, G.S. (2001) Cytokine pathways
and joint inflammation in rheumatoid arthritis. New Eng-
land Journal of Medicine, 344, 907-916.
http://dx.doi.org/10.1056/NEJM200103223441207
[4] Feldmann, M. and Maini, R.N. (2008) Role of cytokines
in rheumatoid arthritis: An education in pathophysiology
and therapeutics. Immunological Reviews, 223, 7-19.
http://dx.doi.org/10.1111/j.1600-065X.2008.00626.x
[5] Brennan, F.M. and McInnes, I.B. (2008) Evidence that
cytokines play a role in rheumatoid arthritis. The Journal
of Clinical Investigations, 118, 3537-3545.
http://dx.doi.org/10.1172/JCI36389
[6] Kokkonen, H., Soderstrom, I., Rocklov, J., Hallmans, G.,
Lejon, K. and Dahlqvist, S.R. (2010) Up-regulation of
cytokines and chemokines predates the onset of rheuma-
toid arthritis. Arthritis & Rheumatism, 62, 383-391.
http://dx.doi.org/10.1002/art.27186
[7] Dixon, W.G., Suissa, S. and Hudson, M. (2011) The asso-
ciation between systemic glucocorticoid therapy and the
risk of infection in patients with rheumatoid arthritis:
Systematic review and meta-analyses. Arthritis Research
& Therapy, 13, R139. http://dx.doi.org/10.1186/ar3453
[8] Singh, G., Wu, O., Langhorne, P. and Madhok, R. (2006)
Risk of acute myocardial infarction with nonselective
non-steroidal anti-inflammatory drugs: A meta-analysis.
Arthritis Research & Therapy, 8, 153-162.
http://dx.doi.org/10.1186/ar2047
[9] Masso Gonzalez, E.L., Patrignani, P., Tacconelli, S. and
Garcia Rodriguez, L.A. (2010) Variability among non-
steroidal antiinflammatory drugs in risk of upper ga-
strointestinal bleeding. Arthritis & Rheumatism, 62, 1592-
1601. http://dx.doi.org/10.1002/art.27412
[10] O’Neil, C.K., Hanlon, J.T. and Marcum, Z.A. (2012)
Adverse effects of analgesics commonly used by older
adults with osteoarthritisFocus on non-opioid and opi-
oid analgesics. American Journal of Geriatric Pharma-
cotherapy, 10, 331-342.
http://dx.doi.org/10.1016/j.amjopharm.2012.09.004
[11] Ruff, K.J., Winkler, A., Jackson, R.W., DeVore, D.P. and
Ritz, B.W. (2009) Eggshell membrane in the treatment of
K. J. Ruff, D. P. DeVore / Modern Research in Inflammation 3 (2014) 19-25
Copyright © 2014 SciRes. OPEN ACCESS
25
pain and stiffness from osteoarthritis of the knee: A ran-
domized, multicenter, double-blind, placebo-controlled
clinical study. Clinical Rheumatology, 28, 907-914.
http://dx.doi.org/10.1007/s10067-009-1173-4
[12] Ruff, K.J., DeVore, D.P., Leu, M.D. and Robinson, M.A.
(2009) Eggshell membrane: A possible new natural the-
rapeutic for joint and connective tissue disorders. Results
from two open-label human clinical studies. Clinical In-
terventions in Aging, 4, 235-240.
http://dx.doi.org/10.2147/CIA.S5797
[13] Danesch, U., Seybold, M., Rittinghausen, R., Treibel, W.
and Bitterlich, N. (Unpublished) NEM® brand eggshell
membrane effective in the treatment of pain associated
with knee and hip osteoarthritis: Results from a six-center,
open-label german clinical study.
[14] Wong, M., Hendrix, M.J.C., von der Mark, K., Little and
C., Stern, R. (1984) Collagen in the egg shell membranes
of the hen. Developmental Biology, 104, 28-36.
http://dx.doi.org/10.1016/0012-1606(84)90033-2
[15] Baker, J.R. and Balch, D.A. (1962) A study of the organic
material of hen’s-egg shell. Biochemical Journal, 82,
352-361.
