G ENER A L

INFORMATION

biologically active principle of

CYTOKINES

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Scientific BioTech
Content
Introduction 3

Cytokines 4
Effect 6
Listing and Significance 8
Treatment with Cytokines 10
The Cytokine Network 14
Summary 15

Growth Factors 16
Autologous Peptides 17
Definition of Growth Factors 19

Collagen 22
Main Component of the Skin 23

Detection 26
Observational study 29

Main 30
List of scientific papers 31
Introduction
The history of cytokines is an integral part of the most recent developments in the regulation of the immune system
and hematopoiesis.

The immune system’s response to infections is swift and firm. This is owed, inter alia, to a group of proteins which
transmits signals among various immune cells and also intervenes in many other areas of the organism. For exam-
ple, cytokines (sometimes also called “lymphokines” or “interleukins”) control the temperature centers in our brain,
they activate inflammatory substances or influence the messengers of the nervous system.

The cytokine family has a bewildering number of members which are active virtually everywhere in the human
organism. They are created by specific cells and released into their surroundings. There, they will then regulate the
respectively local interactions between neighboring cells, partly through inhibiting and partly through stimulating
effects — similar to hormones which, however, usually bridge larger distances for signal transmissions in the body.
A specific cytokine may be formed by entirely different cells and then act upon many different tissues.

The human organism has two mechanisms to defend against any type of invaders. The first cellular half of this
immune defense is mounted by specifically equipped leukocytes; for example, the T-lymphocytes or “killer cells”.
Cytokines are used as signal transmitters between such immune cells. When a messenger is released by one cell
type, it activates, for example, a second cell type and inhibits a third one at the same time. Frequently, cascading
signal chains will thus result.

The other half of the immune defense (the humoral defense) is based on large molecules — the antibodies. They are
formed by so-called B-cells. Here as well, cytokines take on the role of messengers among the cells involved. And
they are impacting their target objects quite differently. They change especially the number of receptors (“receiving
stations”) on the surface of these cells: This is where all cytokines have to dock on to transmit their signal.

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 CYTOKINES
Cytokines are autologous messengers of cells.
There are cytokines that promote inflammation
as well as anti-inflammation.

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About 60 years ago, the first cytokine was discovered — the interferon alpha. Since then, the molecule family has
grown significantly: In addition to three interferons (alpha, beta and gamma), 35 different interleukins (IL) were
discovered; also two tumor necrosis factors (TNF), three different colony-stimulating factors (CSF) as well as the
transforming growth factor beta (TGF-ß).

It has become known only more recently that some cytokines — such as interleukin-1, for example — stimulate
nerve cells in the brain. This will increase the readiness level of the defense against infectious agents or pathogens:
The body’s temperature increases — its metabolism, different brain cell messenger substances and neurotransmit-
ters are affected. Interleukin-1 is also always active in case of an injury. It will then trigger a sort of emergency plan
to contain or control the damage.

Today, we know that each cytokine may have different effects — depending on which target cell it will dock on. If
one of these substances is used as a remedy in the entire body, it will actually have the desired effect in one area;
however, in other areas of the body, it might have undesirable, possibly even dangerous side effects.

It is only the proper combination of specific cytokines in the right place that will effect an adequate response of
the immune system. Researchers have lately only begun to uncover what this combination exactly looks like in the
different situations.

These results explain an already long known phenomenon: The two parts of the immune defense based on cells
or antibodies, respectively, have a fixed relationship to each other: The more antibodies are generated, the fewer
immune cells are activated — and vice-versa. This balance is especially provided by cytokines: IL-4 stimulates the
humoral defense and suppresses the cellular defense; and it is exactly the opposite with interferon gamma.

Whether an organism is able to cope well or not with a specific infection will thus considerably depend on the type
of immune cells in its body. Someday, it might be possible to predict on the basis of a person’s cytokine profile which
diseases that person will be immune to and which the person will be sensitive to.

Likewise, interleukins are still only at the dawn of any therapeutic use. A few years ago, there were hopes that IL-2
might reduce the number of metastases of a specific form of skin cancer. However, initial euphoria gave way to a
skeptic attitude. In Germany, the active substance was approved two years ago for treating the “metastasizing renal
cell carcinoma”. However, the success of this therapy is limited; side effects are frequent.

