Auto immune diseases: Case Studies on Rheumatoid Arthritis and Crohn's Disease: Therapeutics - Past, Present and Future.

Analytical Science Literature Review BE493

Elizabeth Gallagher

12378191

B.Sc. Analytical Science

2015 - 2016

Supervisor: Prof. Christine Loscher

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DCU Science & Health Assignment Submission

Student Name: Elizabeth Gallagher

Student Number: 12378191

Programme: AS4 - BSc in Analytical Science

Project Title: Analytical Science Literature Survey

Module code: BE493

Lecturer: Prof. Christine Loscher (supervisor)

Project Due Date: 11/12/2015

Declaration

I the undersigned declare that the project material, which I now submit, is my own work. Any

assistance received by way of borrowing from the work of others has been cited and

acknowledged within the work. I make this declaration in the knowledge that a breach of the

rules pertaining to project submission may carry serious consequences.

I am aware that the project will not be accepted unless this form has been handed in along

with the project.

Signed:

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TABLE OF CONTENTS

SECTION ONE......................................................................................................................................................4

1.1 Abstract.....................................................................................................................................................4

SECTION TWO.....................................................................................................................................................4

2.1 INTRODUCTION...............................................................................................................................................4

SECTION THREE.................................................................................................................................................5

3.1 THE INFLAMMATORY RESPONSE: WHAT IS THE INFLAMMATORY RESPONSE?..............................................53.2 MEDIATORS OF THE INFLAMMATORY RESPONSE...........................................................................................53.3 DEREGULATION OF THE INFLAMMATORY RESPONSE......................................................................................8

SECTION FOUR...................................................................................................................................................9

4.1 WHAT IS TNF?...............................................................................................................................................94.2 BRIEF HISTORY OF TNF.................................................................................................................................94.3 TNF AND ITS RECEPTORS TNFR1 AND TNFR2..........................................................................................104.4 TNFR-INDUCED INFLAMMATION AND ALTERNATIVE SIGNALLING PATHWAYS...........................................10

SECTION FIVE...................................................................................................................................................11

5.1 WHAT IS RHEUMATOID ARTHRITIS?............................................................................................................115.2 MOLECULAR MECHANISMS OF RHEUMATOID ARTHRITIS...........................................................................125.3 THERAPEUTICS FOR RHEUMATOID ARTHRITIS.............................................................................................14

SECTION SIX......................................................................................................................................................18

6.1 WHAT IS CROHN’S DISEASE?.......................................................................................................................186.2 MOLECULAR MECHANISMS OF CROHN’S DISEASE.......................................................................................186.3 THERAPEUTICS FOR CROHN’S DISEASE........................................................................................................20

SECTION SEVEN...............................................................................................................................................23

7.1 OVERLAP OF CROHN’S DISEASE AND RHEUMATOID ARTHRITIS: CYTOKINES.............................................237.2 OVERLAP BETWEEN THE THERAPEUTICS USED FOR CROHN’S DISEASE AND RHEUMATOID ARTHRITIS......257.3 EMERGING FUTURE THERAPEUTICS..............................................................................................................28

SECTION EIGHT................................................................................................................................................32

8.1 CONCLUDING REMARKS...............................................................................................................................328.2 REFERENCES.................................................................................................................................................33

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Section One

1.1 AbstractDiseases such as Rheumatoid Arthritis (RA) and Crohn’s Disease (CD) are widespread

throughout today’s society, constant research continues into finding the blockbuster

therapeutic to treat autoimmune diseases such as RA and CD and evidence of this can be seen

as a common topic in related literature. The objectives of this study were to explore the

overlapping characteristics of RA and CD, regarding the mechanisms of operation and

therapeutics and by doing so the usage of various search engines such as ScienceDirect and

PudMed were explored in an attempt to improve and strengthen original researching skills.

Throughout this study the involvement of the proinflammatory cytokines, TNFɑ (tumor

necrosis factor alpha) and IL-6 (interleukin-6) in both CD and RA was observed and an

additional overlap was noted in the therapeutics used to treat both disorders. In various

studies detailed below adalimumab was investigated as both a therapeutic in CD and RA and

presented a loss of response in both cases. Advanced search engines were the primary

resource of all studies carried out in the area of autoimmunity over the last three years;

publications exceeding ten years were not included. By exploring various publications the

conclusion was drawn that whilst adalimumab showed efficacy of a specific level. Additional

studies on therapeutics such as tocilizumab and rituximab as present and emerging

therapeutics showed desirable efficacy in RA and insignificant efficacy in CD.

Section Two

2.1 IntroductionEven in today’s constantly advancing, dynamic, highly educated world there still exists

disease. Throughout an individuals life it is undeniable that at some stage they will encounter

a disease of some significance whether that be a common dose of influenza or a more severe

disease such as cancer.

A startling amount of resources are pumped into combating diseases of all levels.

Unfortunately it is still a reality that diseases of unknown etiology exist. Diseases such as

Rheumatoid Arthritis (RA) and Crohn’s Disease (CD) fall under this category and effect

people of all age, race and gender.

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Sadly, as of yet, there is no specific target that can be met in order to completely alleviate

patients from the symptoms of either RA or CD. A potential relapse is always a possibility

and fear among patients who are fortunate enough to achieve remission. Both RA and CD are

caused by an over-stimulation of the inflammatory response. The inflammatory response can

cause colossal damage when over-stimulated by cytokines such as TNFɑ (tumour necrosis

factor). Therefore it is of no surprise that combating diseases of the immune system is a

multi-billion dollar industry, with drugs such as Enbrel®, Remicade®, HUMIRA® and

Cimzia® producing enormous profit turnovers for their mother companies each year. Anti-

TNF therapeutics continue to be widely used in the treatment of both RA and CD and in

many cases their utilisations overlap. The benefit of this overlap has been noted as a dual

treatment for CD patients presenting arthritis as an extraintestinal manifestation. The

treatment of RA and CD with anti-TNF therapeutics has be documented throughout literature

and by exploration of this documentation the link between RA and CD can be drawn closer

through both its cytokines and therapeutic overlap.

Section Three

3.1 The Inflammatory Response: What is the Inflammatory Response?Inflammation is defined by the Medical Dictionary as “A localised protective response

elicited by injury or destruction of tissues, which serves to destroy, dilute, or wall off both the

injurious agent and the injured tissue” [1]

3.2 Mediators of the Inflammatory ResponseInflammation is a mechanism of defence carried out by the adaptive immune response. Upon

activation inflammation can trigger a series of complex cascades. This activation may be

caused by an antigen defeating the first line of defence, a bacteriological infection or

deregulation of the inflammatory response, known as an auto-inflammatory disease.

Inflammation is induced and controlled by various cells displaying defence mechanisms and

in doing so causing further migration of immune cells. Monocytes and macrophage cells play

an imperative role in the onset of inflammation. Derived from the bone marrow, macrophage

cells are found widely distributed throughout the body where they maintain homeostasis.[2]

Macrophage cells are divided into two categories of polarized phenotypes when involved in

the immune response, these phenotypes are M1 and M2.[2]

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M1 macrophages are the classically polarized macrophages which are proinflammatory. M1

macrophages also act as effectors of TH1 and TH17 (helper T- cells one and seventeen)

mediated immune response.[2] M1 macrophages act as effectors of both TH1 and TH17 when

they are stimulated by a cell signalling cytokines such as GC-CSF (granulocyte monocyte

colony stimulating factor) which is a cytokine secreted by various immune cells upon contact

with an antigen and IFN-γ (interferon- γ) which is a dual expression cytokine involved in the

regulation of inflammation.[3][4] Stimulation due to the presence of LPS (lipopolysaccharide)

may also result from pathogenic invasion.[2] M1 macrophages, upon stimulation will produce

vast quantities of TNF (Tumour Necrosis Factor), IL-12 (interleukin-12) and IL-23

(interleukin-23) which will attract both TH1 and TH17 to the target site.[2] The presence of TH1

and TH17 at the target site will enhance inflammation. Cytokines, alike M1 and M2

macrophage can be classified into pro inflammatory or anti inflammatory.