[16] Long, F.D., Adams, R.G., DeVore, D.P., Inventors (2005)
Preparation of hyaluronic acid from eggshell membrane.
US Patent No. 6,946,551.
[17] Benson, K.F., Ruff, K.J. and Jensen, G.S. (2012) Effects
of natural eggshell membrane (nem) on cytokine produc-
tion in cultures of peripheral blood mononuclear cells:
increased suppression of tumor necrosis factor-α levels
after in vitro digestion. Journal of Medicinal Food, 15,
360-368. http://dx.doi.org/10.1089/jmf.2011.0197
[18] Systat Software, Inc. http://www.systat.com
[19] Abramson, S.B. and Attur, M. (2009) Developments in
the scientific understanding of osteoarthritis. Arthritis
Research & Therapy, 11, 227.
http://dx.doi.org/10.1186/ar2655
[20] Sellam, J. and Berenbaum, F. (2010) The role of synovitis
in pathophysiology and clinical symptoms of osteoarthri-
tis. Nature Reviews Rheumatology, 6, 625-635.
http://dx.doi.org/10.1038/nrrheum.2010.159
[21] Roach, H.I., Aigner, T., Soder, S., Haag, J. and Welkerling,
H. (2007) Pathobiology of osteoarthritis: Pathomechan-
isms and potential therapeutic targets. Current Drug Tar-
gets, 8, 271-282.
http://dx.doi.org/10.2174/138945007779940160
[22] Sakkas, L.I. and Platsoucas, C.D. (2007) The role of T
cells in the pathogenesis of osteoarthritis. Arthritis &
Rheumatism, 56, 409-424.
http://dx.doi.org/10.1002/art.22369
[23] Bondeson, J., Wainwright, S.D., Lauder, S., Amos, N. and
Hughes, C.E. (2006) The role of synovial macrophages
and macrophage-produced cytokines in driving aggreca-
nases, matrix metalloproteinases, and other destructive
and inflammatory responses in osteoarthritis. Arthritis
Research & Therapy, 8, R187.
http://dx.doi.org/10.1186/ar2099
[24] Wenham, C.Y.J. and Conaghan, P.G. (2010) The role of
synovitis in osteoarthritis. Therapeutic Advances in Mus-
culoskeletal Diseases, 2, 349-359.
http://dx.doi.org/10.1177/1759720X10378373
[25] Martel-Pelletier, J., Tardif, G., Laufer, S. and Pelletier, J.P.
(2005) Cytokines and growth factors in the treatment of
osteoarthritis: What could be the best disease modifying
drugs. Current Medicinal ChemistryAnti-Inflammatory
& Anti-Allergy Agents, 4, 235-249.
[26] Yuan, G.H., Masuko-Hongo, K., Kato, T. and Nishioka, K.
(2003) Immunologic intervention in the pathogenesis of
osteoarthritis. Arthritis & Rheumatism, 48, 602-611.
http://dx.doi.org/10.1002/art.10768
[27] Yuan, G.H., Masuko-Hongo, K., Sakata, M., et al. (2001)
The role of C-C chemokines and their receptors in os-
teoarthritis. Arthritis & Rheumatism, 44, 1056-1070.
http://dx.doi.org/10.1002/1529-0131(200105)44:5%3C10
56::AID-ANR186%3E3.0.CO;2-U
[28] Alaaeddine, N., Olee, T., Hashimoto, S., Creighton-
Achermann, L. and Lotz, M. (2001) Production of the
chemokine RANTES by articular chondrocytes and role
in cartilage degradation. Arthritis & Rheumatism, 44,
1633-1643.
http://dx.doi.org/10.1002/1529-0131(200107)44:7%3C16
33::AID-ART286%3E3.0.CO;2-Z
[29] Vergunst, C.E., van de Sande, M.G.H., Lebre, M.C. and
Tak, P.P. (2005) The role of chemokines in rheumatoid
arthritis and osteoarthritis. Scandinavian Journal of Rheu-
matology, 34, 415-425.
http://dx.doi.org/10.1080/03009740500439159
[30] Tanaka, E., Aoyama, J., Miyauchi, M., et al. (2005) Vas-
cular endothelial growth factor plays an important auto-
crine/paracrine role in the progression of osteoarthritis.
