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Abcream
A topical treatment of psoriasis with monoclonal antibodies
that neutralize interleukin 8 (IL-8)
1. Introduction:
Psoriasis is a chronic, inflammatory dermatitis induced by multiple factors that affects people
with a specific genetic background. The incidence of the disease varies by population.
According to the National Institute of Health (NIH), approximately 2.2% of the United States
population is affected by psoriasis. Internationally, the incidence of psoriasis is approximately
120-180 million people. The rates of incidence for various nations/regions are as follows:Scandinavia 7-8%Denmark 5-6%Germany 4%Canada 2-3%Russia 2-3%Northern
Europe 2-3%Great Britain 2%China 0.47%and Kuwait 0.11%.
It is interesting to note that the more developed the country, the higher percentage of people
affected by psoriasis. About 4% of the population in the most developed countries suffers from
psoriasis. Some exceptions are Japan and Australia.
The etiology of psoriasis might involve the over-expression of a number of cytokines, amongwhich Interleukin-8 (IL-8) plays a pivotal role. Research has shown that IL-8 levels could elevate
100-fold in psoriasis-affected tissue when compared to normal skin tissue. In addition to
contributing to the inflammation process, IL-8 is also a growth factor for skin cells that proliferate
in psoriatic tissue. Finally, IL-8 is a potent angiogenesis factor, so it may contribute to theingrowth of blood vessels that nourish psoriatic tissue.
Based on these findings, Anogen-Yes Biotech Laboratories Ltd. was the first company in the
world to carry out the research and development of the revolutionary anti-IL-8 treatment of
psoriasis in 1993. Abcream (Anti-IL-8 monoclonal antibody topical cream) reverses the
inflammatory pathological changes by neutralizing the excessive IL-8 in the psoriatic tissue and
other skin conditions.
2. The pharmacological mechanism2.1.The biological functions of interleukin-8 (IL-8):
Interleukin-8IL-8) is a member of the chemokine superfamily with 72 residues (MW=8,000).
Interleukin-8 has been cited as a pro-inflammatory mediator in gingivitis and psoriasis. IL-8
acts as neutrophil activator and chemotactic factor. In particular, there is compelling evidence
showing that IL-8 plays a pivotal role in the inflammatory process. IL-8 is secreted by several
cell types, including macrophage, neutrophil, monocyte, endothelial cells, keratinocyte etc., and
affects the cells to induce inflammatory response.
Most likely, the effect of IL-8 in promoting or causing tissue damage is by inducing the
infiltration of neutrophilic leukocytes as well as by triggering the release of lysosomal enzymes
and superoxide anions from leukocytes. The excess amount of IL-8 level in affected tissues
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has been found to be associated with a number of disease states. Among these diseases are
psoriasisarthritis deformans, idiopathic fibrosis of the lung, enteritisadult respiratory
distress syndrome, and septic shock etc.
2.2.The association of IL-8 over expression with psoriasis:Psoriasis is characterized by abnormal keratinocyte growth and differentiation. The functional
abnormality of keratinocytes is believed to be triggered by T lymphocytes, and various
cytokines. In addition, polymorphonuclear neutrophil (PMN) infiltration of the skin and
Munro microabscesses are characteristic histological findings in psoriasis, confirming that
neutrophils have a role in the pathogenesis of this disease. It has been postulated that in
addition to influencing keratinocyte growth and differentiation of neutrophils in the epidermis,
IL-8 might also trigger T-lymphocyte activation by inducing cell-surface expression of HLA-Dr.
The accumulation of neutrophils in the outermost layer of the epidermis has been associated
with the presence of highly inflammatory, treatment-refractory psoriasis plaques.
IL-8 plays a crucial role in the pathogenesis of psoriasis:
2.2.1. IL-8 is a strong chemotactic factor. IL-8 over-expression in the skin gathers largeamount of neutrophils, T-lymphocytes and other inflammatory cells. The
infiltration of these cells damages skin tissue and causes blisters. The gathered
neutrophils and T-lymphocytes also produce large amount of IL-8 and aggravate
local pathological changes, resulting in inflammation of skin and accumulation of
debris and scales formed by necrotic cells and dead tissue.