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The effect of positive autologous
cytokines
Important interleukins and their effect

IL-1Ra Blocks IL-1, anti-inflammatory, analgesic and neuroprotective

IL-4, -10, -11 Prevent excessive inflammatory reactions; important for homeostasis of the immune system

Important growth factors and their effect

PDGF (Platelet derived growth factor) regulates the migration of fibroblasts, osteoblasts and smooth
muscle cells

FGF (Fibroblast growth factor) stimulates vascularization, collagen synthesis and wound healing

TGF-ß (Transforming growth factor) reduces scarring, coordinates wound healing

VEGF (Vascular endothelial growth factor) increases the permeability of blood vessels and effects migra-
tion of various plasma proteins into the wound area

Catabolic Cytokines Anabolic Cytokines

e.g. IL-1, TNFα, e.g. growth factors
IL-17, IL-18 IGF-1, TGF-βs, BMPs,

Anti-Catabolic Modulators
Cell
Cytokines e.g. IL-6, IL-11
e.g. IL-1RA,
IL-4, IL-10, IL-13

Balance of cytokines and growth factors

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Interleukins - messengers of the immune
system
Interleukins are cytokines, i.e. they are endogenous messengers of cells of the immune system. They are used for
the communication among the immune defense cells (leukocytes) to thus combat pathogens or even tumor cells in
a coordinated fashion. The word interleukin has its roots in Latin — inter = between — and Greek — leukos = white.
Furthermore, certain interleukins — such as IL-1β and IL-6 — also have systemic effects, together with the tumor
necrosis factor.

Following the chronological order of their discovery, they are subdivided into several subgroups designated by
numbers (IL-1 to IL-35; as of November 2009).

The effect of interleukins is highly differentiated. While IL-2 is released by T-cells and has a positive effect on the
growth of these cells, IL-10 inhibits the activity of macrophages and contains the defense reaction. Thus, interleu-
kins specifically stimulate certain cells of the immune system to grow, mature and multiply by division, or they will
prevent precisely these processes of activation.

Scientists discovered a crucial triggering factor for arthrosis: the interleukin-1 protein (IL-1).

The scientists wanted to use the human body’s natural mechanisms in order to be able to intervene — in a more
causative approach then so far — in the pathology of arthrosis. Endogenous proteins were supposed to be used, for
the first time, for an efficient treatment of arthrosis.

Which body cells produce the “bad” proteins and which ones the “good” proteins?

Who or what provides the order for production? What exactly are they doing in our body; how do they act in the
pathological joint?

How can the “good proteins” prevent the destructive attacks of the “bad proteins”?

It was demonstrated that the “arthrosis-inhibiting proteins” are generated by specific blood cells. These are various
immune cells from the leukocyte family. Since the joint cartilage is not supplied with blood, the inhibiting agent
produced by the blood cells cannot get to the cartilage tissue via the blood system but would have to be locally
administered into the joint.

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The most important interleukins and
there significance
Interleukin-1

Interleukin-1 is produced by macrophages, endothelial cells, fibroblasts and some other cells and is a pro-inflamma-
tory signaling substance. Endothelial cells react to it by incorporating special receptors (e.g. e-selectin) into the cell
membrane. Now, a leukocyte in the blood will bind to this receptor and can thus migrate into the damaged tissue.
It thereby displaces endothelial cells and cuts its way through the basal lamina.

Displacement and destruction of cells and connective tissue is necessary to combat pathogenic agents and defec-
tive or degenerated cells outside the bloodstream. However, this reaction might get out of control in case of chronic
inflammations. Interleukin-1 docks to the chondrocytes and effects the release of cartilage-destroying enzymes,
thus resulting in the breakdown or decomposition of cartilage substance and, in the final analysis, in the destruction
of joints. Since about 1998, the orthopedic field has been using a therapy with an interleukin-1 antagonist obtained
from the body’s own blood and injected in an enriched form into a pathological joint. Studies conducted until now
suggest a therapy option for early forms of arthrosis.

Interleukin-1 receptor antagonist

Interleukin-1 had first been written about in 1940. It was quickly discovered that this protein can be detected in
all body cells and that antagonists exist. Subsequently followed the discovery of an autologous antagonist — the
interleukin-1 receptor antagonist (IL-1Ra for short).
While considering the biological effects, scientists detected its involvement in the etiology and chronic inflamma-
tions as well as the destruction of tissue.