Histochemistry and Cell Biology, 123, 275-281.
http://dx.doi.org/10.1007/s00418-005-0773-6
[31] Iannone, F., De Bari, C., Dell’Accio, F., et al. (2001)
Interleukin-10 and interleukin-10 receptor in human os-
teoarthritic and healthy chondrocytes. Clinical & Expe-
rimental Rheumatology, 19, 139-145.
[32] Ishiguro, N., Ito, T., Ito, H., et al. (1999) Relationship of
matrix metalloproteinases and their inhibitors to cartilage
proteoglycan and collagen turnover: Analyses of synovial
fluid from patients with osteoarthritis. Arthritis & Rheu-
matism, 42, 129-136.
http://dx.doi.org/10.1002/1529-0131(199901)42:1%3C12
9::AID-ANR16%3E3.0.CO;2-4
[33] US Food and Drug Administration (2005) Guidance for
industry: estimating the maximum safe starting dose in
initial clinical trials for therapeutics in adult healthy vo-
lunteers.
http://www.fda.gov/downloads/Drugs/GuidanceComplian
ceRegulatoryInformation/Guidances/ucm078932.pdf
... 24 ESM is known to reduce the expression of various pro-inflammatory cytokines, including the key mediators of inflammation interleukin-1 beta (IL-1β) and tumor necrosis factor-alpha (TNF-α) both in vitro 25 and in vivo. 26 ...
... This observed clinical effect is also consistent with the proposed mechanism of action of NEM. That is, the reduction in proinflammatory cytokines demonstrated in previous mechanistic investigations 25,26,30 would be expected to have a direct correlation to joint stiffness, which is a likely result of localized inflammation. Although NEM has also been shown to affect pain signal transduction via reducing prostaglandin E 2 (PGE 2 ) in an OA rat model, 30 the corresponding clinical effect would be expected to lag behind any anti-inflammatory effect as PGE 2 synthesis is amplified by the inducible enzyme, cyclooxygenase-2 (COX-2), that is induced by and responsive to proinflammatory cytokines. ...
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Currently, in the United States, every fifth adult dog or horse suffers from arthritis. The two most common types of arthritis are osteoarthritis (OA) and rheumatoid arthritis (RA). OA occurs with greater frequency than RA. OA is an inflammatory heterogeneous chronic degenerative joint disease (DJD) characterized by chronic and progressive degradation of the articular cartilage, osteophyte formation, thickening and sclerosis of the subchondral bone, bone marrow lesions, hypertrophy of bone at the margin, synovitis, synovial fluid effusion, and fibrosis. Common clinical signs and symptoms associated with OA in dogs and horses include limping, immobility, stiffness of joints, crepitus, periarticular swelling, palpable effusion, and pain upon manipulation of the joint and limb. The pathophysiology of OA is very complex because there are multiple etiologies for this disease, and as a result, treatment is complicated. Pain and inflammation associated with OA are often managed by pharmacological suppression or surgery among a few other modalities. NSAIDs are known to have severe side effects, and surgery is very expensive, so the use of nutraceuticals appears to be a viable alternative for prevention and treatment of OA. This chapter describes various nutraceuticals that have the potential to exert antioxidative, anti-inflammatory, antinociceptive, and chondroprotective effects in osteoarthritis.
