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In The Name Of God
The Most Compassionate
The Most Merciful
Cytokines and Growth Factors
By
Ali Jahanian Najafabadi
Email: [email protected]
Introduction
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•
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A diverse group of regulatory proteins or glycoproteins
Produced in minute quantities by the body
Act as chemical communicators
Intracellular signal transduction
The major proteins/protein families that constitute the cytokine group of regulatory molecules
Introduction
• Mostly produced by, or act upon leukocytes
IgG FcR
Opsonized antigen
IgER
Hyper sensitivity
IgER
Parasitic worms
Kupffer cells, microglia, and alveolar macrophages
Introduction
• Mostly produced by leukocytes
 Play a central role in:
 Regulating both immune and inflammatory function
 Processes such as haematopoiesis (the production of blood cells from
haematopoietic stem cells in the adult bone marrow)
 Wound healing
Introduction
• Nomenclature:
 Cytokines: Firstly introduced in the mid 1970s for the
polypeptide growth factors controlling the differentiation and
regulation of cells of the immune system
 Additional classification:
 Lymphokines (cytokines such as IL-2 and IFN-γ, produced by lymphocytes)
 Monokines (cytokines such as TNF-α, produced by monocytes)
 On the basis of the specific biological activity by which the
cytokine was first discovered
 TNF exhibited cytotoxic effects on some cancer cell lines
 CSFs promoted the growth in vitro of various leukocytes in clumps or
colonies).
Introduction
• Nomenclature:
 Primary sequence analysis of cytokines coupled to determination of
secondary and tertiary structure  one of six families:
Many are known by more than one name:
IL-1: lymphocyte activating factor (LAF), endogenous pyrogen, catabolin and
mononuclear cell factor
Introduction
• The effects of recombinant DNA technology and monoclonal
antibody technology on the understanding of cytokine biology:
 Genetic engineering allowed production of large quantities of most cytokines.
These could be used for structural and functional studies of the cytokine itself, and its
receptor
 Analysis of cytokine genes established the exact evolutionary relationships between
these molecules
 Detection of cytokine mRNA and cytokine receptor mRNA allowed identification of the
full range of sources and target cells of individual cytokines
 Hybridoma technology facilitated development of immunoassays capable of
detecting and quantifying cytokines
 Inhibition of cytokine activity in vivo by administration of monoclonal antibodies (and,
more recently, by gene knockout studies) continues to elucidate the physiological and
pathophysiological effect of various cytokines.
Introduction
• The following generalizations may be made with regard to most
cytokines:
 Very potent regulatory molecules, nanomolar to picomolar concentrations
 Produced by a variety of cell types, which may be leukocytes or nonleukocytes: e.g. IL-1: leukocytes, non-leukocytes (such as smooth muscle cells,
vascular endothelial cells, fibroblasts cells and collagen fibre, astrocytes and
chondrocytes
 Many cell types can produce more than one cytokine:
 Lymphocytes: wide range of interleukins, CSFs, TNF, IFN-αs and IFN-γ
 Fibroblasts can produce IL-1, -6, -8, and -11, CSFs, IFN-β and TNF
Introduction
• The following generalizations may be made with regard to most
cytokines:
 Most cytokines are pleiotropic, i.e. can affect a variety of cell types:
 IFN-γ stimulates activation and growth of

T- and B lymphocytes,

macrophages,

NK cells,

fibroblasts

endothelial cells

It also displays weak anti-proliferative activity
with some cell types.
Introduction
• The following generalizations may be made with regard to most
cytokines:
 Many cytokines play a regulatory role in processes other than immunity and
inflammation: EPO
 Most cytokines are inducible, and are secreted by their producer cell:
IL1IL2
 potent cytokine inducers : infectious agents, tissue injury and toxic stimuli
 some cytokines appear to be expressed constitutively: EPO, CSFs
 Many cytokines exhibit redundancy: i.e. two or more cytokines can induce a
similar biological effect: TNF-α and -β
Introduction
• The following generalizations may be made with regard to most
cytokines:
 Cascade induction
Introduction
• Not all polypeptide regulatory factors are classified as cytokines:
– Classical polypeptide hormones are not considered members of the cytokine
family: Insulin, FSH and GH
•
•
•
•
•
Hormones
Secreted by one types of
specialized cells
Unique action
Restricted target cells and a
limited spectrum of actions
(excp insulin)
Act at a distant site
•
•
•
•
Cytokines
Made by more than one type of
cells
Overlap actions (redundancy)
Multiple target cells, multiple
actions
Short action radius
EPO is produced in the kidney and liver and acts in an endocrine manner, promoting production of red
blood cells in the bone marrow. EPO could thus also be considered to be a true hormone.
Introduction
• Autocrine,Paracrine and Endocrine
Introduction
• Cytokine receptors
Introduction
• Cytokine receptors
 Based upon amino acid sequence homology, receptors are usually classified as
belonging to one of 5 known superfamilies:
 Immunoglobulin superfamily receptors
 Class I cytokine receptor family (also known as the hematopoietin receptor family)
 Class II cytokine receptor family (also known as the interferon receptor family)
 TNF receptor family
 Chemokine receptor family
Introduction
• Cytokine receptors
 Based upon amino acid sequence homology, receptors are usually classified as
belonging to one of 5 known superfamilies:
 Immunoglobulin superfamily receptors
Introduction
• Cytokine receptors
 Based upon amino acid sequence homology, receptors are usually classified as
belonging to one of 5 known superfamilies:
 Class I cytokine receptor family (also known as the hematopoietin receptor family)
Introduction
• Cytokine receptors
 Based upon amino acid sequence homology, receptors are usually classified as
belonging to one of 5 known superfamilies:
 Class II cytokine receptor family (also known as the interferon receptor family)
Introduction
• Cytokine receptors
 Based upon amino acid sequence homology, receptors are usually classified as
belonging to one of 5 known superfamilies:
 TNF receptor family
Introduction
• Cytokine receptors
 Based upon amino acid sequence homology, receptors are usually classified as
belonging to one of 5 known superfamilies:
 Chemokine receptor family
Introduction
• Cytokine receptors
Introduction
• Cytokine receptors
 Individual members of any one superfamily characteristically display 20–50 per cent
homology
 In some cases a single receptor may contain domains characteristic of two or
more superfamilies. e.g. IL-6: both the haematopoietic and immunoglobulin
superfamilies
 Some cytokine receptors are composed of a single transmembrane polypeptide
(e.g. receptors for IL-8, -9 and -10). Many contain two polypeptide components
(including the IL-3, -4, and -5 receptors), and a few contain three or more polypeptide
components (e.g. the IL-2 receptor contains three polypeptide chains)
 In some instances a single cytokine may be capable of initiating signal transduction by
binding two or more distinct receptors (e.g. IL-1 has two distinct receptors (types
I and II), both of which are transmembrane glycoproteins).
Introduction
• Cytokine receptors
– In many cases where a receptor consists of multiple polypeptides, one of those polypeptides (which
will be unique to that receptor) will interact directly with the ligand. The additional polypeptide(s),
responsible for initiation of signal transduction, may be shared by a number of receptors.