The pro inflammatory cytokines such as TNF-ɑ are a focal point of study in the area of

autoimmune diseases as the levels of these cytokines is consistent within these diseases

showing increased levels in affected patients. The tumour necrosis factor (TNF) and its

receptor superfamily consist of ~50 membrane and soluble proteins which can regulate

cellular activity and function. [2] TNF and its receptors possess an extensive range of

functions including the promotion of cell differentiation and production of the cytokines and

chemokines of the inflammatory response.[2] TNF-ɑ is a cytokine of specific importance as it

triggers a series of pro inflammatory events. Figure one (O’Connor & Nichol 2015) below

shows the various cytokines and their functions in the immune and inflammatory response.

Figure one adapted from O’Connor & Nichol 2015[5] showing the classification of cytokines and their functions in the immune system. TNF, Tumor Necrosis Factor. IL-X, interleukin-X, TGF-beta, Transforming Growth

Factor-Beta. IFN-alpha/beta/gamma Interferon-alpha/beta/gamma.

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As figure one adapted from the work of O’Connor and Nichol 2015[5] outlines there are

numerous cytokines involved in the immune response. TNF-ɑ and IL-1 (IL-1 not shown

above) play fundamental roles in the onset of inflammation and their involvement in

inflammatory disease is heavily studied.[5] Conversely, in order to maintain equilibrium for

regulation of the inflammatory response in unaffected individuals there exists anti-

inflammatory cytokines, IL-4, IL-10 and IL-13 which are all shown above in figure one. The

functions of these cytokines are also examined in an attempt to dampen inflammatory

diseases and in hope of discovery of a new method of treatment for inflammatory disorders.

As a result the involvement and balance between the two categories of cytokine families is

imperative for regulation of the inflammatory response. This importance is also reflected in

the requirement for ongoing balance between the differentiation of pro and anti inflammatory

phenotyping of macrophage cells by cytokines which is discussed further below.

As aforementioned an inverse M1 macrophage cell exists. This macrophage is known as the

M2 macrophage. The M2 macrophage is alternatively polarized and its role is imperative in

the regulation of inflammation. Due to this polarization the phenotype of the macrophage is

changed causing it to induce the endpoint of inflammation. This phenotype change causes a

reduction in the expression of IL-12, as seen expressed in M1 macrophages. Conversely there

is an increased expression of IL-10 and IL-1RA of the TH2 cells.[2] M2 macrophages, due to

their immunosuppressive properties are the mediators behind pathogen sediment clearance,

inflammation regulation (to an extent) and tissue repair following inflammation. [6] Figure 1

below shows the simplified diagrammatic explanation of the process of inflammation.

Figure one showing the simplified steps of inflammation. Again note the obvious inclusion of the macrophage as a primary mediator in the inflammatory response and cytokine secretion [6] (Slonczewski 2009)

When the macrophage cells circling the blood stream detect the presence of an antigen which

has overcome the first line of defence a rapid polarization step occurs. In this polarization

step is controlled by many factors including NF-κb (nuclear factor) and activator protein 1

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(AP1). [7] It is here that the macrophage is classically polarized to become an M1 macrophage

which will, by a sequence of events induce inflammation.[7] M1 macrophages will secrete

TNF (Tumour Necrosis Factor) and various cytokines including IL-1. [7] M1 macrophages

will also secrete chemokines for example CXCL9 induced by IFN-γ which in turn will attract

TH1 cells which, with the presence of TH2, TH17 and other cells with initiate inflammation.[7]

Inflammation was first described in the 1st century by the Roman encyclopaedist, Aulus

Celsus as the Celsus tetrad compromised of:  “calor,  dolor,  tumor  and rubor”  translating to

what we know to be the hallmark symptoms of inflammation, heat pain swelling and redness

respectively.[8]

During inflammation a series of cytokines, chemokines and immune cells respond to an

invading agent. Two main events occur during inflammation: vasodilation and increased

vascular permeability.[5] Vasodilation increases the blood pressure migrating cells of interest

to the target site rapidly and vascular permeability allows these cells to enter across the blood

vessels to the target site. Cells secrete cytokines and compounds, Mast cells secrete histamine

which will contribute to inflammation, cause an allergic reaction and in turn increase vascular

permeability. [5]

3.3 Deregulation of the inflammatory responseInflammation, even when regulated causes immense tissue damage, thus the importance of

the M2 macrophage in the mediation of tissue repair is evident. When inflammation becomes

uncontrolled in cases such as rheumatoid arthritis and Crohn’s disease the tissue damage is

colossal and in most cases completely irreversible.[7] In individuals where the immune system

is regulated inflammation plays a protective role and may protect against infection on both a

minor and fatal level. Unfortunately that is not always the case. Many people suffer from an

imbalance of the inflammatory response. This results in the aforementioned degradation of

tissues and irreversible damage over time. This can be seen in many inflammatory diseases or

diseases which affect the immune system.

Autoimmune and Auto-inflammatory diseases share common overlaps in many areas and

those who suffer can experience mild to severe discomfort as a result. Diseases associated

with the immune system and its functions include Rheumatoid arthritis, Crohn’s disease,

COPD (chronic obstructive pulmonary disease) and Diabetes I (stage II diabetes is in the

process of being redefined as an autoimmune disease [9]). In all auto immune diseases the

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proinflammatory cytokine population of the inflammatory response is increase and results in

patient discomfort. Seen particularly in autoimmune diseases such as RA and CD, the over

production of the proinflammatory cytokine TNF is the mechanism behind the initiation and

continuation of the inflammatory response.

Section Four

4.1 What is TNF?TNF is an abbreviation of Tumor Necrosis Factor. TNF is a cytokine whos function is

describes by Rajput and Ware 2016[10] as a cytokine which “activates essential cellular

pathways initiating inflammation providing imperative pathways controlling the body’s

response to invasion.” TNF, like most other cytokines, has two receptors through which it

directs inflammatory cell function. [10] These receptors are aptly labelled TNFR1 (p55) and

TNFR2 (P75). [10] When bound by TNF, TNFR1 and TNFR2 activate additional cytokine and

inflammatory signalling pathways.” [10]

4.2 Brief History of TNFTumor necrosis factor alpha (TNFα) was discovered in the year 1975 and interestingly, was

originally identified as an endotoxin inducible molecule which caused the destruction of

tumors in vitro.[11] Now it is considered a major proinflammatory cytokine involved in the

innate immune response.[12] Throughout autoimmune disorders a trend is visible and that

trend is of the involvement of TNFα. Thus the importance of TNFα in the pharmaceutical

industry and drug development is invaluable. TNFα, although extensively studied is still a

cytokine with many properties which are not completely understood.

TNFα has a synergistic relationship with other proinflammatory cytokines promoting the

secretion of cytokines such as IL-6, IL-1β and IL-2.[11] and is thought to be a strong mediator

in the onset of inflammation. Following its discovery in 1975, TNF was used as an anti-

cancer therapeutic however following lengthy, extensive clinical trials the cytotoxic

properties of TNF did not outweigh the side effects occurring in patients. In many cases the

side effects included shortness of breath, vomiting and hypertension.[11] Even during

administration with the interleukin IL-2 TNF still did not produce significant results as

regards its anti-cancer properties. [11] During the clinical trials it was observed that the mice

used for the initial testing of TNF were protected against TNF-mediated endotoxemia which

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was caused by the use of neutralizing antibodies on TNF. It was here that the potential

therapeutic functions of TNF were noted. [11]

4.3 TNF and its Receptors TNFR1 and TNFR2Two forms of TNF exist, proTNF which is defined as the membrane TNF expressed on the

cell surface and sTNF which is the soluble form of TNF (sTNF) found in the blood plasma.[11]

Soluble TNF is formed by the cleavage of proTNF with a metalloprotease TACE (TNFɑ-

converting enzyme) thus releasing sTNF the soluble form of TNF. [11] Both of these ligands,

proTNF and soluble TNF require receptors. These receptors are conveniently called TNFR1

and TNFR2 (Tumor necrosis factor receptor one and two). It has been documented that both

proTNF and soluble TNF can bind to either receptor although it has been observed in the

work of Sedger and Mcdermott 2014[11] that proTNF is a more dominant ligand for TNFR2,

which is selectively expressed on some cells whereas TNFR1 is expressed on the majority of

cells on some level. [11] The level of TNFR expression is closely controlled by cytokines

specifically the interleukins hence the constant trend in literature of their synergistic

relationship. [11]