Typical clinical disorders where the relationship between interleukin-1 and its antagonist plays a huge part are
arthrosis and rheumatoid arthritis — especially in terms of the destruction of cartilage.

Various investigations have shown that certain clinical disorders show an imbalance in favor of IL-1. Since both are
able to dock on the receptor, occupation by IL-1 and IL-1 Ra seems to be in a competitive environment significantly
influencing the pathological process. Thus, the development of a substantially increased number of antagonists is
required to successfully displace IL-1.

It has been known for a long time that interleukin-1 and its antagonist are to be found in specific cells of the human
blood, especially in monocytes.

IL-1 Ra intervenes in the inflammatory processes and in the breakdown processes that cause degenerative joint
disorders.

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Interleukin-4

Interleukin-4 acts (like IL-10 and IL-11) as a so-called anti-inflammatory cytokine such that it prevents excessive
inflammatory reactions and thus is important for the homeostasis of the immune system. Moreover, IL-4 stimulates
B-cell activation and the IgE production.

Interleukin-4 is a cytokine with anti-inflammatory properties which reduces, inter alia, the body’s own production
of Th1-cells and macrophages and of IFN-gamma and IL-12. It is thus important for the homeostasis of the immune
system. IL-4 differentiates naive CD4+ T cells (Th0) to Th2 cells which are generally attributed an anti-inflammatory
role. IL-4 stimulates activated B-cells and leads to an immunoglobulin class switch towards IgE and IgG4. The main
source of IL-4 are Th2 cells themselves.

Interleukin-10

Interleukin-10 acts (like IL-4 and IL-11) as a so-called anti-inflammatory cytokine such that it inhibits the macro-
phage function and thus prevents excessive inflammatory reactions. It is formed especially by TH2 cells and regu-
latory T cells.

It is especially secreted by monocytes and furthermore by TH2 lymphocytes. It has numerous functions in the regu-
lation of the immune system. It has a limiting and inhibiting function with regard to defense processes which can
lead e.g. to septic shock and thus protects the organism from destroying itself by excessive inflammatory processes.
Together with TGF-β, it is one of the most essential anti-inflammatory cytokines and important in the development
of immune tolerance. It inhibits the development of cytokines of the Th1 response and has a prolonging effect
on the survival, proliferation and antibody production of B-lymphocytes. It blocks the nuclear factor NFκB and
is involved in the regulation of the JAK-STAT signal pathway. Interleukin-10 is a crucial immune modulator in the
intestinal tract. Any deficiency is concomitant with a disposition to chronic-inflammatory intestinal diseases, such
as morbus Crohn or ulcerous colitis.

Interleukin-11

Interleukin-11 acts (like TGF-b and IL-10) as a so-called anti-inflammatory cytokine such that it prevents excessive
inflammatory reactions and thus is important for the homeostasis of the immune system.

Interleukin-13

Interleukin-13 is produced by T-lymphocytes and stimulates the production and differentiation of B-lymphocytes.
Moreover, IL-13 inhibits the activation of macrophages.

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Treatment with autologous cytokines

The systemic drug treatment of arthrosis is mainly based on the application of non-steroid antirheumatic drugs
(NSAR). However, these often have serious side effects which limit their application or may lead to therapy being
discontinued. This applies in particular to older persons who have more frequently negative interactions with other
drugs due to their higher rate of concomitant diseases. Intra-articularly applied glucocorticoids are mostly indicated
only in case of an “activated” arthrosis and to be used cautiously because of potentially joint-damaging effects. The
effect of other intra-articular preparations, such as glucosamine or hyaluronic acid, has not yet been conclusively
clarified.

Since the mid 1990s, a number of experimental and clinical studies have indicated a decisive role of interleukin-1
(IL-1) regarding the progression and manifestation of arthrosis in animals and humans. By means of an experimen-
tally induced arthrosis — through a cruciate ligament resection — the importance of the IL-1 receptor blockade for
the progression of arthrosis could be shown in the horse and in the dog. The intra-articular implantation of trans-
fected synovial cells producing the interleukin-1 receptor antagonist brought to a standstill the artificially induced
arthrosis which was highly progressive on the side of controls. In this respect, the IL-1 triggered release of metal-
loproteinases and collagenase 1 as well as the resistance of cartilage versus the insulin-like growth factor (IGF)
are directly responsible for the damage and degeneration of cartilage. Convincing results were also obtained with
humans in a study with genetic-therapy manipulated IL-1Ra producing synovial cells. However, a different route was
taken for practical applications since the risks of somatic gene therapy are currently not yet completely clarified.