Leukaemia inhibitory factor (LIF)
Introduction
• Cytokines as biopharmaceuticals
As coordinators of the immune and inflammatory response
EPO in anemic person
Growth factors and wound healing
Neurotrophic factors in the abatement of certain neurodegenerative diseases
Defeating certain viral pathogens:
 Cowpox: IL1 binding protein
 EBV: IL10 homologous protein
 Overproduction of certain cytokines
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


 Pro-inflammatory cytokines: IL6, IL8 and TNF: septic shock and
rheumatoid arthritis
 Administration of mABs or soluble receptors
The Interferons
The Interferons
 The first family of cytokines to be discovered
 History: In 1957, researchers observed that susceptible animal cells, if they were
exposed to a colonizing virus, immediately became resistant to attack by other
viruses. This resistance was induced by a substance secreted by virally infected
cells which was named interferon
 It has been shown that most species actually produce a whole range of interferons.
Humans produce at least three distinct classes, IFN-α, IFN-β and IFN-γ.
The Interferons
• Biological Effects
 Induction of cellular resistance to viral attack
 Regulation of most aspects of immune function
 Regulation of growth and differentiation of many cell types
 Sustenance of early phases of pregnancy in some animal species
No one interferon will display all of these biological activities
Effects are initiated by the binding of the interferon to its specific cell surface receptor
present in the plasma membrane of sensitive cells
The Interferons
• IFN-α and INF-β
 Significant amino acid sequence homology (30 per cent)
 Bind to the same receptor
 Induce similar biological activities
 Acid stable
 IFN-α and IFN-β are sometimes collectively referred to as type I interferons, or acid-stable
interferons.
 IFN-γ  Type II interferon
The Interferons
• Producer cells
The Interferons
• Medical conditions in which IFNs could be used:
 Augmentation of the immune response against infectious agents (viral, bacterial,
protozoal, etc.)
 Treatment of some autoimmune conditions
 Treatment of certain cancer types
The Interferons
• The biochemistry of interferon-α
 In human, 24 related genes or pseudo-genes, code for the production of at least 16
distinct mature IFN-αs
 15 type I: 166 aa,
 1 type II: 172 aa
 23 aa Signal peptide
 Predicted mass of 19-20 kD, but observed up to 27 kD (why?):
 70% aa homology, Rich in leucine and glutamic acid
 Conserved cysteines (usually at positions 1, 29, 99 and 139), two disulfide bonds
 Each have an identifying name: In most cases the names were assigned by placing a
letter after the ‘α’ (i.e. IFN-αA, IFN-αB, etc.)
 Some exceptions exist which contain a number or a number and letter, e.g. IFN-α7,
IFN-α8, IFN-α2B.
 To ensure total confusion, several are known by two different names, e.g. IFN-α7 is also
known as IFN-αJ1
The Interferons
• The biochemistry of interferon-β
 Normally produced by fibroblasts
 The first interferon to be purified.
 In human, a single IFN-β molecule
 166 aa
 30 per cent sequence homology to IFN-αs
 A single disulfide bond
 Glycoprotein of molecular mass in excess of 20 kDa: N-gly, Asn 80
The Interferons
• The biochemistry of interferon-γ
 “Immune” interferon
 Initially purified from human peripheral blood lymphocytes
 Is produced predominantly by lymphocytes
 IL-2 and -12, can induce IFN-γ production under certain circumstances
 143 aa
 No significant sequence homology to type I family
 Predicted mass of 17 kD, but observed three bands: 16-17, 20 and 25 (Why?): N-gly, Asn 25,
97
 Its biologically active form appears to be a homodimer in which the two subunits are
associated in an antiparallel manner
The Interferons
• The interferon receptors: type I IFN receptors
 Two type I interferon receptor polypeptides
 Belonging to the class II cytokine receptor family
 Both are transmembrane N-linked glycoproteins
 α/β: capable of binding all type I interferons
 αβ: specific for IFN-α-B (a specific member of the IFN-α family)
 Present on most cell types
The Interferons
• The interferon receptors: type II IFN receptors
 The IFN-γ receptor
 More limited cellular distribution
 a transmembrane glycoprotein of molecular mass 50 kDa
 Function as a homodimer
 The extracellular IFN-γ binding region consists of approximately 200 amino acid
residues
 AF-1 (accessory factor 1, a transmembrane glycoprotein) associates with the
extracellular region of the receptor
The Interferons
• The interferon receptors: type II IFN receptors
 Cell types which display an IFN-γ receptor on their surface
The Interferons
• Interferon signal transduction
 The intracellular events triggered upon binding of type I or II interferons to their
respective receptors are quite similar
 The JAK–STAT pathway
 signal transducers and activators of transcription (STAT1-STAT6)
 Janus kinases: Janus, a Roman god with two faces: two potential active sites
The Interferons
• The JAK–STAT pathway
Ligands which, upon binding to their
cell surface receptors, are known to
promote activation of one or more
STATs. (The STATs activated are also
shown.)
The Interferons
• The JAK–STAT pathway
The Interferons
• The biological effects of interferons:
 Type I IFNs:
 Antiviral activity
 Anti tumor effects
 Anti-proliferative effect
 by increasing NK and T-cytotoxic cell activity
The Interferons
• The biological effects of interferons:
 Type I IFNs: Antiviral activity
 2–5 oligoadenylate synthetase (2,5-An synthetase) and the eIF-2α protein kinase.
(A) The OAS/RNase L system, an innate immunity pathway that acts against viral infections.
Silverman R H J. Virol. 2007;81:12720-12729
The Interferons
• The biological effects of interferons:
 IFN-γ:
 Weak antiviral and anti-proliferative activity
 Potentates the IFN-α/β activities
 Regulating most aspects of the immune and inflammatory responses
 main macrophage-activating factor
 Destruction of invading microorganisms;
 Destruction of intracellular pathogens;
 Tumour cell cytotoxicity;
 Increased major histocompatibility complex (MHC) antigen expression,
leading to enhanced activation of lymphocytes via antigen presentation
The Interferons
• The biological effects of interferons:
 IFN-γ:
 Binding to its receptor on Neutrofils  increasing in expression of a surface
protein capable of binding to Fc portion of IgG
 Direct modulation of immune responses by affecting growth, differentiation and
function of both T- and B-cells
The Interferons
• Interferon Biotechnology
 The antiviral and anti-proliferative activity of interferons, as well as their ability to
modulate the immune and inflammatory response renders obvious their potential
medical application
 Large-scale purification from sources such as blood was non-viable.
 Interferons exhibit species preference and, in some cases, strict species specificity.
 Transfused blood supplies: only 1% pure
 Mammalian cell culture: cancer cell lines; the namalwa cell line (8000 L) +sendai virus
 Recombinant DNA technology
 E. coli
 Fungi
 Yeast
 Some mammalian cell lines, such as CHO cell lines and monkey kidney cell
lines
The Interferons
• Interferon Biotechnology
The Interferons
• Medical uses of interferon-α
 Anti cancer agent



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Breast cancer
Certain lymphomas (malignant tumour of the lymph nodes)
Multiple myeloma (malignant disease of the bone marrow)
Delay recurrence of tumour growth after surgery in osteogenic sarcoma (cancer of connective
tissue involved in bone formation)
 Approved indications of different INF-αs:
 rhIFN-α2a and -α2b (Intron A): Hairy cell leukemia
 More than 16 medical conditions
The Interferons
• Medical uses of interferon-α
 Antiviral agent: Mostly approved for viral hepatitis
 PEGylated interferons
 PEG IntronA (Pegylated rhIFN-α2b): HCV
 Viraferon Peg (Pegylated rhIFN-α2b): HCV
 PEGasys (Pegylated rhIFN-α2a): HCV
Synthetic Interferon:
 Infergen (interferon alfacon-1 or consensus interferon):
 The most frequently occurring amino acid residue in each corresponding position of the
native interferons
 Cloning and expression of the synthesized coding sequenceby E. coli
 Approved for Hepatitis C
 When compared on a mass basis, the synthetic interferon displays higher antiviral,
antiproliferative and cytokine-inducing activity than do native type I interferons.