The biological interactions between TNF and its receptors are extremely complex. The

movement of TNF between receptors has been proposed through a number of complex

models. [11] An example of the level of complexity of the TNF and its receptors is seen in the

theory of reverse signalling. [11] It has been observed the TNFR which binds to proTNF has

the capability of what is known as reverse signalling. [11] Reverse signalling being in this case

the signalling back into the proTNF producing cells, the resultant activation is of NF-κB as

the intracellular regions of the proTNF become phosphorylated. [11] This is one of the ways in

which the complexity of TNF and its receptors can result in altered cytokine expression by

the same TNF producing cells, a further indication of their synergistic relationship. [11]

4.4 TNFR-induced inflammation and alternative signalling pathwaysOutlined in the work by Sedger and McDermott 2014[11] the effects of TNF induced TNFR

signalling pathways are diverse. During TNFR-induced inflammation a number of signally

pathways are possible. Ligation of TNFR with TNF results in pathways that are non-

apoptotic and non-proliferation – these pathways include acidic and neutral

sphingomyelinase, activation of a 5-lipoxygengase and phospholipid A2 enzymes the

resultant production is of 5-HETE (5-hydroxyeicosatetraenoic acid) and the inflammatory

molecules, leukotrienes. [11] In the sphingomyelinase pathway the production of diacyl

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glycerol and the activation of the kinase C protein occur. [11] This is followed subsequently by

the activation of NF-κB. [11] The result of this is again the expression of proinflammatory

cytokines and chemokines, continuing the initiation of inflammation. [11] Involvement of TNF

is widespread throughout the inflammatory response hence its apparent value as a therapeutic

target in autoimmune diseases such as RA and CD.

As aforementioned the role of TNF in the initiation of inflammation is one of major

importance. As a result anti TNF treatments have been at the forefront of research for many

years and continue to remain there. It has been noted throughout this review that TNF is

secreted by many cells of the immune system specifically macrophage and T-cells and its

secretion is stimulated by interferons which again illustrates their synergistic relationship. It

has also been noted that the blockage of TNF from its receptors has shown significant

symptom improvement in patients.

Section Five

5.1 What is Rheumatoid Arthritis?Rheumatoid Arthritis (RA) is defined as a “systemic, chronic inflammatory, autoimmune disorder that results in joint destruction.” [13]

Rheumatoid Arthritis is associated with progressive disability and multiple systemic

complications.[13] RA can inflict moderate to severe discomfort in individuals affected and is

present in one percent of the population to date. [14] Due to the widespread nature of RA

research is carried out worldwide in a bid to find the block buster pharmaceutical drug to

completely combat RA without rendering the patient immunocompromised. In RA Joint

erosion is known to occur early resulting in bone erosion in about 40% of the patients during

the first year and 90% during the first two years. Thus, early and aggressive treatment is

favoured.[15] Figure three below shows the synovial joints of the body in all of which RA can

occur. [16] Figure four depicts a clear image of the cells involved in the mistaken destruction

of the synovial joint. [17]

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Figure three shows the synovial joints of the body in which rheumatoid arthritis can occur. (Image adapted from Simon 2013[16].) Figure four shows a normal health synovial joint left and a synovial joint of an individual

affected by RA right. (Image adapted from Tilleman 2008)[17]

Immune cells including osteoclasts, dendritic cells, T cells, B cells, mast cells and

macrophage can all be seen in action in figure four above. These cells carry out specific

immune functions secreting cytokines and enabling other immune cells this leads to the onset

and progression of RA. The molecular mechanisms of RA are complex and there are a vast

majority of mediators involved hence there is an extensive variety of possible future

therapeutics for the treatment of RA still yet to be explored.

5.2 Molecular Mechanisms of Rheumatoid Arthritis.As aforementioned in section 3.2 Macrophage play an extensive role in the onset of RA.

Macrophage secrete the cytokine TNFɑ, which is of major importance in the research of the

onset, progression and treatment of RA.[2]

In case in an individual unaffected by RA, the synovial membrane consists primarily of two

types of cells, macrophage like synoviocytes and fibroblast like synoviocytes.

Both of the aforementioned cells carry out functions similar to that of macrophage and

fibroblasts and also have additional functions. [18] Fibroblasts are mesenchymal cells that

constitute the primary resident cell type of connective tissues and secrete collagen.[18]

In cases in which the individual is affected by RA the level of macrophage like synoviocytes

increases beyond that of the fibroblast like synoviocytes.[2] This increase may be caused by

various factors including the increased influx of monocytes and reduced apoptosis and

increased proliferation of cells. The increase in the levels of macrophage results in the

secretion of a many of cytokines including GM-CSF, TNFɑ, IL-1, IL-6, IL-8 and chemokines

such as MIP-1 (macrophage inflammatory protein 1). [2] Monocyte concentration is also

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increased by the secretion of MCP-1 (monocyte chemoattractant protein 1) the presence of

this chemokine increases the level of monocytes which, in turn mature into macrophage cells

thus adding to the already heavily mounting immune response. [2] Figure five below shows the

molecular mechanisms involved in the mounting immune response which chronically attacks

the synovial joints in RA patients.

It has also been observed that osteoclasts and chondrocytes mediate bone erosion in RA, see

figure five. Chrondrocytes are, in unaffected individuals, a cell which mediates the balance of

components in the matrix of the adult articular cartilage which covers all long bone.[19]

Osteoclasts are large multinucleated cells which differentiated from macrophage. In normal

bone they have a regulatory role and are responsible for bone tissue absorption during growth

and healing however in individuals affected by RA they are the mediator for bone desorption.[2] For the formation of osteaoclasts to occur the presence of two factors is required; RANKL

(receptor activator of nuclear κB ligand) and M-CSF (macrophage colony stimulating factor). [2] TNFɑ plays primary roles in M-CSF production, increasing the levels of RANKL through

various aforementioned pathways leading to the activation of RANKL and promotes the

expression of proinflammatory cytokines such as IL-17, IL-6 and IL-1β [2]

The functional properties of TNFɑ continue into the function of macrophage cells induced to

produce ROS (reactive oxygen species) which, in turn increases inflammation in the joints

the result is further damaged to the cartilage and bone of the synovial joint. [2]

The mounting of the inflammatory response due to TNFɑ is in some sense limitless as it can

use aforementioned alternative pathways subsequently activating proinflammatory cytokines.

As the immune cells outlined above and illustrated in figure five carry out their individual

immune responses they attack the synovial joint causing painful inflammation, desorption

and destruction of bone, destruction of cartilage. The result of this heavily mounted immune

response is the severe disfigurement of joints.

Without suitable treatment RA can develop into a systemic disease affecting the entire body

an example of this is outlined by Levesque 2008 [20] whereby other systems in the body are

affected by the increased levels of cytokines, increased inflammation and angiogenesis. This

includes the onset of cardiovascular disease seen in 27% of patients over a seven year period

(1996-2006.)[20] Anaemia is also an abundant extra-articular manifestation of RA however

treatment with anti-TNF treatment is effective and has shown to significantly improve

anaemia as a manifestation of RA. [20]

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Figure five showing the mechanisms of cytokines and immune cells involved in RA (Pope 2002)[21]. MCP1, monocyte chemoattractant protein 1. TNF, tumor necrosis factor. IL-1, 6 and 8, interleukin 1, 6 and 8. GM-

CSF, granulocyte-macrophage colony stimulating factor. MMP, matrix metalloproteinases.

5.3 Therapeutics for Rheumatoid arthritis Throughout literature many signalling cytokines, pathways and immune cells have been

extensively studied and although this work is laudable and may be the future answer to

combating RA there appears to be a trend in literature in relation to a specific cytokine. The

involvement of this cytokine has been noted even in the earliest of publications, this cytokine

is TNFɑ. Many companies have exploited the use of anti-TNF treatments in an attempt to

find the right point of blockage to alleviate the symptoms of RA without rendering the patient

immunosuppressed. Immunosuppression in patients receiving anti-TNF treatment is common

and indeed of serious concern as a level of TNF is necessary to protect patients from infection

by eliciting an immune response against invading antibodies. As a result research is ongoing

into creating an anti-inflammatory therapeutic which will suppress unwanted inflammation

and help the immune cells to differentiate between a need for an immune response and a bias

stimulation involving TNF and other cytokines.