2.2.2. IL-8 is a strong growth factor of epidermal cells. Excessive amount of IL-8production can lead to the overgrowth of abnormal keratinocytes in the focus of
psoriasis.
2.2.3. IL-8 is also a potent angiogenesis factor. It causes the acceleration of blood vesselformation in psoriatic focus, making possible sufficient blood supply for theabnormal growth and proliferation of epidermal cells.
2.2.4. The biological effect of IL-8 is mediated by its receptors on the surface of theinflammatory cells. Both keratinocyte in the focal zone and the infiltrated
neutrophilic leukocyte can express large amount of IL-8 receptors on their surface.
Increased numbers of IL-8 receptors and elevated IL-8 can lead to severe dermatitis
in the vicious cycle.
2.3. The therapeutic effects of anti-human IL-8 monoclonal antibody on psoriasis:
The monoclonal antibody is a high titer neutralization antibody specific to IL-8. It can make an
effective therapy to psoriasis by neutralizing excessive IL-8 production and eliminating
neutrophil recruitment, thus, possessing anti-inflammatory role at the psoriatic skin. In addition,
the antibody can block IL-8s angiogenic effect. Therefore, local microvascular formation and
the abnormal proliferation, differentiation, and necrosis of keratinocytes can be controlled. As a
total result, the symptoms of psoriasis can be reduced.
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2.4. The Specificity of this anti-IL-8 monoclonal antibody:
The specificity of this anti-IL-8 antibody was tested by measuring the cross-reactivity of this
antibody with other cytokines, chemotactic factors and cytokines that share structural similaritywith IL-8. The results (Table 1 & 2) showed that the anti-IL-8 monoclonal antibody was
highly specific to IL-8, and had no cross-reactivity with following factors: GM-CSF, TGF-,
MCAF, TNF-, IL-7, IL-1, b-FGF, IL-16, MCP-3, M-CSF, EGF, and GRO, PF-4, ENA78,
GCP2. The following results were from 2 ELISA assays:
Table 1. Test the cross-reactivity with other cytokines and chemotactic factors.
Cytokine OD reading
IL-8 Over reading range (>2.7)
GM-CSF 0.037
TGF- 0.021
MCAF 0.023
TNF- 0.039
IL-7 0.060
IL-1 0.029
b-FGF 0.027
IL-16 0.044
MCP-3 0.024
MCSF 0.044
EGF 0.044
BSA 0.147
HAS 0.129
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Table 2. Test the cross-reactivity with structurally similar cytokines:
Cytokine OD reading
IL-8 Over reading range (>2.7)
GROa 0.097+ 0.008
PF-4 0.113 + 0.04
ENA78 0.073 +0.009
NAP-2 0.031 +0.004
GCP-2 0.088 +0.007
2.5. Neutralizing the chemotactic effect of IL-8 in vitro
Chemotaxis assay was used to determine the neutralization effect of anti-IL-8 antibody to the
IL-8 induced chemotactic migration of IL-8 receptor expressing 293 cells.
Method:
10ng/ml IL-8 antigen was used to react with anti-IL-8 monoclonal antibody diluted to
different concentrations (50g/ml, 5g/ml, 0.5g/ml, and 0.05g/ml). For negative
control, only antigen existed in the reaction. All the samples were reacted at 37Cfor30mins. Each sample was seeded respectively into the wells at the lower layer of the
chemotaxis chamber. 50l of cell culture was seeded into each well at the upper layer of
the chamber. Between upper and lower layer of wells, 10m pore-size polycarbonate
ester filter membrane was used to separate the two layers. The chamber was incubated in
5 % CO2 at 37C for 5 hours. After that, the filter membrane was taken out and stained
with Dieff-Quick. The number of cells on the membrane was counted and the result was
calculated by the average cell numbers of three wells.