The Interferons
• Medical uses of interferon-β
 Relapsing–remitting multiple sclerosis (MS)
 Betaferon (rIFN-β-1b, mutated form: C17S): E. coli
 Betaseron (rIFN-β-1b, mutated form: C17S): E. coli
 Avonex (rhIFN-β-1a): CHO
 Rebif (rhIFN-β-1a): CHO
 Cinnovex (Iran, (rhIFN-β-1a))
 Mechanism of action:
 Perhaps by inhibiting the production of IFN-γ and TNF-α and hence mediating downregulation of the pro-inflammatory responses against the myelin sheath
The Interferons
• Medical uses of interferon-γ
 Chronic granulomatous disease (CGD)
 Phagocytic cells are poorly capable/incapable of ingesting or destroying infectious agents such
as bacteria or protozoa
 Healthy phagocytes produce highly reactive oxidative substances, such as hydrogen peroxide
and hypochlorous acid, via a multicomponent NADPH oxidase system, which are lethal to
pathogens
 CGD: a genetic defect in any component of this oxidase system
NADPH oxidase system
The Interferons
• Medical uses of interferon-γ
 The recombinant human IFN-γ produced in E. coli: termed IFN-γ1b (Actimmune)
 A potent activator of phagocytes
 Potentiates their ability to generate toxic oxidative products
 As long as the genetic defect has not totally inactivated a component of the system,
it promotes increased synthesis of these oxidative substances
 Promotes increased expression of IgG Fc receptors on the surface of phagocytes
 Destroy opsonized infectious agents via phagocytes
The Interferons
Increased expression of IgG Fc receptors on
phagocytes results in enhanced phagocytosis.
These receptors will retain opsonized (i.e.
antibody-coated) infectious agents at their surface
by binding the Fc portion of the antibody. This
facilitates subsequent phagocytosis
The Interferons
• Interferon toxicity
 Like most drugs, administration of interferons can elicit a number of unwanted side
effects
 In some instances the severity of such effects can limit the maximum recommended
therapeutic dose to a level below that which might have maximum therapeutic effect
 The common adverse effect between the different IFNs is Flu-like symptoms
 Fever
 Headache
 Chills
The Interleukins
The Interleukins
• General aspects
 The interleukins represent another large family of cytokines
 at least 36 different constituent members
 Most of these polypeptide regulatory factors are glycosylated (a notable
exception being IL-1) and display a molecular mass ranging from 15 to 30 kDa
(IL-9: heavily glycosylated, 40 kD)
 Most of the interleukins are produced by a number of different cell types
 IL-1  17 different cell types, IL-8  at least 10 different cell types
 IL-2, IL-9 and IL-13  produced only by T- lymphocytes
The Interleukins
• General aspects
 Most cells capable of synthesizing one interleukin are capable of synthesizing
several, and many prominent producers of interleukins are non-immune
system cells
 In most instances, induction or repression of any one interleukin is prompted by
numerous regulators (mostly additional cytokines)
 IL-1 promotes increased synthesis and release of IL-2 from activated Tlymphocytes
 It is highly unlikely that cells capable of synthesizing multiple interleukins
concurrently synthesize them all at high levels
The Interleukins
• General aspects
 Nearly all of the interleukins are soluble molecules (one form of IL-1 is cell
associated)
 Their biological response is induced by binding to specific receptors on the
surface of target cells
Most interleukins exhibit paracrine activity
Some display autocrine activity : IL-2
Some interleukins display more systematic endocrine effects: IL-1
The Interleukins
 Signal Transduction:
 Tyrosine phosphorylation
 Serine and threonine residues of specific intracellular substrates are also
phosphorylated
 Promoting an increase in intracellular calcium concentration
 Inducing hydrolysis of phosphatidylethanolamine with release of diacyl
glycerol
The Interleukins
• General aspects
Many cell types are capable of producing a whole range of
interleukins
T-lymphocytes are capable of producing all the interleukins, with
the possible exception of IL-7 and IL-15.
Many cell types producing multiple interleukins can also produce
additional cytokines
For example, both macrophages and fibroblasts are capable of
producing several interleukins, CSFs and PDGF.
The Interleukins
• Biological effects
 Interleukins regulate a variety of physiological and pathological conditions, including:
 Normal and malignant cell growth;
 All aspects of the immune response;
 Regulation of inflammation.
 6 interleukin-based products have gained approval for general medical use
 Proleukin: an IL-2
 Neumega: an IL-11
 Kineret: anti IL-1
 Ustekinumab: anti IL-12 and IL23
 Reslizumab: Anti IL-5
 Secukinumab: anti IL-17A
 Ontak: DT-IL2 immunotoxin
The Interleukins
• Interleukin-2
 known as T-cell growth factor
 The first T-cell growth factor to be identified
 It is produced exclusively by T-lymphocytes (especially T-helper cells), in response to
activation by antigen and mitogens
 A single-chain glycoprotein containing 133 amino acids
 The mature molecule displays a molecular mass ranging from 15 to 20 kDa
 The carbohydrate moiety is not required for biological activity
 O-gly to 3rd Thr
The Interleukins
• Interleukin-2 Receptor
 The high-affinity receptor complex consists of three membrane-spanning polypeptide
chains (α, β and γ)
 The α chain binds IL-2 with low affinity
 The γ subunit does not interact directly with IL-2: γc (common): IL-4, 7, 9, 13 and 15
 αγ or βγ can bind IL-2 with intermediate affinity
 The heterotrimeric αβγ complex represents the cytokine’s true high-affinity receptor
Prolonged elevated levels of IL-2
promote the shedding of the IL-2
receptor α subunit from the cell
surface
The Interleukins
• Interleukin-2 Receptor
 The IL-2 receptor is associated with a number of cell types, mainly cells playing a central
role in the immune response
 Binding of IL-2 to its receptor induces growth and differentiation of these cells
Lymphokine Activated Killer
The Interleukins
• Interleukin-2 Receptor
 Quiescent T-lymphocytes are stimulated largely by direct binding to an antigen fragment
presented on the surface of a macrophage in the context of MHC complex:
 This results in the induction of expression of at least 70 genes whose products are
collectively important in immune stimulation
The Interleukins
• Interleukin-2 Receptor
 Several cytoplasmic proteins capable of inducing T-cell growth (i.e. several cellular
protooncogenes, including C-fos and C-myc).
 Various cytokines, most notably IL-2.