Already in 1994, the American Prof. W. P. Arend had shown that the stimulation of monocytes with immune globulin
G leads to a highly increased IL-1Ra production of these cells. However, since the addition of heterologous IgG runs
counter to the autologous concept, other, physiologically inert stimuli were necessary to stimulate the production
of IL-1Ra. The production of autologous IL-1Ra conditioned serum is based on a stimulation of autologous blood
monocytes by specially pre-treated glass surfaces. After taking blood, the average natural concentration of IL-1Ra in
human blood is 100 pg/ml on average; by means of the monocyte induction, it increases in the mean to 2,800 pg/ml
after 24 hours. Test series with different concentrations of cycloheximide showed that, actually, a de novo-synthesis
of IL- 1Ra takes place in the monocytes and that no IL-1Ra possibly present already during blood taking is secreted
into the serum. Depending on the concentration, cycloheximide inhibits the ribosomal protein synthesis.

Pro-inflammatory substances — such as interleukin-1 (IL-1) and the tumor necrosis factor α (TNF-α) — are not
produced. In addition to IL-1Ra, the serum contains larger amounts of IGF and the platelet derived growth factor
(PDGF) which have a synergistic effect on IL-1Ra. The amount of IL-1Ra available in the serum ready for injection is
sufficient even for large joints to achieve a therapeutically effective surplus of IL-1Ra versus the IL-1 there existing
and to completely displace IL-1 from the receptor.

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Interleukin-1 (IL-1) — the “bad” protein

Interleukin-1 belongs to the cytokines released within the scope of the inflammatory reaction which is the central char-
acteristic of the RA pathogenesis.

Our body has very specific “good proteins” which are able to stop the destructive processes in the joint. If every
human has this protective mechanism, why do these proteins not prevent cartilage destruction in patients with
arthrosis as well? In the final analysis, all reactions in our bodies are based on biochemical processes permanently
pursuing an ulterior objective:

Keeping everything in the balance to ensure a certain order guaranteeing our health.

The onset of a disease signals that somewhere in the body something has gone so wrong in such a massive way
that the naturally intended state of balance for maintaining our health can no longer be maintained. Today, modern
technologies enable us to demonstrate which mechanisms are involved in the pathological causes and effects of
arthrosis. In the case of arthrosis, we are primarily dealing with a “natural, bad instigator” — interleukin-1 — which
causes damage to the cartilage, inflammations and pain.

But we have a “natural, good protein“, taking countermeasures and boycotting the negative effects of the malefac-
tor. For a better understanding, we call this “good interleukin-1 blocker” the “arthrosis inhibiting protein”. Moreover,
even more “good proteins” and growth factors are active in fighting arthrosis. In healthy persons, the “bad” as well
as the “good proteins” exist in the same number so that the natural state of equilibrium remains unchanged. On the
other hand, patients with arthrosis have an excess supply of destructive proteins and not enough “good arthrosis-in-
hibiting proteins”. The “damaging forces” have the upper hand in terms of numbers. Accordingly, the body suffering
from arthrosis is no longer able to protect itself and stop the destruction of cartilage.

Interleukin-1 receptor antagonist — the good protein

It was demonstrated that the “arthrosis-inhibiting proteins” are generated by specific blood cells. These are various
immune cells from the leukocyte family. Since the cartilage of the joint is not supplied with blood, the inhibiting
agent produced by the blood cells cannot get to the cartilage tissue via the blood system but would have to be
locally administered into the joint.

IL-1Ra is considered the naturally occurring antagonist within the cytokine family. IL-1Ra is expressed by almost
all cells which also produce IL-1. Both agonist and antagonist bind to the same receptor. However, IL-1Ra does not
trigger any signal transduction.

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There are 2 known receptors for interleukin-1 and IL-1Ra:

IL-1R type 1 and IL-1R type II,

while type II does not seem to have any proven physiological function. Concerning osteoarthritis, it is considered
established that there is an imbalance between the agonist and the antagonist. Since IL-1Ra also acts via the bond
on the interleukin-1 receptor, occupation of the receptor seems to play a crucial part in the course of pathogenesis. It
is a competitive bond where an excess of IL-1Ra must be available to adequately displace IL-1 and to thus antagonize
the biological effects of IL-1.