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As aforementioned anti-TNF treatment is used across the board for autoimmune diseases.

Interestingly there have been many overlaps in the usage of anti-TNF treatments in various

autoimmune diseases; an example of this is the use of Etanercept which trades in the

pharmaceutical industry by the name Enbrel®. Etanercept is used to treat both rheumatoid

arthritis and plaque psoriasis along with many other autoimmune disorders. [11] Etanercept is

an Ig (immunoglobulin) fusion protein which was FDA approved in 1998[11]. It is

administered for the treatment of rheumatoid arthritis once a week subcutaneously. Figure six

below shows the make up of etanercept.

Figure six showing the structures of the protein fusion therapeutic etanercept and the monoclonal antibody therapeutic Infliximab, both therapeutics are anti-TNF. Image adapted from Sedger and McDermott, 2014[11]. Abbreviations used: TNFR2, Tumor necrosis factor receptor two. LTɑ, lymphotoxin-ɑ. IgG1, immunoglobulin

G 1. Fv, Fragment variable.

As illustrated by figure six featured in the work of Sedger and McDermott, 2014, the make up

of etanercept can be seen. Etanercept consists of a human IgG1 Fc fragment and two TNF

two receptors. [22] Etanercept binds to both TNFɑ and TNFβ as TNFβ is considered to have a

similar pro-inflammatory effect to TNFɑ according to the work of Takeshita et al. 2015 [22]

However the role of TNFβ in relation to RA is yet to be defined. Interestingly there has been

cases stated in the work of Takeshita et al[22] whereby the pathogenesis of RA has been more

heavily dependant on TNFβ than TNFɑ however more research is required on this

observation. As aforementioned etanercept works by binding TNFɑ and TNFβ which, in

patients affected by RA will be in excess. By binding to both TNFɑ and TNFβ the

inflammatory response in the body is reduced thus reducing the chronic inflammatory

response in the synovial joints. As can be seen in figure six there exists two long extracellular

portions of TNFR2, these receptors when injected into the body as a component of etanercept

will bind to TNF present in the body. As a result TNF will not be present in the body in

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excess in order to initiate an inflammatory response in the synovial joint which is the basis of

RA.

Another anti-TNF therapeutic used to treat RA is Infliximab (IFX) or known to the

pharmaceutical industry as Remicade®. IFX is a monoclonal antibody which, in its function,

suppresses both TNFɑ and IL-6, which, as aforementioned is an interleukin with major

importance in the proinflammatory element of RA. IFX as outlined by Takeshita et al.

2015[22] binds only to TNFɑ thus rendering it at a disadvantage to fusion proteins like

etanercept. The structure of IFX is shown above in figure six adapted from Sedger and

McDermott 2014.[11]

As aforementioned, IFX is a monoclonal, neutralising antibody used to treat RA and other

autoimmune diseases. As a therapeutic, IFX was discovered and developed in the late 1990’s

and interestingly binds to both soluble (sTNF) and membrane (proTNF) TNF.[11] This binding

of TNF reduces the excess TNF free to bind to its two receptors TNFR1 and TNFR2 thus

reducing the level of unnecessary inflammatory initiation which causes joint and tissue

damage.[11] As outlined in the work of Sedger and Mcdermott, 2014 [11], IFX consists of

human constant regions and variable murine regions[11] these variable murine regions bind

excess TNF reducing the level of TNF binding to its receptors TNFR1 and TNFR2[22]. The

difference in etanercept and infliximab is discussed in work by Takeshita et al. 2015 [22] as

regards to the binding of TNF itself in case of monoclonal antibodies (IFX) or in the case of

fusion proteins, binding TNFɑ and TNFβ as a decoy receptor (etanercept).[22] IFX, similar to

etanercept can be used to treat rheumatoid arthritis and a number of other disorders such as

moderate to severe Crohn’s Disease, Plaque psoriasis and Ankylosing Spondylitis.

In a study carried out by Takeshita et al. 2015[22] two groups of patients were enrolled in a

controlled study to assess the difference in Etanercept and Infliximab as treatment for

rheumatoid arthritis. Sixty-eight patients in total were enrolled and all of the patients had

their blood serum sampled and stored at weeks 0, 22 and 54 of a 54 week study. [22] No

patients changed their treatment for the duration of the study.[22] Figure seven below shows

the decrease in disease activity during the progression of the treatment. Both groups were

administered with methotrexate for the duration of the study although the exact affect of

methotrexate in rheumatoid arthritis is still undefined methotrexate has been shown to be

potent in observational studies. The use of methotrexate in patients with early rheumatoid

arthritis illustrated short term clinical improvement in forty-eight to ninety percent of

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patients.[23] Etanercept appears to lower the disease activity when compared to Infliximab, by

observation of figure seven below it is creditable to say that in the patient base enrolled in the

study the most effective therapeutic for the alleviation of rheumatoid arthritis disease activity

is the ETN+MTX group (Etanercept and Methotrexate).[22]

Figure seven showing the chart containing the results for the disease activity recorded for the two groups of patients enrolled in the study. Image adapted from work by Takeshita et al.2015 [22]. Abbreviations used:

ETN+MTX, Etanercept and methotrexate. IFX+MTX, Infliximab and methotrexate. HDA, high disease activity. MDA, moderate disease activity. LDA, low disease activity.

Upon analysis of the cytokine level in the serum samples it was found that in both the

ETN+MTX group and the IFX+MTX group the level of IL-6, a cytokine heavily associated

with the promoting of the symptoms of rheumatoid arthritis was decreased in both groups

however it is observed in the study carried out by Takeshita et al. 2015[22] that the role of

methotrexate may lie in the decrease of the level of IL-6.[22] This observation is made on the

basis that in an additional group studied (not shown above in figure seven) the level of IL-6

did not decrease with treatment using Etanercept only.[22]

Unfortunately due to interferences caused by drug-cytokine complexes the study carried out

by Takeshita et al. 2015[22] did not include results on the TNFɑ levels in the patients at the

sample timepoints. It was observed however that the level of TNFβ increased in the serum

samples during the IFX+MTX treatment.[22] An interesting observation of this is in the

aforementioned case reported by Buch et al. 2004 [24] in which the pathogenesis of RA in a

patient was more dependent on TNFβ stimulation than that of TNFɑ. The study carried out by

Takeshita et al. 2015[22] poses many interesting observations on the combination of anti-TNF

17

therapeutics with additional treatments such as methotrexate in the search for an effective,

low impact source of relief of the symptoms and progression of rheumatoid arthritis. This

combination of treatments may be a focal point of research for emerging therapeutics in the

treatment of patients affected by rheumatoid arthritis however the observation itself remains

in its infancy as the patient base used in the study by Takeshita et al. 2015[22] was minute.

Section Six

6.1 What is Crohn’s Disease?Crohn’s Disease (CD) is a disease of unknown etiology, categorised as an autoimmune

disease CD can affect any area of the gastrointestinal tract which spans the area from the

mouth to the anus. [25]

CD falls under the category of inflammatory bowel diseases which is compromised of two

major types, CD and ulcerative colitis (UC). Whilst UC affects the mucosal surface of the

colon CD can affect any area along the gastrointestinal tract. [25] Crohn’s disease is shown to

affect men and women equally.[25] CD general strikes people in their early teens or after the

age of fifty. [25] Interestingly the incidence of CD is shown to be highest in Caucasians and

individuals of Jewish descent. [25] As aforementioned CD can affect any area along the

gastrointestinal tract however it primarily involves the ileocolic region of the intestines. [25]

Crohn’s Disease can be subdivided in three categories including inflammatory, obstructive

and fistulating, generally inflammatory and obstructive present together. [25] This occurs

because the inflammatory response causes a thickening of the intestinal mucosal wall thus

causing bowel obstructions. Similar to rheumatoid arthritis CD is categorised as a systemic

disease as it has be observed to manifest in extraintestinal areas. CD can manifest

extraintestinally causing erthema nodosum and peripheral arthritis. [25] Patients affected by

CD display various symptoms including diarrhoea, painful abdominal cramping and anorexia.[26]

6.2 Molecular mechanisms of Crohn’s DiseaseCrohn’s Disease is caused by damaged to the gastrointestinal tract as a result of chronic

inflammation much the same as RA is caused by chronic inflammation of the synovial joint.