 Cytokine receptors, most notably the IL-2 receptor α subunit. (The T-lymphocytes
appear to constitutively express the β and γ IL-2 receptor polypeptides. Induction of the α
gene leads to formation of a high-affinity αβγ receptor complex, thereby rendering the
activated T cell highly sensitive to IL-2)
The Interleukins
• Interleukin-2 Biological Activities
 IL-2 acts as a critical autocrine growth factor for T-cells, and the magnitude of the T-cell
response is largely dependent upon the level of IL-2 produced
 IL-2 serves as a growth factor for activated B-lymphocytes. In addition to promoting
proliferation of these cells, IL-2 (as well as some other interleukins) stimulates enhanced
antibody production and secretion  humoral immune response
 IL-2 promotes the growth and differentiation of NK cells: lymphokine activated killer
(LAK) cells (with an enhanced ability to kill tumour cells or virally infected cells
directly). NK cells express the β and γ IL-2 receptor subunits only: elevated
concentrations of IL-2 is required
The Interleukins
• Interleukin-2 Clinical Applications
 Cancers
 T-cell and other forms of immunodeficiency
 Infectious diseases
The Interleukins
• Interleukin-2 production
 Sources of production
Jurkat leukemia cell line
Proleukin:
 E. coli
 Non-gly
 Lacks N-terminal Ala
 C125S
The Interleukins
Whatever the exact nature of tumour escape, it has been demonstrated, both in
vitro and in vivo, that immunostimulation can lead to enhanced tumor detection
and destruction.
• Interleukin-2 and cancer treatment
 Cancer Specific antigens:
Novel surface antigens not expressed by normal cells
Greatly elevated levels of certain antigens present normally on the cell at
extremely low levels.  so low that they have escaped immunological
tolerance
 In this regard, both a humoral and cell-mediated response can be induced,
although the T-cell response appears to be the most significant
The Interleukins
Whatever the exact nature of tumour escape, it has been demonstrated, both in
vitro and in vivo, that immunostimulation can lead to enhanced tumour detection
and destruction.
• Interleukin-2 and cancer treatment
 Some transformed cells obviously display characteristics that allow them
to evade this immune surveillance:
Most transformed cells do not express class II MHC molecules and express
lower than normal levels of class I MHC molecules
Some tumor-specific surface antigens resemble normal surface antigens
or were expressed previously during the neonatal period and considered as
“self”
Some tumors secrete cytokines and additional regulatory molecules
that can suppress local immunological activity: TGF-β
The glycocalyx
The Interleukins
• Interleukin-2 and infectious diseases
 Numerous pathogens exist for which no effective treatment could be find
 Most of these pathogens are non-bacterial (e.g. viral, fungal and parasitic,
including protozoal) capable of survival within macrophages:
 Mycobacteria (e.g. M. tuberculosis and M. leprae)
 Listeria monocytogenes (causes listeriosis which is characterized by flu-like
symptoms, but can cause swelling of the brain and induce abortions)
 Legionella pneumophila (the bacterium that causes legionnaire’s disease)
 The immunological response raised against intracellular pathogens is largely a
T-cell response. IL-2’s ability to stimulate T-cells may render it useful in the
treatment of a wide range of such conditions
The Interleukins
• Inhibition of interleukin-2 activity
 A variety of medical conditions exist that are caused or exacerbated by the
immune system itself:
Autoimmune diseases
Tissue/organ transplantation
The Interleukins
• Inhibition of interleukin-2 activity
 Selective immunosupression by preventing the synthesis or functioning of IL2:
Cyclosporin A, one of the foremost immunosuppressive agents currently
in use, functions by preventing IL-2 synthesis
Administration of soluble forms of the IL-2 receptor
Administration of monoclonal antibodies capable of binding the IL-2
receptor (without signal transduction activity)
Administration of IL-2 variants that retain the ability to bind the
receptor, but which fail to initiate signal transduction
Administration of IL-2 coupled to bacterial or other toxins: Immunotoxins
The Interleukins
• Immunotoxins
• Ontak (denileukin diftitox)
The Interleukins
• Immunotoxins
The Interleukins
• Interleukin-1
 It is also known as lymphocyte-activating factor (LAF), endogenous pyrogen and
catabolin
 Two distinct forms: IL-1α (membrane anchored) and IL-1β (soluble)
 20% amino acid homology, different genes, but bind the same receptor
 Different cell types produce the different IL-1s in varying ratios
• Fibroblasts and endothelial cells: similar ratios
• IL-1β is produced in larger quantities than IL-1α in monocytes
• Activated macrophages appear to represent the major cellular source for IL-1
The Interleukins
• Interleukin-1
 Two distinct receptors:
 Type I: fibroblasts, keratinocytes, hepatocytes and endothelial cells
 Type II: B-lymphocytes, bone marrow cells and polymorphonuclear leukocytes
Both IL-1s could bind both receptor types
The Interleukins
• Interleukin-1 Biological Effects
 It is a pro-inflammatory cytokine: major biological function of IL-1
 It plays a role in activating B-lymphocytes
 It acts as a co-stimulator of haematopoietic cell growth/differentiation
 Along with IL-6, it induces synthesis of acute-phase proteins in hepatocytes
• These various biological activities depends largely upon the quantities of IL-1
produced in any given situation
 At low concentrations: paracrine, induction of local inflammation.
 At elevated concentrations: endocrine, inducing systematic effects, such as the
hepatic synthesis of acute-phase proteins, induction of fever (hence the name,
endogenous pyrogen) and general body wasting, such as that associated with some
cancers
The Interleukins
• IL-1-like protein or IL-1 receptor antagonist (IL-1Rα)
 This molecule appears to be capable of binding to the IL-1 receptors without
triggering an intracellular response
 Clinical application: Because of the IL-1 role in mediating acute/chronic
inflammation, (downward) modulation of IL-1 levels may prove effective in
ameliorating the clinical severity of these conditions
anti-IL-1 antibodies
soluble forms of the IL-1 receptor
Native IL-1 receptor antagonist
The Interleukins
• IL-1-like protein or IL-1 receptor antagonist (IL-1Rα)
 Kineret: an approved product based on the latter strategy. Indicated in the
treatment of rheumatoid arthritis
 rhIL-1 receptor antagonist
 17.3 kD, E. coli, Non-gly, add. Met.