The data (Table 3) below showed that this anti-IL-8 monoclonal antibody strongly
inhibited the chemotactic migration of 293 cells by neutralizing IL-8.
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Table 3. Inhibition of the chemotactic migration of 293 cells by IL-8 antibody.
Anti-IL-8
antibody
IL-8
antigen
Ratio of antibody to
antigen
Inhibition in
chemotaxis50g/ml 10ng/ml 5000:1 118.25%
5g/ml 500:1 99.81%
0.5g/ml 50:1 83.90%
0.05g/ml 5:1 17.09%
% Inhibition in chemotaxis = 1 (the number of migrated cells in anti-IL-8 group
the number in medium control group) / (the number of migrated cells in IL-8 group
the number in medium control group).
2.6. Cytokine profile in the skin tissue of psoriasis patients
2.6.1. Material and method:
2.6.1.1. Patients: 11 patients with active plaque-type psoriasis (male: 6, female: 5, the
median age was 39.2, and the age span was 21~68) were selected for this study. The
conditions of these psoriasis patients were determined by the same clinical doctor on the
standard of PASI. The median of the conditions was 18.6, and the span was 5.7~34.6.
All of them didnt have any neopathy or received treatment 21 days prior to the
experiment. The control group was 8 normal volunteers (male: 4, female: 4, the median
age was 40.5, and the age span was 22~64).
2.6.1.2. Biopsy and organ culture: 40mm2 biopsy skin samples were collected from the
lesion region and the surrounding non-inflammatory skin of the patients (at least 10cmaway from the lesion), and the healthy skin tissue were collected from the normal
volunteers as control. The subcutaneous tissue was removed and the skin samples were
cultured in 24 well plates containing KGM medium (GIBCO, Invitrogen) at 37C for 48
hours. The cell culture supernatant was collected respectively and stored in -80C for
further analysis.
2.6.1.3. Concentrations of IL-8 and other pro-inflammatory cytokines in supernatant were
determined by quantitative ELISA kits (supplied by Anogen and R&D).
2.6.1.4. Statistical analysis: Kruskall-wallis, Mann-whiteny and Wilcoxon paired test.
2.6.2. The results:
2.6.2.1. Skin thickness varies in different type of skin tissue and affects the weight:
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Table 4. Tissue weight of psoriasis skin and normal skin
Psoriatic skin weight
(40mm2 area)Patient
Surrounding non-inflammatory
skin weight (40mm2 area)patient
Healthy skin weight
(40mm2 area)normal person
Median Span Median Span Median Span
43.5mg 18~103mg 38.8mg 20.1~78.6mg 34.7mg 16~76mg
2.6.2.2. Quantitative determination of cytokines expressed by psoriatic skin tissue and
normal skin tissue:
The cytokine concentration in the supernatant of the tissue culture was determined by
quantitative ELISA. Considering that the weights of the tissue were different, cytokinelevels in the supernatant were calculated by picogram per milligram of tissue (pg/mg).
Table 5. Cytokine expression in the skin tissue culture supernatant
Cytokine Psoriasis skin Non-inflammatory skin Normal skin
IL-1
(pg/mg)
Median 4.0 0.7 0.6
Span 0.3-15 0-3 0-2
IL-6
(pg/mg)
Median 226 63 51
Span 147-550 18-287 0-135
IL-8
(pg/mg)
Median 570 38 35
Span 198-2131 0-677 0-109
GM-CSF
(pg/mg)
Median 7.3 2.4 1.7
Span 0.4-21 0-13 0.3-6
TNF-
(pg/mg)
Median 343 87 29
Span 38-929 0-401 0-93
INF-
(pg/mg)
Median 197 25 3.4
Span 78-625 0-108 0-19
MCAF
(pg/mg)
Median 102 16 1.7
Span 0.9-310 0-99 0-5.7
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Above results showed that cytokine production in psoriasis skin was significantly higher
than in non-inflammatory skin and normal control. In particular, the concentration of IL-
8 was the highest.