This is the starting point for novel therapy approaches. Since, in case of joint destruction, there is a relative inter-
leukin-1 excess in relation to the receptor antagonist, the efforts are aimed at supplying IL-1Ra from the outside and
to thus neutralize the effects of IL-1. Results from animal studies provide additional information for this approach.
Cartilage deterioration is induced by intra-articular injection of IL-1.

In contrast, interleukin-1 receptor antagonists (IL-1Ra) inhibit the loss in cartilage. Blocking IL-1 by IL-1Ra resulted
— in collagen-induced arthrosis — in the prevention of cartilage and bone destruction while the antagonization of
other pro-inflammatory cytokines — TNF-α, for example — was less able to influence cartilage destruction.

The role of the tumor necrosis factor alpha (TNF-α)

The tumor necrosis factor is a signaling substance (cytokine) of the immune system which is involved in inflamma-
tory processes. The tumor necrosis factor is part of a group of cytokines which all activate the acute phase proteins.
It is primarily released by macrophages and may trigger various processes including apoptosis, cell proliferation, cell
differentiation and the release of other cytokines.

The tumor necrosis factor is primarily released by macrophages but also, to a minor degree, by mast cells, lympho-
cytes, endothelial cells, myocardial cells, lipocytes, fibroblasts and nerve cells. It has different effects:

• On the hypothalamus:
• Distribution of corticotropin-releasing hormone (CRH)
• Appetite reduction
• Triggering fever
• On the liver:
• Acute phase proteins are increasingly released
• Insulin resistance is increased by phosphorylating insulin receptors
• Chemotaxis of neutrophilic granulocytes
• On macrophages: Stimulating phagocytosis
• On other tissue: Increases insulin resistance

An excessive concentration of TNF over a longer period of time may result in cachexia (emaciation). This may be
observed in some tumor patients. An overproduction of TNF is also found in some tumor patients which is why it is
also used as a so-called tumor marker in diagnostics.

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Since TNF triggers inflammatory processes, autoimmune reactions may clinically result which may be evident in the
form of rheumatoid arthritis (RA), spondylitis ankylosans (joint stiffening), morbus Crohn, psoriasis and bronchial
asthma. These diseases and syndromes may be therapied by means of an TNF α inhibitor. Either monoclonal anti-
bodies such as infliximab or golimumab are used or cytokine-binding substances, such as etanercept.

Anti-TNF shows similar effects as IL-1 in chondrocyte activation, including the stimulation of production of
matrix-destroying proteinases and the suppression of cartilage buildup. On a molecular level, IL-1 is between 100
and 1,000 times more potent than TNF-α. Yet, they have considerably potentiating effects. If TNF-α and IL-1 are
simultaneously injected into a joint, the result will be a heightened cartilage deterioration which far exceeds the
effect of one cytokine alone. The concept that TNF-α causes the acute inflammation while interleukin-1 has the key
role in maintaining the inflammation and cartilage erosion is derived in animal trials with rheumatoid arthritis. IL-1
and TNF-α stimulate the chondrocytes to synthesize prostaglandin E2 (PGE2) and nitric oxide radicals (NO) and a
number of other products and thus regulate the catabolic or anabolic activities of the chondrocytes. Additionally,
TNF-α and interleukin-1 can mutually stimulate their production in the sense of positive feedback regulation.

The key factors of IL-1 and TNF-α interact with other substances, such as growth factors, interferons, leukotrienes
or other interleukins within the framework of a complicated network. Despite the heterogeneity of the individual
substances, this system is in a delicate balance which finally has a major impact on the tissue structure; in terms of
feedback regulations, the functions of cartilage and synovial cells are thus controlled, among others.

The metabolism of joints is very low under physiological conditions. The interaction of cytokines and the increase in
catabolic and anabolic activities of the cells by means of IL-1 and TNF-α will result, in osteoarthritis, in an increased
metabolic rate of metabolic products, with the catabolic metabolism dominating in the final analysis.