The Interleukins
• IL-11
 Produced largely by IL-1-activated bone marrow stromal cells and
fibroblasts
 functions as a haematopoietic growth factor
 It stimulates:
 Thrombopoiesis (the production of platelets formed by the shedding of fragments
of cytoplasm from large bone marrow cells called megakaryocytes
 Growth/differentiation of bone marrow cells, derived from stem cells that have
become committed to differentiate into macrophages
The Interleukins
 The rationale for assessing IL-11 as a potential therapeutic agent relates mainly
to its ability to induce platelet synthesis
 Neumega is the tradename given to the IL-11-based product approved for the
prevention of thrombocytopenia
Growth Factors: Haematopoietic growth factors
86
Oprelvekin
Trade name: Neumega™
Approval Date: 1997
Description:The protein has a molecular mass of approximately 19kD, and is
non-glycosylated. The polypeptide is 177 amino acids in length and differs from
the 178 amino acid length of native IL-11 only in lacking the amino-terminal
proline residue. This alteration has not resulted in measurable differences in
bioactivity either in vitro or in vivo
Production system: E. coli
Dosage form: Single-use vials containing 5 mg of oprelvekin
Therapeutic Indications: Is indicated for the prevention of severe
thrombocytopenia and the reduction of the need for platelet transfusions
following myelo-suppressive chemotherapy in adult patients with nonmyeloid
malignancies who are at high risk of severe thrombocytopenia
The Tumor Necrosis
Factors
The Interleukins
• Tumour necrosis factor (TNF)
 Two types: α and β
 Bind to same receptor
 Induce very similar biological activities
 Display limited sequece homology
 Chromosome 6, adjacent, 1100bp distance
The Interleukins
• Tumour necrosis factor (TNF)
 TNF-α : macrophage cytotoxic factor, macrophage cytotoxin and necrosin
 As some of these names suggest, activated macrophages appear to represent
the most significant cellular source of TNF-α
 Producer cells do not store TNF-α, but synthesize it de novo following
activation
 Macrophages appear to express TNF-α mRNA constitutively, which is
translated only upon their activation
 Monomeric TNF is biologically inactive; the active form is a homotrimer in
which the three monomers associate non-covalently
The Interleukins
• Tumour necrosis factor (TNF)
 TNF-α: mostly studied, simply as TNF
 Sources of TNF-α:
 Inducers of TNF-α:
The Interleukins
• Biological Activities of TNF- α
 Activation of certain elements of both non-specific and specific immunity, in
particular in response to Gram-negative bacteria
 Induction/regulation of inflammation
 Selective cytotoxic activity against a range of tumor cells
 Mediation of various pathological conditions, including septic shock, cachexia
and anorexia
 The extent of the activity depends on the released amount:
 Low amount: locally, affecting WBCs and ECs
 High amount: Endocrine, is largely detrimental
The Interleukins
• TNF receptors
 2 types:
 Type I; 55kDa (TNF-R55): more distributed, almost all cells
 Type II; 75 kDa (TNF-R75): restricted, most prominent on hematopoietic cells
 Both binds to both TNF types
TNF-R55
and
TNF-R75
Trimerisation following binding of the ligand
to the receptor
The Interleukins
• Clinical Applications of TNF- α
 The initial interest in utilizing TNF as a general anti-cancer agent has
diminished, largely due to the realization that:
• Many tumours are not susceptible to destruction mediated by TNF (indeed, some
tumours produce TNF as an autocrine growth factor)
• Tumour cell necrosis is not TNF’s major biological activity
• Severe side effects usually accompany systemic administration of therapeutically
relevant doses of this cytokine
 Beromun™ (tasonermin): TNF-α-1a
 Soft Tissue Sarcoma (STS) in the limbs to prevent or delay amputation;
 Used in addition to melphalan
The Interleukins
• Clinical Applications of TNF- α
 Most clinical interest in TNF centers around neutralizing its biological effects
in situations where overexpression of TNF induces negative clinical effects
 TNF has been firmly implicated in mediating many of the adverse effects
associated with dozens of diseases
The Interleukins
• Clinical Applications of TNF- α
 Enbrel: an engineered hybrid protein consisting of the extracellular domain of
the TNF p75 receptor fused directly to the Fc (constant) region of human IgG
 Enbrel functions as a competitive inhibitor of TNF, a major pro-inflammatory
cytokine
 Binding of TNF to Enbrel prevents it from binding to its true cell surface
receptors
 The antibody Fc component of the hybrid protein confers an extended serum
half-life on the product, increasing it by fivefold relative to the soluble TNF
receptor portion alone
96
Tumor Necrosis Factors
TNF dependent drugs
Etanercept
Trade name: Enbrel™
Approval Date: 1998
Description: Enbrel (etanercept) is a dimeric fusion protein consisting of the
extracellular ligand-binding portion of the human 75 kilodalton (p75) tumor
necrosis factor receptor (TNFR) linked to the Fc portion of human IgG1. It consists
of 934 amino acids and has an apparent molecular weight of approximately 150
kilodaltons
Production system: E. coli
Therapeutic Indications: Enbrel is indicated for reducing signs and symptoms,
inducing major clinical response, inhibiting the progression of structural damage,
and improving physical function in patients with moderately to severely active
rheumatoid arthritis (RA). Enbrel can be initiated in combination with
methotrexate (MTX) or used alone
The Growth Factors
The Growth Factors
The differentiation, growth and division of eukaryotic cells is modulated by
various influences, of which growth factors are amongst the most important for many
cell types
A wide range of polypeptide growth factors have been identified and more,
undoubtedly, remain to be characterized
Each growth factor has a mitogenic (promotes cell division) effect on a
characteristic range of cells
Some factors affect only a few cell types, most stimulate growth of a wide range of
cells.
The Growth Factors
Some growth factors may be classified as cytokines (e.g. interleukins, TGF-β and
CSFs)
Others (e.g. IGFs) are not members of this family
The ability of growth factors to promote accelerated cellular growth, differentiation
and/or division has predictably attracted the attention of the pharmaceutical industry
Several such products, most notably a range of haematopoietic growth factors, have
now gained approval for general medical use
The Growth Factors
Overview of some polypeptide growth factors
Many can be grouped into families on the basis of amino acid sequence homology, or the cell
types affected. Most growth factors are produced by more than one cell type and display endocrine,
paracrine or autocrine effects on target cells by interacting with specific cell surface receptors
The Growth Factors
Growth factors approved for general medical use
Growth Factors: Haematopoietic growth factors
Blood consists of red and white cells which, along with platelets, are all suspended
in plasma
Derived from Stem cells (also known as a pluripotential, pluripotent or haemopoietic
stem cell)
Stem cells reside in the bone marrow, alongside additional cell types, including
(marrow) stromal cells
Pluripotential stem cells have the capacity to undergo prolonged or indefinite
self-renewal
They also have the potential to differentiate, thereby yielding the range of cells
normally found in blood
102
Growth Factors: Haematopoietic growth factors
The process, by which a fraction of stem cells is continually ‘deciding’ to
differentiate (thus continually producing new blood cells and platelets to replace aged
cells), is known as haemopoiesis
During the haemopoietic process, the stem cells differentiate, producing cells that
become progressively more restricted in their choice of developmental options
103
Growth Factors: Haematopoietic growth factors
The production of many mature blood cells begins when a fraction of the stem cells
differentiates, forming a specific cell type termed CFU-S (where CFU refers to colonyforming unit)
These cells, in turn, differentiate to yield CFU-GEMM cells, a mixed CFU that has
the potential to differentiate into a range of mature blood cell types, including
granulocytes, monocytes, erythrocytes, platelets, eosinophils and basophils
Note that lymphocytes are not derived from the CFU-GEMM pathway, but
differentiate via an alternative pathway from stem cells
104
Growth Factors: Haematopoietic growth factors
105
Growth Factors: Haematopoietic growth factors
107
Two questions arises in this regard:
How is the correct balance between stem cell self-renewal and
differentiation maintained?
What forces exist that regulate the process of differentiation?
The answer to both questions, in particular the latter, is beginning to
emerge in the form of a group of cytokines termed ‘haemopoietic growth
factors’
108
Growth Factors: Haematopoietic growth factors
The group of haemopoietic growth factors includes;
Several interleukins, which primarily affect production and
differentiation of lymphocytes
CSFs, which play a major role in the differentiation of stem-derived
cells
into
neutrophils,
macrophages,
megakaryocytes,
eosinophils and basophils
EPO, which is essential in the production of red blood cells
TPO, which is essential in the production of platelets
Growth Factors: Haematopoietic growth factors
109
Most of these haemopoietic growth factors are glycoproteins,
displaying a molecular mass in the region of 14–24 kDa
Most are produced by more than one cell type
Several such regulators can stimulate proliferation of any one
haemopoietic cell lineage
Receptor numbers for any one growth factor are low (less than 500
per cell), and proliferation can be stimulated even when only a small
proportion of these are occupied
IL3!