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Eggshell membrane (ESM) is a relatively new ingredient on the list of ingredients that have been used to mitigate the effects of joint inflammation in animals. Eggshells contain a thin membrane that coats the inner surface of the shell that is comprised of a wide number of proteins, amino acids, collagen-like proteins, enzymes, and glycosaminoglycans. When ESM is consumed by an animal, the compounds found in ESM will play a role in maintaining joint health. However, ESM also has antimicrobial effects. The antimicrobial effects of ESM can be utilized in animal feeds as an alternative to antibiotics for growth promotion in production animals. Thus, ESM represents a new ingredient for veterinarian use for not only fighting inflammation in joints but also as an alternative to antibiotics for growth promotion activities.
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Soluble eggshell membrane protein (SEP) and polysaccharides extracted from algae exhibited immunomodulatory, presenting a potential for inflammatory bowel disease (IBD) treatment. However, the bioavailability of proteins is limited because of harsh gastrointestinal pH environment, enzymatic degradation and limitation of intestinal epithelial transport. This study investigates the release of SEP from chitosan/fucoidan nanoparticles (CS/F NPs) and evaluates the protective effect of released SEP on intestinal epithelial cells (IECs) damage. The SEP was successfully extracted and encapsulated by CS/F NPs. When the SEP/CS/F weight ratio was 0.2/3/1, the NPs were spherical with 100 nm in diameter and 10 mV in zeta potential. The NPs were stable in simulated gastric acid (pH = 1.2), and released 50% of SEP when disintegrated in imitating intestinal environment (pH = 7.4). The SEP-CS/F NPs had good biocompatibility and presented high antioxidant activities. Using co-culture Caco-2 and RAW264.7 cells for immunomodulatory assessment, the SEP-CS/F NPs effectively reduced the amount of NO and inhibited the TNF-α and IL-6 production. Moreover, the SEP-CS/F NPs could protect the IECs from disruption caused by lipopolysaccharide (LPS) evidenced by measuring the transepithelial electrical resistance and paracellular permeability of fluorescein isothiocyanate-dextran. Briefly, the CS/F NPs are potential carriers for SEP delivery to ameliorate LPS-induced intestinal epithelial inflammation.
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®) is a novel dietary supplement that contains naturally occurring glycosaminoglycans and proteins essential for maintaining healthy joint and connective tissues. Two single center, open-label human clinical studies were conducted to evaluate the efficacy and safety of NEM ® as a treatment for pain and inflexibility associated with joint and connective tissue disorders. Methods: Eleven (single-arm trial) and 28 (double-arm trial) patients received oral NEM ® 500 mg once daily for four weeks. The primary outcome measure was to evaluate the change in general pain associated with the treatment joints/areas (both studies). In the single-arm trial, range of motion (ROM) and related ROM-associated pain was also evaluated. The primary treatment response endpoints were at seven and 30 days. Both clinical assessments were performed on the intent-to-treat (ITT) population within each study. Results: Single-arm trial: Supplementation with NEM
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Osteoarthritis (OA) is the most common form of arthritis worldwide yet there is still a lack of effective treatments for this condition. Increasingly, attention has turned to the role of the synovium in OA as it is now recognized, in part from the use of modern imaging techniques, that synovitis is both common and associated with pain. This offers a target for treatment, for both symptom and potential structure modification. In this review we discuss the evidence for histological and imaging-detected synovitis and the current role of antisynovial therapies in OA.
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Tumor necrosis factor-α (TNF-α) plays an important role in inflammatory processes. This study examined the effects of natural eggshell membrane (NEM(®)) (ESM Technologies, LLC, Carthage, MO, USA) on interleukin (IL)-2, IL-4, IL-6, IL-10, interferon-γ (IFN-γ), and TNF-α cytokine production by 4-day peripheral blood mononuclear cell (PBMC) cultures exposed to serial dilutions of either an aqueous extract of natural eggshell membrane (NEM-AQ) or NEM subjected to in vitro digestion (NEM-IVD). The effects on cytokine production were also assessed in the presence of phytohemagglutinin (PHA) and pokeweed mitogen (PWM) where exposure to NEM-AQ resulted in reduced levels of proliferation and statistically significant effects on IL-6, IL-10, IFN-γ, and TNF-α cytokine production. NEM-AQ reduced levels of IL-6, IL-10, IFN-γ, and TNF-α in cultures exposed to PHA. In cultures containing PWM, NEM-AQ reduced production of IL-10 and at the highest dose tested increased IL-6 and decreased TNF-α cytokine levels. NEM-IVD, at the two lowest concentrations of product, significantly reduced TNF-α production by PBMC cultures exposed to PWM compared with the in vitro digest control or native NEM. Taken together, these results suggest that NEM-AQ can influence signaling events in response to the T cell-specific mitogen PHA as well as to the mitogen PWM that require cellular cross-talk and that these effects may be partially mediated through a reduction in level of the pro-inflammatory cytokine TNF-α. The suppression of TNF-α production in the presence of NEM-IVD is promising for the use of NEM as a consumable anti-inflammatory product.