Table 6. The IL-8 concentrations in skin tissue culture supernatant:
IL-8 (pg/mg)
Psoriasis skin tissue Non-inflammation
skin tissue
Normal control
1 2131 284 1 109
2 1852 35 2 63
3 1289 677 3 51
4 860 0 4 35
5 794 12 5 35
6 570 110 6 15
7 519 82 7 0
8 486 0 8 0
9 380 38
10 355 40
11 198 0
Statistical analysis:
Psoriasis skin --- Non-inflammatory skin P=0.006 (Wilcoxon rank test)
Psoriasis skin ---Normal control P=0.003(Mann-whiteny test)
Non-inflammatory skin--- Normal control P=0.8(Mann-whiteny test)
2.7. The anti-IL-8 monoclonal antibody is able to penetrate into psoriasis-damaged tissue:
The skin permeability is the ability of foreign substances to penetrate and diffuse through the
skin. The skin barrier naturally has a low permeability, thus protects the body from particles and
foreign toxins by not allowing them to penetrate through the surface. However, studies have
shown that nanoparticles of 40nm in diameter and smaller can penetrate the skin, and
dermatosis such as psoriasis and measles can weaken the skin barrier.
A study (Table 7) by the medical school of university of California showed that the skin
permeability in psoriasis plaque can reach 11 times, 5 times, and 2 times of the normal skin, as
measured by Transepidermal Water Loss (TEWL).
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Table 7. Transepidermal water loss increased in psoriasis-damaged skin
Lesion Skin
TEWL( g//h)
Significance Normal Skin
TEWL( g//h)
Erythrodermic
psoriasis
36.42.26 P0.001 3.51.0
Active plaque
psoriasis
16.10.97 P0.001 3.90.4
Chronic plaque
psoriasis
9.01.9 P< 0.05 4.10.5
Anti IL-8 antibody is a large molecule of 150 kilo-Dalton. Its entry into the skin is more
difficult than small molecules. To assess if or not the antibody can penetrate the psoriasis plaque
in sufficient amount, in vivo experiments have been conducted independently in two labs on
voluntary patients.
2.7.1 Biopsy result from Beijing Union Hospital:2.7.1.1. Case information: 10 patients were recruited for the study. There were 5 males
and 5 females; the average age was 34 years old. The patients were treated with Abcream
for 1-2 weeks. 5 psoriatic samples were collected from upper limb, 4 psoriatic samples
from abdomen, and 1 psoriatic sample from lower limb. Sample from 1 patient not
treated with Abcream was used as negative control. All samples were studied by
immunohistochemical detection.
2.7.1.2. Method: To detect the mouse anti-IL-8 antibody in Paraffin specimen, ABC
method and SPTM immunohistochemistry kits (supplied by LAB VISION) were used.
The samples were treated with 10% formalin, fixed into paraffin. The de-waxed paraffin
section was washed with water, and then incubated with biotinylated secondary antibody.
After washing, Avidin-HPR was added and incubated with the specimen. The specimenwas stained with DAB substrate for 5~10 min, and counter-stained with hematoxylin.
The positive samples should show brown color in epidermic cell space.
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2.7.1.3. Results:
Fig1. Scattered brown stain was observed in aceratosis horny layer (1040)
Fig 2. Brown stain was observed in aceratosis horny layer and superficial acanthocyte
layer where the inflammatory cells gathered (1010)
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Fig 3. No coloration in negative control (1010)
2.7.1.4. Discussion: In this study, whether anti- IL-8 in the cream was able to penetrate
into psoriasis-damaged tissue was studied using ABC method. Scattered brown flake in
aceratosis layer was observed in 7 psoriasis samples. Among them, 2 samples showed
brown color in the superficial acanthocyte layer, indicating that anti-IL-8 antibody
infiltrated the epidermis and accumulated in the stratum spinosum of the epidermis. In
contrast, anti-IL-8 antibody was not detectable in the deepest layer of the epidermis.
These results indicated that the anti-IL-8 antibody didnt penetrate the epidermis or the
amount of this antibody penetrated was under detection limit of this method.