Growth Factors: Haematopoietic growth factors
110
The interleukins
A number of inteleukins are known to influence haemopoiesis
The IL-3 receptor, for example, is found on a wide variety of progenitor
haemopoietic cells
IL-3 appears to stimulate not only CFU-GEMM, but also the precursor
cells of basophils, eosinophils and platelets
The role of IL-11, which also plays a role was discussed before
Growth Factors: Haematopoietic growth factors
111
Granulocyte colony-stimulating factor (G-CSF)
G-CSF is also known as pluripoietin and CSF-β
Two slight variants are known
One consisting of 174 amino acids, and the other of 177
The smaller polypeptide predominates and also displays significantly
greater biological activity than the larger variant
Growth Factors: Haematopoietic growth factors
112
Granulocyte colony-stimulating factor (G-CSF)
G-CSF is a glycoprotein
It displays a single O-linked glycosylation site and an apparent
molecular mass in the region of 21 kDa
Growth Factors: Haematopoietic growth factors
113
Granulocyte colony-stimulating factor (G-CSF)
It functions as a growth and differentiation factor for neutrophils and
their precursor cells
It also appears to activate mature neutrophils (which are leukocytes
capable of ingesting and killing bacteria)
G-CSF also appears to act in synergy with additional haemopoietic
growth factors to stimulate growth/differentiation of various other
haemopoietic progenitor cells
In addition, this cytokine promotes the proliferation and migration of
endothelial cells
Growth Factors: Haematopoietic growth factors
114
Macrophage colony-stimulating factor (M-CSF)
M-CSF serves as a growth, differentiation and activation factor for
macrophages and their precursor cells
It is also known as CSF-1
The mature form is a glycoprotein containing three potential N-linked
glycosylation sites
Growth Factors: Haematopoietic growth factors
Macrophage colony-stimulating factor (M-CSF)
This cytokine is produced by various cell types
115
Growth Factors: Haematopoietic growth factors
116
Macrophage colony-stimulating factor (M-CSF)
The biologically active form of M-CSF is a homodimer (two identical
subunits)
These homodimers can exist as integral cell surface proteins, or may
be released from their producer cell by proteolytic cleavage, thus yielding
the soluble cytokine
Growth Factors: Haematopoietic growth factors
117
Granulocyte Macrophage colony-stimulating factor
GM-CSF is also known as CSF-α or pluripoietin-α
It is a 127 amino acid, single-chain, glycosylated polypeptide, exhibiting
a molecular mass in the region of 22 kDa
It is produced by various cells
Growth Factors: Haematopoietic growth factors
118
Granulocyte Macrophage colony-stimulating factor
Its biological activities include:
Proliferation/differentiation factor of haemopoietic progenitor cells,
particularly those yielding neutrophils (a variety of granulocyte) and
macrophages, but also eosinophils, erythrocytes and megakarycytes
Activation of mature haemopoietic cells, resulting in:
Enhanced phagocytic activity;
Enhanced anti-microbiocidal activity;
Augmented anti-tumour activity;
Enhanced leukocyte chemotaxis
Growth Factors: Haematopoietic growth factors
119
Clinical application of colony-stimulating factors
G-CSF and GM-CSF have proven useful in the treatment of
neutropenia
All three CSF types are (or are likely to be) useful also in the treatment
of infectious diseases, some forms of cancer and the management of
bone marrow transplants, as they stimulate the differentiation/activation
of white blood cell types most affected by such conditions
Growth Factors: Haematopoietic growth factors
120
Clinical application of colony-stimulating factors
Neutropenia is a condition characterized by a decrease in blood
neutrophil count below 1.5×109 cells per liter
A normal blood count is (2.0–7.5)×109 cells per liter
Its clinical symptoms include the occurrence of frequent and usually
serious infections, often requiring hospitalization
Neutropenia may be caused by a number of factors at least some of
which are responsive to CSF treatment
Growth Factors: Haematopoietic growth factors
121
Clinical application of colony-stimulating factors
Neutropenia may be caused by a number of factors at least some of
which are responsive to CSF treatment
Particularly noteworthy is neutropenia triggered by administration of
chemotherapeutic drugs to cancer patients
When administered at therapeutically effective doses, often induce the
destruction of stem cells and/or compromise stem cell differentiation
Growth Factors: Haematopoietic growth factors
122
Clinical application of colony-stimulating factors
Neutropenia may be caused by a number of factors at least some of
which are responsive to CSF treatment
Growth Factors: Haematopoietic growth factors
Clinical application of colony-stimulating factors
Drugs reported to induce neutropenia
123
Growth Factors: Haematopoietic growth factors
124
Clinical application of colony-stimulating factors
Filgrastim
Trade name: Neupogen™
Approval Date: 1991
Description: Filgrastim is a recombinant human granulocyte-colony stimulating
factor (G-CSF) that regulates the production of neutrophils and differs from the
naturally occurring molecule in having an additional methionine residue at the Nterminus and in lacking glycosylation
Production system: Escherichia coli
Dosage form: Neupogen is provided as a solution for subcutaneous or
intravenous administration in vials and in prefilled syringes
Therapeutic Indications: Neupogen is indicated for the treatment of neutropenia
associated with various medical conditions
Growth Factors: Haematopoietic growth factors
125
Clinical application of colony-stimulating factors
Pegfilgrastim
Trade name: Neulasta™
Approval Date: 2002
Description: Neulasta is a recombinant human granulocyte-colony stimulating
factor (G-CSF), conjugated to a single 20-kDa
methoxypolyethylene glycol-propionaldehyde (PEG) molecule at the N terminus
Production system: Escherichia coli
Dosage form: Neulasta™ is provided as a solution for subcutaneous or
intravenous administration in vials and in prefilled syringes
Therapeutic Indications: Neulasta™
is
indicated for the treatment
of
neutropenia and febrile neutropenia in patients with malignancies treated with
cytotoxic chemotherapy.
Growth Factors: Haematopoietic growth factors
126
Clinical application of colony-stimulating factors
Sargramostim
Trade name: Leukine™
Approval Date: 1991
Description: Sargramostim is a recombinant human granulocyte-macrophage
colony stimulating factor (rhuGM-CSF), a 127-amino acid glycosylated
hematopoietic growth factor. It differs from the native human molecule by a
substitution of leucine at position 23
Production system: Saccharomyces Cerevisiae
Dosage form: both liquid (500 mg/ml) and lyophilized (250 mg) formats
Therapeutic Indications: Leukine was originally indicated for use following
induction of chemotherapy in adult patients with acute myelogenous leukemia
(AML), in order to shorten time to neutrophil recovery and reduce the incidence of
severe infection; Neutrophil recovery after bone marrow transplantation
Growth Factors: Haematopoietic growth factors
127
Erythropoietin
Responsible for stimulating and regulating erythropoiesis in mammals
The erythron
Mature erythrocytes, along with all stem-cell-derived progeny that
have committed to developing into erythrocytes.