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Infection is a major cause of morbidity and mortality in patients with rheumatoid arthritis (RA). The objective of this study was to perform a systematic review and meta-analysis of the effect of glucocorticoid (GC) therapy on the risk of infection in patients with RA. A systematic review was conducted by using MEDLINE, EMBASE, CINAHL, and the Cochrane Central Register of Controlled Trials database to January 2010 to identify studies among populations of patients with RA that reported a comparison of infection incidence between patients treated with GC therapy and patients not exposed to GC therapy. In total, 21 randomised controlled trials (RCTs) and 42 observational studies were included. In the RCTs, GC therapy was not associated with a risk of infection (relative risk (RR), 0.97 (95% CI, 0.69, 1.36)). Small numbers of events in the RCTs meant that a clinically important increased or decreased risk could not be ruled out. The observational studies generated a RR of 1.67 (1.49, 1.87), although significant heterogeneity was present. The increased risk (and heterogeneity) persisted when analyses were stratified by varying definitions of exposure, outcome, and adjustment for confounders. A positive dose-response effect was seen. Whereas observational studies suggested an increased risk of infection with GC therapy, RCTs suggested no increased risk. Inconsistent reporting of safety outcomes in the RCTs, as well as marked heterogeneity, probable residual confounding, and publication bias in the observational studies, limits the opportunity for a definitive conclusion. Clinicians should remain vigilant for infection in patients with RA treated with GC therapy.
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Osteoarthritis (OA), one of the most common rheumatic disorders, is characterized by cartilage breakdown and by synovial inflammation that is directly linked to clinical symptoms such as joint swelling, synovitis and inflammatory pain. The gold-standard method for detecting synovitis is histological analysis of samples obtained by biopsy, but the noninvasive imaging techniques MRI and ultrasonography might also perform well. The inflammation of the synovial membrane that occurs in both the early and late phases of OA is associated with alterations in the adjacent cartilage that are similar to those seen in rheumatoid arthritis. Catabolic and proinflammatory mediators such as cytokines, nitric oxide, prostaglandin E(2) and neuropeptides are produced by the inflamed synovium and alter the balance of cartilage matrix degradation and repair, leading to excess production of the proteolytic enzymes responsible for cartilage breakdown. Cartilage alteration in turn amplifies synovial inflammation, creating a vicious circle. As synovitis is associated with clinical symptoms and also reflects joint degradation in OA, synovium-targeted therapy could help alleviate the symptoms of the disease and perhaps also prevent structural progression.