Throughout literature a reoccurring trend is visible and that is of the involvement of cytokines

such as TNFɑ and IL-6 which can play the role of both an anti and a pro inflammatory

cytokines.[25]

18

Crohn’s disease falls under the category of inflammatory bowel diseases. The two main types

of inflammatory bowel diseases are Crohn’s disease and Ulcerative Colitis. Cytokines play a

major role in CD throughout the onset and continuation of inflammation and in the inducing

of other cytokines.[2] The differentiation of naïve T-cells into proinflammatory cytokine

producing TH1 and TH17 T-cells is an important contributing factor in the onset and

continuation in CD.[25]

In the immune response of an unaffected individual the T-cell population is controlled by a

process known as apoptosis. Unfortunately in patients affected by CD apoptosis is not carried

out efficiently and thus the resultant injury is the mucosal lesions as seen in CD.[25] Due to the

extension of the lifespan of the T-cells in those affected by CD the abnormal population of T

cells remains in the designated mucosal compartments, these T-cells which have had their

lifespan extended produce cytokines like those seen in RA including IL-6 and IL-17.[25] As

well as T-cell playing an extensive role in the onset of inflammation dendritic cells also play

a large role in the continuation of the inflammatory response. Dendritic cells do so by

secretion IL-12, an interleukin which induces T-cells to produce more IL-6 and IL-17 thus

aiding the continuation of the inflammatory response. Dendritic cells also carry out a role of

most interest; these cells secrete IL-12. [25] The IL-12 cytokine is the cytokine which aids the

differentiation of T-cells into TH1 cells. TH1 cells are of interest here as they are the primary

cells responsible for the secretion of TNFɑ and INFγ. [25] IL-12 is also referred to as a master

cytokine in driving the TH1 response. [25] IL-12 stimulates the differentiation of naïve T-cells

into TH1 cells which are responsible for the production of TNFɑ and IFNγ.[25]

By the reviewing of literature regarding IBDs it is plausible to say that as well as TNF, other

cytokines play an imperative role in the onset and continuation of the chronic inflammatory

characteristics of IBDs such as CD or UC. Cytokines such as IL-6 play a major role in the

prevention of apoptosis. As aforementioned the T-cells in an individual affected with CD do

not undergo appropriate apoptosis thus the extended life of the pro-inflammatory T-cells is

one of the causative elements of CD. As outlined by Neurath 2014[28] the role of IL-6 is seen

as one of the major elements in CD. Produced by the macrophage and dendritic cells present

in the gastrointestinal tract, IL-6 binds to its soluble IL-6 receptor forming the complex IL-6-

sIL-6R.[28] This complex will prevent the correct apoptosis of cells mainly mucosal T-cells

which will continue secreting pro-inflammatory cytokines, thus aiding the chronic

inflammation seen in conditions like CD.[28] Again, as seen as a common trend in literature

huge emphasis is placed on the presence of macrophage and dendritic cells as they are

19

classed as the main antigen presenting cells (APCs).[28] These APCs are found throughout the

mucosal inflamed tissue in patients affected with CD.[28] Figure eight below shows a

diagrammatic illustration of the cells involved in the chronic inflammatory bowel disease,

CD.

Figure eight showing the cells involved in the onset and continuation of inflammatory bowel diseases such as CD and UC. Note the reoccurring secretion of TNF by macrophage cells TH1 cells, TH2 cells and

differentiation of naïve T-cells into TH1 cells which will secrete more TNF. Abbreviations used; TH1-17, Helper T-cells 1-17. Treg, regulatory T-cell. IFNγ, Interferon gamma. IL-1-23, interleukin 1-23. TNF, Tumor

necrosis factor. [28]

As can be seen in figure eight above many cytokines and cells play a pro-inflammatory role

in inflammatory bowel diseases such as CD, The chronic nature of CD implies that the

aforementioned mechanisms of action are triggered by many external agents and as a result

the process of inflammation repeats itself due to this trigger. Throughout literature many

cytokines are discussed in detail as seen in the work of Bandzar et al. 2013 [25] and Singh et al.

2014[2] many cells and cytokines in literature are discussed in literature but their direct

linkage is still somewhat unexposed. In the therapeutic nature of CD many cytokine

blockades have been tested and display disappointing results however the use of anti-TNF

treatment produces exceptional results.[28] Evidence of this in literature will be discussed

further in section 6.3 – Therapeutics for Crohn’s Disease.

6.3 Therapeutics for Crohn’s DiseaseCrohn’s disease and rheumatoid arthritis show a common overlap in the usage of anti-TNF

therapeutics in their treatment. It is a given circumstance that not 100% of patients will

20

respond advantageously to the therapeutics used on them failure of a patient in response to

therapy is known as primary and secondary response failure. Primary response failure is

defined as an initial lack of improvement of the clinical signs during the first phase of

treatment. [29] Whereas secondary response failure is defined as the event in which the patient

meets the initial criteria showing a clinical sign improvement however they begin to lose

response to anti-TNF treatment. [29]

In most cases, CD in patients is treated with an anti-TNF treatment. Unfortunately for some

patients there is a 25% failure rate in the treatment of CD with anti-TNF therapeutics [27] This

failure rate is not extensively proven to be attached to any additional factors i.e genetics,

biochemical or virologic.[27] Higher response failures are seen however in patients with CD

who smoke. Furthermore a secondary response failure is also seen in up to 50% of CD

patients. [29] Generally if the initial anti-TNF treatment is unsuccessful a further anti-TNF is

tested, additionally the mode of administration is also changed in order to evoke a successful

anti-TNF result. [25] The aforementioned change in mode of administration may simply be a

switched from intravenous to subcutaneous drug delivery. [27]

In the treatment of CD anti-TNF therapeutics are mainly used. The therapeutics which will be

discussed in relation to CD are HUMIRA® (Adalimumab) and Cimzia® (Certolizumab

Pegol). Figure nine below shows the structure of both Certolizumab Pegol and Adalimumab.[11]

Figure nine showing the structure of the fully humanised therapeutic Adalimumab(HUMIRA®) and the TNF monoclonal antibody Certolizumab Pegol (Cimzia®) Abbeviations used: Fv, fragment variable. IgG1,

immunoglobulin G1. PEG, polyethylene glycol. [11]

As shown above in figure nine Certolizumab Pegol is a monoclonal antibody. Cimzia was

first approved for use in CD in 2008.[30] Cimzia was developed by Union Chimique Belge

21

(UCB) initially approve for use in CD in Switzerland in 2007 and then for use in rheumatoid

arthritis in 2009.[30] The structure of Cimzia consists of a Fab region which is a humanised

antigen-binding fragment attached to a polyethylene glycol. The difference between Cimzia

and the other TNFα-inhibitors is the lack of an Fc region, which is advantageous to the

patient as the absence of an Fc region results in the minimising of potential Fc-mediated

effects.[30] An example of the potential Fc-mediated effects includes complement-dependent

cytotoxicity (CDC) and additionally antibody dependent cell-mediated cytotoxicity

(ADCC). [30] The method in which Cimzia acts is in its binding to both transmembrane and

soluble TNFɑ. The binding of the TNFɑ results in the inhibition of its binding to its receptors

TNFR1 and TNFR2.[30] In a study carried out by Goel et al. 2010[30] the efficacy and induction

of a clinic response of patients with CD was evaluated. This was carried out in two separate

studied referred to as PRECiSE 1 and PRECiSE 2.[30]

In the case of PRECiSE 1, a phase three trial of the use of Ccertolizumab pegol in patients

with moderate to severe active CD a twenty six week clinical trial was carried out involving