The erythron can thus be viewed as a disperse organ whose primary
function relates to transport of oxygen and carbon dioxide, as well as
maintaining blood pH
An average adult contains in the region of 2.3×1013 erythrocytes
(weighing up to 3 kg)
Growth Factors: Haematopoietic growth factors
128
Erythropoietin
Erythrocytes are synthesized at a rate of about 2.3 million cells per
second and have a circulatory life of approximately 120 days, during
which they travel almost 200 miles
EPO is an atypical cytokine, in that it acts as a true (endocrine)
hormone and is not synthesized by any type of white blood cell
Growth Factors: Haematopoietic growth factors
129
Erythropoietin
A single copy gene, located on (human) chromosome 7
The mature EPO gene product contains 165 amino acids and exhibits
a molecular mass in the region of 36 kDa
EPO is a glycoprotein, almost 40 per cent of which is carbohydrate
Three N-linked and one O-linked glycosylation sites are evident
The O-linked carbohydrate: No essential role in the EPO
Removal of the N-linked sugars destroys its in vivo activity
The roles of sugar components: solubility, cellular processing
and secretion, as well as its in vivo metabolism
Growth Factors: Haematopoietic growth factors
130
Erythropoietin
Incomplete (N-linked) glycosylation prompts decreased in vivo activity
due to more rapid hepatic clearance of the EPO molecule
The reported plasma t1/2 value for native EPO is 4–6 h
The t1/2 for desialated EPO is 2 min
EPO in the human adult is synthesized almost exclusively by
specialized kidney cells
Minor quantities are also synthesized in the liver, which represents the
primary EPO-producing organ of the foetus.
Growth Factors: Haematopoietic growth factors
131
Erythropoietin
EPO stimulates erythropoiesis by:
Increasing the number of committed cells capable of differentiating
into erythrocytes
Accelerating the rate of differentiation of such precursors
Increasing the rate of hemoglobin synthesis in developing cells
Growth Factors: Haematopoietic growth factors
Erythropoietin
characterized stages in the process of erythropoiesis
CFU-E cells display the greatest density of
EPO cell surface receptors. These cells
also display the greatest biological
response to EPO
More mature erythrocyte precursors
display progressively less EPO receptors
on their cell surfaces.
Erythrocytes themselves are devoid of
EPO receptors
132
Growth Factors: Haematopoietic growth factors
133
Therapeutic applications of erythropoietin
A number of clinical circumstances have been identified which are
characterized by an often profoundly depressed rate of erythropoiesis
Renal failure
Rheumatoid arthritis
Cancer
AIDS
Infections
Bone marrow transplantation
Growth Factors: Haematopoietic growth factors
Therapeutic applications of erythropoietin
EPO has been approved for use to treat various forms of anemia
Anaemia associated with chronic disease
Anaemia associated with cancer/chemotherapy
To reduce transfusion requirements after surgery
To prevent anaemia after bone marrow transplantation
134
Growth Factors: Haematopoietic growth factors
135
Therapeutic applications of erythropoietin
Epoetin alfa
Trade name: Epogen™ and Procrit™
Approval Date: 1990
Description: It exhibits an amino acid sequence identical to the naturally
occurring erythropoietin but differs somewhat from this in terms of their exact
glycosylation pattern.
Production system: Chinese hamster ovary
Dosage form: a lyophilized form, to be reconstituted prior to subcutaneous use
Therapeutic Indications: Epogen/Procrit is indicated for the end-stage renal
disease, treatment of anemia associated with renal disease and cancer
chemotherapy, anemia and leukemia in HIV-infected patients, and anemia in
patients undergoing elective, noncardiac, nonvascular surgery
Growth Factors: Haematopoietic growth factors
136
Therapeutic applications of erythropoietin
Epoetin beta
Trade name: NeoRecormon™
Approval Date: 1997
Description: NeoRecormon is identical to the natural molecule, as derived from
the urine of anemic patients
Production system: Chinese hamster ovary
Dosage form: a lyophilized form, to be reconstituted prior to subcutaneous use
Therapeutic Indications: NeoRecormon is indicated for the treatment of renal
anemia in patients undergoing dialysis or prior to initiation of dialysis. It is also
indicated for prevention and treatment of anemia in premature infants and in
adults with solid tumors, multiple myeloma, non- Hodgkin’s lymphoma, and
chronic lymphocytic leukemia who are undergoing antitumor therapy
Growth Factors: Haematopoietic growth factors
137
Therapeutic applications of erythropoietin
Darbepoetin alfa
Trade name: Nespo (trade name E.U.), Aranesp (trade name U.S.)
Approval Date: 2001
Description: It is a recombinant 166-amino acid human erythropoietin which
differs from the natural molecule in the occurrence of two additional sugar chains.
It has five N-linked carbohydrate side chains. This leads to an increased halflife (21h vs 4-6h) and, therefore, a coupled, reduced frequency of administration is
necessary.
Production system: Chinese hamster ovary
Dosage form: is presented as a solution for intravenous or subcutaneous
administration
Therapeutic Indications: Nespo is indicated for the treatment of anemia in
patients with chronic renal failure, and for patients with anemia associated with
malignancies
Growth Factors: Haematopoietic growth factors
138
Therapeutic applications of erythropoietin
Methoxy polyethylene glycol-epoetin beta
Trade name: Mircera™
Approval Date: 2007
Description: It is methoxy polyethylene glycol-epoetin beta, which differs from
erythropoietin through formation of a chemical bond between either the N-terminal
amino group or the e-amino group of any lysine present in erythropoietin,
predominantly Lys52 and Lys45 and methoxy polyethylene glycol (PEG)
butanoic acid
Production system: Chinese hamster ovary
Dosage form: formulated as a sterile, preservative-free protein solution for IV or
SC administration
Therapeutic Indications: It is indicated for the treatment of anemia associated
with chronic renal failure (CRF) in adults, including patients on dialysis and not
on dialysis
Growth Factors: Haematopoietic growth factors
Thrombopoietin (TPO)
332 amino acid
60 kDa glycoprotein
containing six potential N-linked glycosylation sites, all localized
towards the C-terminus of the molecule
The N-terminal half exhibits a high degree of amino acid homology
with EPO and represents the biologically active domain of the
molecule
139
Growth Factors: Haematopoietic growth factors
140
Thrombopoietin (TPO)
TPO is the haemopoietic growth factor now shown to be the primary
physiological regulator of platelet production
This molecule represent an important therapeutic agent in combating
thrombocytopenia, a condition characterized by reduced blood platelet
levels
Growth Factors: Haematopoietic growth factors
141
Clinical application of TPO or its mimetics
Romiplostim
Trade name: Nplate™
Approval Date: 2008
Description: TPO mimetic, an Fc-peptide fusion protein (peptibody) that
activates intracellular transcriptional pathways leading to increased platelet
production via the TPO receptor. The peptibody molecule contains two identical
single-chain subunits, each consisting of human immunoglobulin IgG1 Fc
domain, covalently linked at the C-terminus to a peptide containing two
thrombopoietin receptor-binding domains. Romiplostim has no amino acid
sequence homology to endogenous TPO
Production system: E. coli
Dosage form: Nplate is supplied as a sterile, preservative-free, lyophilized, solid
white powder for subcutaneous injection
Therapeutic Indications: treatment of thrombocytopenia in patients with
chronic immune thrombocytopenia (ITP) who have had an insufficient
response to corticosteroids, immunoglobulins or splenectomy
Growth Factors: Haematopoietic growth factors
142
Clinical application of TPO or its mimetics
Eltrombopag
Trade name: Promacta™
Approval Date: 2008
Description: TPO mimetic, a small molecule thrombopoietin receptor
agonist for oral administration. Eltrombopag interacts with the transmembrane
domain of the TPO receptor.