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Objective: NEM® brand eggshell membrane is a novel dietary supplement ingredient that contains naturally occurring glycosaminoglycans and proteins essential for maintaining healthy joints. A six center, open label clinical study was conducted to evaluate the efficacy and safety of NEM® as a treatment for pain and inflexibility associated with osteoarthritis of the knee and/or hip in a European population. Methods: Forty-four subjects received oral NEM® 500 mg once daily for eight weeks. The primary outcome measure was to evaluate the mean effectiveness of NEM® in relieving general pain associated with moderate osteoarthritis of the knee and/or hip at 10,30 and 60 days utilizing a 10-question abbreviated questionnaire based on the WOMAC osteoarthritis questionnaire. Results: Supplementation with NEM® produced a significant treatment response from baseline at 10 days (Q1-6 and Q9) (8.6% to 18.1% improvement) and at 30 and 60 days for all nine pain-related questions evaluated (22.4% to 35.6% improvement) and at 30 and 60 days for stiffness (Q10)(27.4% to 29.3% improvement). In a Patient’s Global Assessment, greater than 59% of patients rated the efficacy of NEM® as good or very good following 60 days of supplementation. Physicians also rated the treatment effective in subjects, with greater than 75% having moderate or significant improvement from baseline after 60 days. There were no serious adverse events reported during the study and the treatment was reported to be well tolerated. Conclusions: Supplementation with NEM® significantly reduced pain, both rapidly (10 days) and continuously (60 days) demonstrating that it is a safe and effective therapeutic option for the treatment of pain associated with osteoarthritis of the knee and/or hip. Results from previous clinical studies on NEM® can likely be extended to the broader European population.
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Osteoarthritis (OA) is by far the most prevalent arthritic disease affecting around 10% of the world's population and approximately 60% of 60-year-olds. It is also one of the most common arthritic diseases seen by general practitioners and rheumatologists. The increased frequency of OA with age, makes it a growing social health concern, as it is a disease associated with disability and pain. In the US today, the immediate cost of the disease is estimated at approximately 60 billion dollars a year. Despite the clinical success of non-steroidal anti-inflammatory drugs (NSAIDs) and of anti-cyclooxygenase (COX)-2 in treating symptoms, there is no cure for the disease to date. However, our knowledge of its etiopathogenesis has progressed significantly in the past few decades. Based on studies of human cells and animal models, targets for therapeutic intervention have been put forward and major efforts are underway to bring about new therapies that can reduce or stop the progression of the disease. This review should help the reader better understand the most recent advances regarding inflammatory and growth factors as new targets in reducing or stopping OA.
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Background: Osteoarthritis (OA) is the most common cause of disability in older adults, and although analgesic use can be helpful, it can also result in adverse drug events. Objective: To review the recent literature to describe potential adverse drug events associated with analgesics commonly used by older adults with OA. Methods: To identify articles for this review, a systematic search of the English-language literature from January 2001 to June 2012 was conducted using PubMed, MEDLINE, EBSCO, and the Cochrane Database of Systematic Reviews for publications related to the medical management of OA. Search terms used were "analgesics," "acetaminophen," "nonsteroidal anti-inflammatory drugs" (NSAIDs), "opioids," "pharmacokinetics," "pharmacodynamics," and "adverse drug events." The search was restricted to those articles that concerned humans aged ≥65 years. A manual search of the reference lists from identified articles and the authors' article files, book chapters, and recent reviews was conducted to identify additional articles. From these, the authors identified those studies that examined analgesic use in older adults. Results: There are limited data to suggest that non-frail elders are more likely than their younger counterparts to develop acetaminophen-induced hepatotoxicity. However, decreased hepatic phase II metabolism in frail elders may result in increased risk of hepatotoxicity. It is now well established that older adults are at higher risk of NSAID-induced gastrointestinal toxicity and renal insufficiency. Insofar as opioids, the data that suggest an increased risk of falls, fractures, or delirium need to be tempered by the potential risk of inadequately treating severe chronic OA-related pain. Conclusions: Acetaminophen is the mainstay frontline analgesic for treating OA-related pain in older adults. NSAIDs should be limited to short-term use only, and for moderate to severe OA-related pain, opioids may be preferable in individuals without substance abuse or dependence issues.