662 patients.[30] For the duration of this trial the patients received 400mg of certolizumab

pegol or a placebo at weeks 0, 2 and 4 and every 4 weeks thereafter.[30] The use of

immunosuppressants and low dosage oral glucocorticoids was also permitted as a means of

background therapy.[30] In this study outlined by Goel et al. 2010[30] a defined response was a

decrease in the CD activity index score of at least 100 points and as this study was

completely randomised to drug or placebo from the baseline it therefore cannot be compared

to any other study regarding certolizumab pegol.[30]

The resultant observations were that 37% of treated patients had a response at week six and

were then enrolled in the study PRECiSE 2 whereas 26% of placebo patients showed a

response by week six.[30] In a comparison of patient response at weeks 6 and 26, 22% of

treated patients had a response compared to 12% of placebo patients, however the rates of

remission at weeks 6 and 26 did not differ significantly. [30] The conclusion of the study

PRECiSE 1 was that the induction and maintainence therapy in affected patients evoked a

modest improvement when compared to that of the placebo group.[30]

The individuals involved in PRECiSE 2 where the individuals which showed a clinical

response at weeks six.[30] During an open label treatment the efficacy of the ability of

cetolizumab pegol to maintain a response in affected individuals was evaluated.[30] In the

works detailed by Geol et al. 2010[30] it was observed that 64.7% of patients in PRECiSE 1

22

responded to treatment in week six. These individuals were then treated every four weeks

with 400mg of certolizumab pegol or a placebo.[30] The conclusion of PRECiSE 2 was as

follows; In those patients whom continued with treatment of certolizumab pegol where more

likely to maintain this response throughout treatment or to achieved remission when

compared to those patients switched to a placebo treatment.[30] Thus the efficacy of

certolizumab pegol as a therapeutic for treating moderate to severe Crohn’s disease is

observed in the study detailed by Geol et al. 2010[30].

Another well established therapeutic used in the treatment of CD is adalimumab

(HUMIRA®). As seen by the structure of adalimumab, shown above in figure nine,

adalimumab is a fully human monoclonal antibody. Adalimumab works by binding soluble

and membrane TNFɑ, thus rendering it unable to bind to its receptors P55 (TNFR1) and P75

(TNFR2) respectively.[11] In a study detailed by Cordero-Ruiz et al. 2011[31] twenty-five

patients with moderate to severe CD who had previously been treated with Infliximab

(Remicade®) with response failure were then treated with adalimumab.[31] Twenty-four of the

twenty-five patients were treated with a 160/80/40mg dosage whereas one patient was treated

with a lower dosage due to additional factors.[31] The treatment using adalimumab also

included a 40mg every two weeks to sustain the response.[31] Prior to the treatment with

adalimumab five of the patients displayed normal CD activity index however of the

aforementioned five patients, four of them presented perianal and enterocutaneous fistulae.[31]

The results of the study outlined by Cordero-Ruiz et al. 2011[31] were as follows, by week

twenty-four 72% of patients had shown clinical remission and by week forty-eight 60% of

patients had shown clinical remission.[31] It was observed however that in 72% of these

patients the intervals between treatments had to be shortened in order to maintain desired

response.[31] The study detailed by Cordero-Ruiz et al. 2011[31] displayed the efficacy of

adalimumab to maintain a response in patients with CD initially treated with infliximab with

a failed response.

Section Seven

7.1 Overlap of Crohn’s Disease and Rheumatoid arthritis: CytokinesIt is in this section that the overlap of the cytokines involved in CD and RA will be discussed.

It has been observed throughout literature that often arthritis is seen as an extraintestinal

manifestation of RA. Furthermore it has been noted that although CD and RA present

23

extensively different symptoms in the patient they overlap in the same hyper activity of the

inflammatory response which leads to destruction of the body tissues.

In an article detailed by Reimold 2005[32] it is stated that “up to 40% of IBD patients present

extraintestinal manifestations” including arthritis. Arthritis associated with CD presents in ~

26% of CD patients.[32] This is followed by the statement that the connection between RA and

CD is often noticed by the presentation of joint degeneration symptoms at or after the initial

onset of bowel symptoms.[32] Although this paper is quite dated it is interesting to observe the

research and consequent documentation of arthritis associated with CD. It is also interesting

that Reimold 2005[32] detailed that when CD associated arthritis does occur it generally occurs

in the first year of CD and this is seen especially in juvenile CD. The author also continues to

outline that in almost 70% of CD associated arthritis joint flares coincide with a worsening of

CD. Interestingly it is also stated in the study by Reimold 2005[32] that CD may actually lie

undetected for an extended period of time even after the onset of arthritis especially if

patients do not display the hallmark signs of CD outlined in section 6.1 “What is Crohn’s

Disease?”

As outlined above in observations made by Reimold 2005[32] arthritis is associated with CD as

one of its many possible extraintestinal manifestations. Furthermore it is also the cytokines

and cells involved in both RA and CD that bring their association that bit closer.

In particular the pro inflammatory cytokine IL-17 has been observed by Pöllinger 2011 [33] as

a major cytokine in both RA and CD. IL-17 was cloned by a “subtractive hybridization

screen” in the T-cell library of a rodent in the year 1993 and has since been referred to a

major proinflammatory cytokine produced by the differentiated TH17 cells which descended

from the T-cell lineage.[33] IL-17 has been observed to have been up-regulated in the affected

areas in inflammation moreover IL-17 works to amplify inflammation in the affected area

through its synergistic relationship with other proinflammatory cytokines such as TNF and

IL-6.[33]

Pöllinger 2011[33] goes on to describe the promotion of IL-17 by IL-23, another

proinflammatory cytokine involved in the inflammatory response. Elevated levels of both IL-

23 and IL-17 can be seen in patients with active CD at protein and mRNA levels. [33] As is

outlined by Pöllinger 2011[33] the role of IL-17 is a shared trend among the autoimmune

disease category. The synergistic relationships that exist between the proinflammatory

cytokines are documented continuously to be the driving force in what can be an extremely

24

aggressive response from the immune system moreover the involvement of each cytokine is

researched and a common cytokine is seen in every autoimmune disorder and that cytokine is

TNF. Anti-TNF therapeutics are heavily used in the treatment of autoimmune diseases. The

pharmaceutical markets sees drugs such as HUMIRA®, Enbrel®, Remicade® and Cimzia®

produce billion dollar turnovers for their mother companies.

7.2 Overlap between the therapeutics used for Crohn’s Disease and Rheumatoid Arthritis

- Case study: Adalimumab.

As aforementioned anti-TNF therapeutics such as Enbrel®, Remicade®, HUMIRA® and

Cimzia® are blockbuster pharmaceutical drugs, producing a large segment of the profitable

turnover for their mother companies. The aforementioned drugs are all used to treat both

rheumatoid arthritis and crohn’s disease and as mentioned above the incidence of arthritis in

patients with CD is estimated to be up to 26%.[32] Hence the overlap in treatment used for

both RA and CD is beneficial to those patients who suffer from both autoimmune conditions.

The therapeutic to be discussed in detail in relation to both CD and RA is Adalimumab

(marketed as HUMIRA®).

As detailed above in section 6.3- Therapeutics for Crohn’s Disease adalimumab is a fully

human monoclonal antibody. Adalimumab works by binding soluble and membrane TNFɑ,

thus rendering it unable to bind to its receptors P55 (TNFR1) and P75 (TNFR2) respectively.[11] Unfortunately although an extensive search was carried out it has proven quite difficult to

retrieve a scientific paper in which both CD and RA treatment with adalimumab have been

detailed, henceforth two separate papers detailing treatment with adalimumab will be

discussed in order to show the clinical results and long term efficacy of adalimumab as a

treatment for autoimmune diseases such as RA and CD.

In a study detailed by Peters et al. 2014[34] 438 patients, all of which were affected by

moderate to severe crohn’s disease were treated with adalimumab. This study however was

carried out in contrast to a generally clinical study. This study was carried out to mimic real-

life long term clinical response and assess the effectiveness of adalimumab as a long term

treatment for CD.[34] As a result the trial spanned over an eight year period from 2003 to

2011, involving patients from northern Holland affected by CD.[34] The study was assessed

after the induction phase which was defined as a timepoint three months after the first

treatment was administered.[34] Following the induction phase adalimumab was documented

25

to have produced a 92.5% response rate by Peters et al. 2014[34]. Initially this appeared to be

an extremely efficient and highly effective treatment for patients with CD however the

response rate was reported by Peters et al. 2014[34] to have decreased at an average of 10%

each year following. One year after the initial treatment 83.3% of patients remained

responsive showing sustained benefit of maintenance treatments, two years after the initial

treatment the number of responsive patients had again dropped to 74.0%. [34] The percentage

of patients still responding following two years of treatment shows significant efficacy as a

therapeutic in the treatment of CD however it also illustrates the need for a long-term

treatment with a lesser annual decrease in response.