Production system: Chemical entity
Dosage form: Tablets equivalent to 12.5 mg, 25 mg, 50 mg, 75 mg, or 100 mg
of eltrombopag free acid
Therapeutic Indications: treatment of thrombocytopenia in patients with
chronic immune thrombocytopenia (ITP) who have had an insufficient
response to corticosteroids, immunoglobulins or splenectomy
Growth Factors and wound healing
143
The wound healing process is complex and not yet fully understood
The area of tissue damage becomes the focus of various events, often
beginning with immunological and inflammatory reactions
The various cells involved in such processes, as well as additional cells
at the site of the wound, also secrete various growth factors
These mitogens stimulate the growth and activation of various cell
types, including fibroblasts (which produce collagen and elastin
precursors, and ground substance), epithelial cells (e.g. skin cells) and
vascular endothelial cells
Growth Factors and wound healing
144
The growth factors that appear most significant to this process include:
FGFs
PDGF
IGF-1
EGF
Growth Factors and wound healing
145
Wounds can be categorized as:
Acute (healing quickly on their own)
Chronic (healing slowly, and often requiring medication)
Chronic wounds occur if some influence disrupts the normal healing
process
Diabetes
Malnutrition
Rheumatoid arthritis
Ischaemia
Elderly people are particularly susceptible
to developing chronic wounds, often
resulting in the necessity for hospitalization
Growth Factors and wound healing
146
Various types of ulcers along with their underlying cause. An ulcer may simply
be described as a break or cut in the skin or membrane lining the digestive
tract which fails to heal. The damaged area may then become inflammed
Growth Factors and wound healing
147
The fluid exuded from a fresh or acute wound generally exhibits high
levels of various growth factors
The concentration of such mitogens present in chronic wounds is
usually several-fold lower
The fluid exuded from chronic wounds harbours high levels of
proteolytic activity (almost 200-fold higher than associated with acute
wounds)  rapid proteolytic degradation of mitogens or their receptors
A range of growth factors may demonstrate potential in wound
management/healing, or for other therapeutic indications
Growth Factors and wound healing
148
Insulin-like growth factors
Also termed ‘somatomedins’
Constitute a family of two closely related (small) polypeptides
IGF-I
IGF-II
As the names suggest, these growth factors bear a strong structural
resemblance to insulin (or, more accurately, proinsulin)
149
Growth Factors and wound healing
Insulin-like growth factors
IGFs display pluripotent activities, regulating the growth, activation,
differentiation (and maintenance of the differentiated state) of a wide
variety of cell and tissue types
Promotes cell cycle progression in most cell types
Foetal development: promotes growth and differentiation of foetal
cells and organogenesis
Promotes longitudinal body growth and increased body weight
Promotes
enhanced
functioning
of
the
male
and
reproductive tissue
Promotes growth and differentiation of neuronal tissue
female
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Insulin-like growth factors
Sources
Liver represents the major site of synthesis of the IGFs, from
where they enter the blood stream, thereby acting in a classical
endocrine fashion. A wide variety of body cells express IGF
receptors
IGFs are also synthesized in smaller quantities at numerous sites in
the body and function in an autocrine or paracrine manner at these
specific locations
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Insulin-like growth factors
Virtually all mammalian cell types display surface IGF receptors
Biological effects
Stimulate cellular growth and differentiation
Most of the growth-promoting effects of GH are actually
mediated by IGF-I
IGFs also play a core role in tissue renewal and repair
IGF-I influences (usually enhances) renal function by a
number of means
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Clinical application of IGF
Mecasermin
Trade name: Increlex™
Approval Date: 2005
Description: Contains human insulin-like growth factor-1 (rhIGF-1) produced
by recombinant DNA technology. IGF-1 consists of 70 amino acids in a single
chain with three intramolecular disulfide bridges and a molecular weight of 7.6
kD. Its amino acid sequence is identical to that of endogenous human IGF-1.
Production system: E. coli
Dosage form: a sterile, aqueous, clear and colorless solution intended for
subcutaneous injection
Therapeutic Indications:
Growth failure in children with severe primary IGF-1 deficiency
Growth hormone (GH) gene deletion who have developed neutralizing
antibodies to GH
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Clinical application of IGF
Mecasermin rinfabate
Trade name: IPLEX™
Approval Date: 2005
Description: It is a binary protein complex of human insulin-like growth
factor-1 (rhIGF-1) and human insulin-like growth factor-binding protein-3
(rhIGFBP-3), which are produced by two separate E. coli strains.
Production system: E. coli
Dosage form: It is an aqueous solution for injection
Therapeutic Indications:
Growth failure in children with severe primary IGF-1 deficiency
Growth hormone (GH) gene deletion who have developed neutralizing
antibodies to GH
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Platelet-derived growth factor
Osteosarcoma-derived growth factor
Glioma-derived growth factor
It is the major growth factor synthesized by platelets
PDGF exhibits a mitogenic effect on fibroblasts, smooth muscle cells
and glial cells
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Platelet-derived growth factor
Range of cells producing PDGF, and its major biological activities
155
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Platelet-derived growth factor
It plays an important role in the wound healing process
It is released at the site of damage by activated platelets, and
acts as a mitogen/chemoattractant for many of the cells responsible
for initiation of tissue repair
It tends to act primarily in a paracrine manner
It represents an autocrine/paracrine growth factor for a variety of
malignant cells
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Platelet-derived growth factor
Active PDGF is a dimer. Two constituent polypeptides, A and B, have
been identified, and three active PDGF isoforms are possible: AA, BB and
AB
PDGF is of value in wound management, particularly with regard to
chronic wounds
In vitro studies, using various cell lines, suggest that PDGF AB or BB
dimeric isoforms are the most potent
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Platelet-derived growth factor
Normal skin appears to be devoid of PDGF receptors
Animal studies illustrate that rapid expression of receptor subunits is
induced upon generation of an experimental wound (e.g. a surgical
incision)
Receptor expression is again switched off following re-epithelialization
and complete healing of the wound
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Platelet-derived growth factor
Various clinical trials showed higher healing rates of chronic wounds
following topical application of PDGF
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Becaplermin
Trade name: REGRANEX ™
Approval Date: 1997
Description: It is a recombinant human PDGF for topical administration.
Becaplermin has a molecular weight of approximately 25 KD and is a homodimer
composed of two identical polypeptide chains (BB) that are bound together by
disulfide bonds.
Production system: Saccharomyces cerevisiae
Dosage form: It is a non-sterile, low bioburden, preserved, sodium
carboxymethylcellulose-based (CMC) topical gel
Therapeutic Indications: It is indicated for the treatment of lower extremity
diabetic neuropathic ulcers that extend into the subcutaneous tissue or beyond
and have an adequate blood supply, when used as an adjunct to, and not a
substitute for, good ulcer care practices including initial sharp debridement,
pressure relief and infection control.