2.7.2. Biopsy results from the First Hospital of Beijing University:
2.7.2.1 Material and method
Mouse anti-human IL-8 antibody cold cream and blank control cream were supplied by
YES Biotech Laboratory Inc. of Canada. ABC staining KIT was supplied by Huamei
Biotechnology Company. Microtome was from BRIGHT Instrument Inc. Microscope
was manufactured by OLYMPUS.
A psoriasis patient with stable plaque lesion was selected for the experiment. The cream
was applied once to the plaque with gentle massage each day for 2 weeks. The test
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sample was collected from Abcream treated plaque lesion at the left abdomen. The
control sample was collected from cream ingredients (Blank cream) treated plaque lesion at
the right abdomen. These 4mm diameter biopsy samples were frozen and cut with
microtome to obtain frozen sections. The anti-IL-8 antibody in these samples was
detected by ABC method. The experimental design included blank controls (B1, B2)
which were treated with blank cream (without IL-8 Ab), and a self coloration control that
obliterated biotinylated anti-mouse IgG during the immuno-staining (A3). Counter-
staining was used for specimen A2 and B2.
2.7.2.2. Result:
Table 8. The immuno-staining results of the treated group and control group.
AbcreamBlank
cream
Biotinylation
anti-mouse IgG
Counter-
staining
Brown
colorationFigure #
A1 + + + Fig 4
B1 + + - Fig 5
A2 + + + + Fig 6
B2 + + + - Fig 7
A3 + + - Fig 8
Fig 4. Tan coloration in epidermis without counter-staining
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Fig 5. No tan coloration in epidermis for blank cream
Fig 6. Tan coloration in epidermis with counter-staining
Fig 7. No tan coloration in epidermis for blank cream.
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Fig. 8. No tan coloration in epidermis without biotinylation anti-mouse IgG
The epidermis of the Abcream treated specimens (A1 & A2) was brown colored by the
immuno-staining with (Fig. 7) or without (Fig. 4) counter-staining. Removal of the
biotinylated anti-mouse IgG from the immuno-staining procedure resulted in non-
coloration in Abcream treated specimen (A3, Fig.9). The blank controls (B1 & B2) were
not colored by the immuno-staining with (Fig. 5, 6) or without counter-staining (Fig. 8).
The data indicated that the staining was specific. The immunohistochemical study
showed that the anti-IL-8 antibody in Abcream reached the epidermal basal layer ofpsoriatic skin lesion.
3. Summary of phase II/ phase III clinical study:
The Therapeutic Effect of Abcream Against Psoriasis, - a Multicenter Clinical Evaluation
Authors: Lu Lin1, Xiangsheng Chen1, Jiabi Wang2, Fanqin Zeng3, Yijie Bai4, Kanghuaug Liao5,
Chuanchao Pang6, Peiying Jing1
1. The Institute of Dermatology of the Chinese National Institute of Medicine2. Beijing Union Hospital3. The Sun-Yat-Sen Memorial Hospital of Zhongshan Medical University4. University Hospital of Bethune Medical University5. Huashan Hospital of Shanghai Medical University6. Peoples Hospital of Liaoning Province
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3.1. Introduction
Psoriasis is a chronic skin disease of unknown etiology. Multiple cytokines and inflammatoryfactors may play a role in the pathogenesis of this disease. Abcream, which contains an anti-
IL-8 monoclonal antibody, is a topical cream developed by Yes Biotech Laboratories Ltd. for
the treatment of this disease. We studied the therapeutic effect of this cream on psoriasis from
October 1999 to June 2000.
3.2. Methodology:
Participants: The recruits were from the Institute of Dermatology of the Chinese National
Institute of Medicine, Beijing Union Hospital, the Sun-Yat-Sen Memorial Hospital of
Zhongshan Medical University, University Hospital of Bethune medical University, Peoples
Hospital of Liaoning Province, Huashan Hospital of Shanghai Medical University. The
majority of the patients were in progressive phase and some were in quiescent phase. All the
patients possess typical psoriasis symptoms.