This conclusion provided by Peters et al. 2014[34] was support by a conclusion detailed by

Chaparro et al. 2012[35] in which it was stated “Adalimumab is an effective treatment for

Crohn's disease (CD), but may also be associated with loss of response. Few reports provide

insight into the durability of treatment of CD with adalimumab for periods longer than 12

months in clinical practice.” In the study outlined by Peters et al. 2014 [34] in which only one

third of the patients treated over the two year period were in steroid free remission by their

last follow up illustrated that the long term use of adalimumab, assessed over a two year

period, that adalimumab does not show significant efficacy as a life-long treatment for CD.

In the study documented by Chaparro et al. 2012[35] 380 patients which moderate to severe

CD were enrolled in a study to assess the clinical use and loss of response of adalimumab.

The conclusion of the work by Chaparro et al. 2012[35] was that within the 380 patients, 28%

of which were completely anti-TNF treatment naïve and 72% of which were anti-TNF

treatment experienced the higher loss of response was evaluated to have come from the anti-

TNF experienced patients. It was reported by Chaparro et al. 2012 [35] that in the anti-TNF

naive patients 8% of patients showed a loss of response following the induction phase

whereas in the anti-TNF experienced patients 22% of patients showed a loss of response

following the induction phase.[35] This shows a significant indication that long term exposure

to anti-TNF therapeutics may have an effect on the patients sensitivity to anti-TNF treatment.

The two aforementioned studies also highlight the significant unsuitability of adalimumab for

life-long treatment of CD in patients.

As a method of comparison, various papers detailing trials in which adalimumab was used to

treat patients with active RA were studied. In a report detailed by Frazier-Mironer et al.

2014[36] a study was held for four years, from 2005 to 2009 in an effort to compare the use of

26

various TNFi (Tumour necrosis factor inhibitors) in RA. The study involved 706 patients 203

of which received adalimumab as a first line of biotherapy for active RA. [36] Two objectives

were given for the purpose of this study, those objectives were to assess and compare the

retention rates of adalimumab, etanercept and infliximab which were administered as a first

line of biotherapy to those enrolled in the study. Secondly the retention rates of a patient

receiving a second treatment with a TNFi were assessed and compared.[36] No patient enrolled

had had any pervious treatment with biotherapies.[36]

The data for the paper detailed by Frazier-Mironer et al. 2014[36], was collected at a 3, 6 and

12 months period and every 12 months thereafter until 2009. As outlined above in the usage

of adalimumab for the treatment of CD, a loss of response is associated with adalimumab.

This is seen again in the utilisation of adalimumab for the treatment of active RA in the paper

detailed by Frazier-Mironer et al. 2014.[36] In those 203 patients whom received adalimumab

treatment as a first line of biotherapy the average retention rate was 31 months, interestingly

the retention rate for etanercept and infliximab was 45 and 23 months respectively.[36]

Moreover, when administered with adalimumab as a second line of biotherapy the average

retention rate was 11 months for adalimumab, 43 months for etanercept and 19 months for

Infliximab.[36] Thus illustrating adalimumab’s associated loss of response detailed above in

relation to CD. The study documented by Frazier-Mironer et al. 2014[36] concluded that

etanercept has a better retention rate than adalimumab and interestingly also states that in

both studies carried out by Tubach et al. 2009[37] and Rose et al. 2012[38] the utilisation of

monoclonal antibodies such as adalimumab and infliximab show a higher risk of tuberculosis

than the usage of soluble receptors such as etanercept.

Furthermore the paper by Frazier-Mironer et al. 2014[36] a trial is detailed which was carried

out in May 2004 by Keystone et al.[39] In the work of Keystone et al. 2004[39] a 52 week,

double blinded, placebo controlled study was carried out with spanned 89 sites in both the US

and Canada. Of the 619 patients who were enrolled in this study, 207 patients received a

40mg dosage of adalimumab every second week, 212 patients received a 20mg dosage every

week and 200 patients received a placebo each week. [39] All patients were consistently

receiving background treatment with methotrexate in addition to adalimumab or placebo.[39]

For this study detailed by Keystone et al. 2004[39] the definition of a clinical response was a

given definition by the American college of Rheumatology core criteria (ACRCC) as a

minimum 20% improvement in RA in patients. By week twenty-four 63% of the 40mg

dosage group had achieved a 20% improvement, 61% of the 20mg weekly group achieved a

27

20% improvement and 30% of the weekly placebo group met the criteria of the ACRCC. [39]

Furthermore by the end of the trial, week 52, the response had decreased to 59%, 55% and

24% in the 40mg, 20mg and placebo groups respectively.[39] This again show the associated

loss of response of adalimumab which was stated in an earlier discussed paper by Chaparro et

al. 2012.[35]

The various studies detailed above all present a common trend: the utilisation of adalimumab

in autoimmune diseases is effective as an immediate method to suppress a hyperactive

immune response. Unfortunately long term the usage of adalimumab shows a significant

decrease in efficacy over a period of time as presented in the work of Peters et al. 2014 [34] in

which patients enrolled in the study showed a average 10% decrease in response each year

following the induction phase. Hence the trend between the usage of adalimumab and lose of

response in both RA and CD stands true to both disorders. This associated loss of response

from the usage of adalimumab and indeed other monoclonal antibodies illustrates the need

for a long-term treatment for autoimmune disorders such as RA and CD which are often

chronic and thus life-altering when a relapse occurs.

7.3 Emerging future therapeutics.As shown above in the usage of adalimumab in autoimmune disorders, most TNFi responses

are short lived and although this may offer rapid alleviation from symptoms the level of

response soon decreases leaving the patient exposed and vulnerable to potential relapses. This

is a trend seen throughout the usage of anti-TNF treatment in patients suffering from auto

immune disorders. As a result there are many alternative therapeutics now in preclinical and

clinical trials. [40]

One of the aforementioned emerging therapeutics being used in patients with moderate to

severe RA since July 31st 2011 is abatacept, which is marketed as Orencia®[41]. Abatacept

was approved for use by the FDA in patients who were defined as non-responsive to anti-

TNF treatment[41]. The supplied dosage for patients with RA is based on body weight,

generally in the recommended range of 10mg/kg[41]. Induction phase consists of drug

administration though IV and additional doses can be administered 2 and 4 weeks following

original administration.[41] Abatacept is a recombinant protein fusion made up of the Fc

region of human IgG1 which is fused to the extracellular domain of cytotoxic T-lymphocyte

antigen-4.[42] The mechanism on which abatacept is based is that the role of T cells in the

onset and continuation of RA is of major importance. In order for a T-cell to be activated and

28

thus initiate an immune response two signals must first reach the cell from an APC [41]. These

signals are the MHC (major histocompatibility complex) and the antigen and the other is the

co-activation of CD28 with the antigen-presenting cell (APC) protein CD80/86 which in turn,

if not inhibited will result in release of pro-inflammatory cytokines.[41] Abatacept works by

binding to CD80/86 on antigen presenting cells thus inhibiting the CD28 co-stimulation on T-

cells preventing the release of pro-inflammatory cytokines. [41]

In a study detailed by Rosman et al. 2013[41], improvement was shown in patients with RA

enrolled in a meta-analysis study comparing phase II and phase III trials. All improvement

was defined by the ACRCC (American college of rheumatology core criteria), this

improvement was defined under ACR20, ACR50 and ACR70.[41] An average of 50% of the

patients treated with abatacept were seen in ACR20 and interestingly 30% of placebo patients

showed improvement by the ACR20 definition.[41] A similar result was presented within the

abatacept and placebo groups for ACR50 and ACR70[41] In an additional long term study

which spanned over 3 years both groups had been being treated with background therapy of

methotrextrate, the group receiving abatacept showed a far more superior improvement than

the placebo group.[41]

