Psoriasis Treatment By Anogen

7/30/2019 Psoriasis Treatment By Anogen

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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 During treatment After treatment

Before treatment

After treatment


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Before treatment

After treatment


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Before treatment

After treatment


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Before treatment

After treatment


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Before treatment

After treatment


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