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Objective To examine the expression of the chemokine RANTES and its receptors in normal and osteoarthritic (OA) human cartilage and to analyze its effects on chondrocyte function.Methods The expression of RANTES and its receptors were examined by reverse transcription–polymerase chain reaction (RT-PCR) and immunohistochemistry. The effect of RANTES on gene expression of other cytokines and on the release of mediators of cartilage degradation was also examined by PCR and enzyme-linked immunosorbent assay.ResultsThe expression of RANTES was undetectable in normal chondrocytes until after stimulation with interleukin-1β (IL-1β) or IL-18. Cultures of normal cartilage also produced RANTES in response to IL-1β, as demonstrated by immunohistochemistry. All OA cartilage samples analyzed expressed RANTES messenger RNA (mRNA); RANTES protein was detected by immunohistochemistry in the superficial and mid zones of the tissue. OA chondrocytes produced elevated levels of RANTES constitutively and after IL-1β stimulation. Normal cartilage expressed the RANTES receptors CCR3 and CCR5, but not CCR1. CCR1 was expressed in OA cartilage, and CCR3 and CCR5 were increased. In normal chondrocytes, RANTES induced the expression of inducible nitric oxide synthase and IL-6. RANTES stimulated the release of matrix metalloproteinase 1 in normal and OA chondrocytes as effectively as IL-1β. Treatment of normal articular cartilage with RANTES increased the release of glycosaminoglycans and profoundly reduced the intensity of Safranin O staining.Conclusion Chondrocytes produce RANTES and express RANTES receptors. RANTES and CCR5 were markedly increased in OA and after in vitro treatment of normal chondrocytes with IL-1. Chondrocyte activation and cartilage degradation were identified as novel biologic and pathogenetic activities of this chemokine.
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Objective: Traditional nonsteroidal antiinflammatory drugs (NSAIDs) increase the risk of upper gastrointestinal (GI) bleeding/perforation, but the magnitude of this effect for coxibs in the general population and the degree of variability between individual NSAIDs is still under debate. This study was undertaken to assess the risk of upper GI bleeding/perforation among users of individual NSAIDs and to analyze the correlation between this risk and the degree of inhibition of whole blood cyclooxygenase 1 (COX-1) and COX-2 in vitro. Methods: We conducted a systematic review of observational studies on NSAIDs and upper GI bleeding/perforation published between 2000 and 2008. We calculated pooled relative risk (RR) estimates of upper GI bleeding/perforation for individual NSAIDs. Additionally, we verified whether the degree of inhibition of whole blood COX-1 and COX-2 in vitro by average circulating concentrations predicted the RR of upper GI bleeding/perforation. Results: The RR of upper GI bleeding/perforation was 4.50 (95% confidence interval [95% CI] 3.82-5.31) for traditional NSAIDs and 1.88 (95% CI 0.96-3.71) for coxibs. RRs lower than that for NSAIDs overall were observed for ibuprofen (2.69 [95% CI 2.17-3.33]), rofecoxib (2.12 [95% CI 1.59-2.84]), aceclofenac (1.44 [95% CI 0.65-3.2]), and celecoxib (1.42 [95% CI 0.85-2.37]), while higher RRs were observed for ketorolac (14.54 [95% CI 5.87-36.04]) and piroxicam (9.94 [95% CI 5.99-16.50). Estimated RRs were 5.63 (95% CI 3.83-8.28) for naproxen, 5.57 (95% CI 3.94-7.87) for ketoprofen, 5.40 (95% CI 4.16-7.00) for indomethacin, 4.15 (95% CI 2.59-6.64) for meloxicam, and 3.98 (95% CI 3.36-4.72) for diclofenac. The degree of inhibition of whole blood COX-1 did not significantly correlate with RR of upper GI bleeding/perforation associated with individual NSAIDs (r(2) = 0.34, P = 0.058), but a profound and coincident inhibition (>80%) of both COX isozymes was associated with higher risk. NSAIDs with a long plasma half-life and with a slow-release formulation were associated with a greater risk than NSAIDs with a short half-life. Conclusion: The results of our analysis demonstrate that risk of upper GI bleeding/perforation varies between individual NSAIDs at the doses commonly used in the general population. Drugs that have a long half-life or slow-release formulation and/or are associated with profound and coincident inhibition of both COX isozymes are associated with a greater risk of upper GI bleeding/perforation.