The efficacy of abatacept is shown in the study detailed by Rosman et al. 2013 [41] and in

numerous other studies. The utilisation of abatacept as a new biotherapy for CD is

disappointing however, as the study detailed by Sandborn et al. 2012[43] shows. In the study

by Sandborn et al. 2012[43] 4 placebo controlled trials were set up to evaluate the efficacy of

abatacept as both an induction and maintenance therapy for patients with moderate to severe

CD. For these trials 451 patients with CD were enrolled and received either 30, 10 or 3mg/kg

(of body weight) of abatacept.[43] The level of dosage given to patients was randomized,

patients were given dosages at weeks 0, 2, 4 and 8.[43] Following the induction phase 90

patients who had been defined as “responsive” to abatacept by week 12 of the induction

phase where again given abatacept in order to evaluate the efficacy of abatacept as a

maintenance therapeutic.[43] This time at a 10mg/kg dosage every month until the end of the

trial at week 52.[43] The results concluded that 17.2%, 10.2% and 15.5% of patients receiving

30, 10 and 3mg/kg of abatacept respectively achieved a defined clinical response at weeks

eight and twelve. In addition to this the study detailed by Rosman et al. 2012[43] also

presented the result that 23.8% of the patients enrolled in the maintenance trial showed

remission at week 52 compared to 11.1% of the placebo group which were supplied with

background therapy only. The study then concluded that abatacept is not a significantly

29

effective therapeutic for moderate to severe CD in patients due to its low scoring as an

induction and maintenance therapeutic.[43]

Additional new therapies are also now being used in the treatment of RA and CD. The

utilisation of monoclonal antibodies in a bid to bind excess TNF has now moved to the

binding of other proinflammatory cytokines such as IL-6. As detailed anti-TNF therapies

were often and remain to be the main treatment for auto immune conditions such as RA and

CD. Monoclonal antibodies such as infliximab and adalimumab are used to bind excess

TNFɑ or its receptors TNFR1 and TNFR2.

A common trend seen in emerging therapeutics discussed in literature is the use of

monoclonal antibodies to target associated interleukins or proteins presented on cells

associated with the inflammatory response. The study detailed by Rosman et al. 2013 [41]

discusses the use of monoclonal antibodies in the binding of cytokines and proteins involved

in additional pathways of inflammation initiation. Monoclonal antibodies such as tocilizumab

and rituximab are discussed in the study by Rosman et al. 2013.[41] Tocilizumab is defined as

a “recombinant monoclonal IgG1 anti-human IL-6 receptor (IL-6R) antibody.”[41] During the

initiation of inflammation IL-6 may bind to either soluble IL-6 receptor or membrane bound

IL-6 receptor. As a result of binding, the bound complex then binds to the 130 gp signal

transducer thus promoting the inflammation related cascades, enhancing adhension molecules

and additionally activation Osteoclasts.[41]

Il-6 plays many important roles in inducing the activation of T and B cells, both B and T cells

are involved in immune responses particularly the inflammatory response thus inhibition of

IL-6 binding has been an interesting target for therapeutics to treat auto immune disorders.[41]

Tocilizumab was approved by the FDA in January 2010 for use in patients with RA in the

USA. [41] In the study by Rosman et al. 2013[41] additional studies were discussed in one of

which tocilizumab was involved in the meta-analysis of a placebo controlled trial. In this trial

tocilizumab was compared to other biotherapies such as abatacept, rituximab and TNFis

(adalimumab, Infliximab and etanercept).[41]

Tocilizumab proven to be interestingly competitive in the trials involving comparison with

other biotherapies, especially in that of ACR70 where tocilizumab showed its superiority.[41]

Furthermore tocilizumab also showed a faster response rate, occurring in some cases

following the initial infusion.[41] This was further discussed in a 24 week study featured in the

work by Rosman et al. 2013[41] in which a group of RA patient were treated with tocilizumab

30

along with methotrextrate and the control group received methotrextrate only, in this case

tocilizumab proved superior by the 24th week of the study.

Additionally another therapeutic discussed in the study by Rosman et al. 2013 [41] was

rituximab. Rituximab is define by Rosman et al. 2013[41] as “a chimeric human monoclonal

antibody against the CD20 protein found on naive, mature, and memory B cells.” The

mechanism by which rituximab operates is the B-cell population decrease, cytotoxicity of

cells and activation of the complement system that it initiates upon administration to the RA

patient.[41] In the study by Rosman et al. 2013[41] two separate trials of rituximab are

discussed. The first being the trial in which 646 patients with active RA whom had previously

displayed a failure of response to anti-TNF treatment were treated for six months with

rituximab, following this a good clinical response was reported and in some cases even

remission was achieved in six months.[41]

The second trial discussed in the study by Rosman et al. 2013 [41] was the SUNRISE trial

(study of retreatment with rituximab in patients with RA receiving background

methotrextrate.) In this study 559 patients with active RA received two cycles of rituximab

following pervious response failure for anti-TNF treatment.[41] Interestingly 475 of the RA

active patients showed improvement based on ACR20.[41]

As an emerging therapeutic for CD the use of tocilizumab and rituximab are limited and

literature is scarce. In a study by Dwivedi et al. 2014[43] a phase I pilot clinical trial of the

utilisation of anti-IL-6R antibody which is the mechanism by this tocilizumab operates

presented positive clinical results in the treatment of active CD. Furthermore in a study

carried out by Ito et al. 2004[44] which features in the study by Dwivedi et al. 2014[43] 36

patients with active CD, which is defined in the study as a CDAI (crohn’s disease activity

index) greater than or equal to 150 were administered biweekly intravenous infusions of

either placebo, human anti-IL-6 monoclonal antibody or a alternate of anti-IL-6

antibody/placebo.[44] In the ten patients who received anti-IL-6 monoclonal antibody

treatment 80% of the patient group achieved the defined clinical response by week 12

compared to 31% of the placebo group. Furthermore 20% of the patient group achieved

remission following treatment with anti-IL-6 monoclonal antibody treatment.

Both aforementioned studies present the inhibition of IL-6 signalling as a promising

therapeutic target. It is stated in the work by Dwivedi et al. 2014 [43] that many other plans

which involve IL-6 signalling have been presented and proposed including the inhibition of

31

IL-6 or Il-6R. Additionally the utilisation of soluble, transmambrane glycoprotein 130 as an

antagonist of the signalling of IL-6 or inhibiting the classical IL-6 and trans IL-6 signalling

has also been proposed.[43] Unfortunately however therapeutic antibodies including these

targets are still in their infancy in development for use in CD.[43]

Section Eight

8.1 Concluding RemarksIn conclusion, the objectives of the literature survey were carried out. Studies, new and old

were researched. The mechanisms by which RA and CD operate were studied and the

therapeutic effects on these mechanisms were explored. The incidence arthritis presenting in

patients with CD was also explored as an additional region of overlap between RA and CD.

The numerous regions of overlap between RA and CD were presented clearly throughout this

text, however it did pose a certain level of difficulty to find scientifically accredited papers in

which both RA and CD and their therapeutic options were discussed. As a result separate

papers were used for the discussion of their overlapping therapeutic options. The usage of

Adalimumab as a case study was assessed in both CD and RA and the resulting conclusion

was that in both RA and CD a loss of response was observed over time. Upon further

exploration into emerging therapeutics, treatments such as tocilizumab and rituximab were

analysed and have shown a significant level of efficacy in RA unfortunately however their

usage in CD has proven insignificant. At present there have been proposals to exploit the IL-

6R and IL-6 in an attempt to combat these autoimmune diseases however there level of

literature available is scare as this category of therapeutics are still in their infancy.

The purpose of this study was to construct a paper on a given topic over a given time period

and by doing so a higher level of researching skills would be achieved. It was noted however

that before the preparation of this paper the level of researching skills was limited and upon

completion it has been observed that significant improvement has been achieved,

identification of accredited papers, usage of relevant information and an in-depth

understanding of current trends in CD and RA was established. Over the last eight weeks it

has been found that the level of researching skills has been immensely strengthened and will

prove to be an extensively useful tool for future researching in the scientific and wider fields.

32

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