Method of the trial: It was randomized, double blinded, placebo controlled clinical trial. An
open-labeled group was also included in the study. In this research, Abcream was applied to
the treatment group while the control group used all other cream ingredients (Blank cream) butwithout the anti-IL8 antibody.
Method of drug application: The cream was applied to the lesion area twice each day with
gentle massage for several minutes. The application lasted for 6 weeks.
Observation: Visit began one week prior to the treatment and continued during the treatment.
The frequency was one visit per week. The diameter of of the lesion, erythoderma, the
thickness, and other symptoms were recorded. The symptoms were scored from 0 to 4
accordingly. Adverse reactions such as irritation, allergic reaction and systemic mal-
absorption were also observed and recorded.
3.3. Result:
The participants that met the design requirement were divided into treatment group (208 cases),
control group (234 cases) and open-labeled group (231 cases). The recruits that actually
completed the procedure were: 202 participants in the treatment group, 221 in the control group,
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and 210 in the open-labeled group. The reason for dropping-out included missing visits,
ineffectiveness, non-complying, local adverse effect and change of treatment protocol. No
significant difference in dropping out rate was observed between treatment group and control
group (X2=3.37, P>0.05).
The treatment group and control group were similar in age distribution, disease progression,
plaque type and clinical phase. Table 12 showed that the symptoms of the treatment group and
control group were comparable. After one week of treatment, the scores for each symptom
changed faster in the treatment group than in the control group (Table 13). After 3-6 weeks,
the decrease in the severity of each symptoms in the treatment group was significant comparing
with the control group (p
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difference was insignificant. Other factors might cause the variation during the 6 week
treatment.
Table 9. Comparison of the average psoriasis symptom scores in the treatment group and
control group prior to the treatment.
Diameter of skin
lesion
Erythroder
ma
Thickness Scales Itching Total score
Treatment group 2.06+1.03 2.86+0.89 2.32+0.81 2.41+0.84 1.86+0.91 11.5+3.06
Control group 2.00+1.00 2.81+0.87 2.35+0.87 2.47+0.79 1.38+1.01 11.5+3.13
Table 10. Weekly score decrease in the treatment group and control group.
Diameter
of skin
lesion
Erythroderm
a
Thickness Scales Itching Total score
decrease
Week 1 Treatment
group
0.02+0.22 0.24+0.52 0.27+0.52 0.52+0.72 0.37+0.68 1.44+1.81
Control group 0.02+0.15 0.14+0.38 0.11+0.34 0.42+0.62 0.21+0.67 0.91+1.42
Week 3 Treatment
group
0.19+0.59 0.75+0.85 0.67+0.78 1.06+0.95 0.91+0.93 3.59+2.97
Control group 0.06+0.35 0.37+0.64 0.38+0.56 0.82+0.75 0.51+0.85 2.15+2.02
Week 5 Treatment
group
0.40+0.79 1.18+1.04 1.08+0.92 1.49+1.07 1.25+1.01 5.40+3.61
Control group 0.11+0.57 0.61+0.86 0.56+0.75 1.17+0.94 0.71+0.96 3.16+2.83
Week 6 Treatment
group
0.52+0.95 1.34+1.16 1.22+0.97 1.61+1.07 1.32+0.99 6.02+3.96
Control group 0.16+0.65 0.70+0.93 0.67+0.85 1.25+1.02 0.76+1.01 3.55+3.23
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Table 11. Recovery rate (%)
Open-labeledgroup
Treatmentgroup
Control group X2
P value
Week 1 0 0 0 -
Week 3 3.8 1.5 0.5 - 0.35
Week 5 6.2 5.9 0.9 6.86
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Affected Areas Before and After Abcream Topical Treatment
Before treatment After treatment
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Before treatment After treatment
Before treatment After treatment
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Before treatment After treatment
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Before treatment During treatment After treatment
Before treatment
After treatment
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Before treatment
After treatment
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Before treatment
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Before treatment After treatment
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Before treatment
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Before treatment
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Before treatment After treatment