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The cytokine network

IFN α
IFN β
TNF α

IL-2 IL-18
IL-12 IL-21
IL-15 TNF α
DENTRITIC
CELL
MACROPHAGE

IL-1 IL-12 IL-1

IL-6 IL-15 IL-8
IL-10 TNF α
TNF α
IL-12 IL-1
IL-15 IL-1 IL-15 IL-8
IL-1 TNF α
TNF α IL-6 IL-18 TNF α
IFN α TNF β
IL-8 IFN α
IFN β
IL-10 IFN γ
BASOPHIL
IL-12 TNF α
IL-10
IL-12 IL-1 IFN α TNF α
TNF α IL-3 IFN β
TNF β IL-5
B-CELL IL-4 IFN γ
IL-10 TNF α
IFN ∈ IL-13 TNF β
IL-1 IFN α
IL-6 IFN β
IL-1
IL-10 TNF α
IL-3
IL-1 IL-14 IL-12 TNF β
IL-4
IL-2 IL-18 EOSINOPHIL
IL-8
IL-4 IFN α NEUTROPHIL
IL-6 IFN β
TNF α
IL-10 IFN γ
IL-13

IL-4
IL-1 IFN γ IL-4
T-CELL
IL-3 TNF α
IL-2 IL-4 TNF β IL-4 IL-13
IL-8 IL-4
IL-2 IL-16
IL-4 IL-17
IL-6 IFN α
IL-8 IFN β
IL-9 IFN γ IL-3 IL-3
IL-9 IL-4
IL-4 IL-10 IL-5 IL-10 IL-12
NK CELL
IL-10 IL-4
IL-5
MONOCYTE
MAST CELL

IL-10
IL-6

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Summary of the cytokines

Cytokines play a key role in the pathogenesis of not only degenerative but also inflammatory joint diseases such as
osteoarthritis (OA) and rheumatoid arthritis (RA). A special role is attributed, in this respect, to interleukin-1 and the
TNF-α. Certain cytokines which inhibit the effect of catabolic cytokines have great therapeutic potential and are
currently the subject of detailed investigations in numerous clinical studies. According to the present state of scien-
tific knowledge, pro-inflammatory cytokines stimulate or enhance the deterioration of cartilage and their blockage
may protect the cartilage.

Findings from basic research regarding the etiology of arthrosis indicate that interleukin-1 (IL-1) has a key function
in activating arthrosis and in the degeneration of the cartilage substance. Antagonization of this key substance
might accordingly be used in the therapy of arthrosis. For some years now, an autologous serum conditioned in the
laboratory — with a high percentage of the IL-1 receptor antagonist (IL- 1Ra) — has been available for intra-articular
arthrosis therapy. In this form of therapy, the synthesis of endogenous IL-1Ra is stimulated in the patient’s blood
serum in the laboratory and the enriched serum is injected into the affected joint in up to five 5 doses.

Studies on the role of interleukin-1 (IL-1) and Interleukin-1

receptor antagonist (IL-1Ra) at cartilage damage

PRO O F I N OS T EOA RT H RITIS S T U DY BY

Proved in the synovial fluid in osteoarthritis Westacott et al.

Proved in the synovial cells in osteoarthritis Smith et al.

Proved in the chondrocytes in osteoarthritis Towle et al.; Melchiorri et al.;
Moos et al. ; Tetlow et al.
Interactions in osteoarthritis with MMP-1,3,8,13 und TNF-α Tetlow et al.

ANIMAL MODEL
Intra-articular injections Henderson u. Pettifer

Stimulates cartilage loss Page-Thomas et al.;

Van Beuningen et al.

Intra-articular IL-1Ra reduced the progression in Caron et al.; Fernandes et al.;

early stages of osteoarthritis in an animal model Frisbie et al.; Pelletier et al.

CLI N IC A L S T U DI E S
Recombinant IL-1Ra improves mood in Goupille et al.
patients with osteoarthritis

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G ROW T H FAC TO R S
Growth factors stimulate repair mechanisms and
healing processes.
Growth factors - autologous pertides
cotrolling manifold cell functions
Peptides are the subject matter of intensive biochemical research since many of them have regulatory functions
in the human body. This also includes the growth factors which control the development of young organisms and
which, in the later course of life, will initiate repair mechanisms and healing processes. It is thus not surprising that
they are of interest for cosmetics.

Peptides are widespread in nature and occur as natural components of our connective tissue, of nails and hair, also
as body messenger elements and hormones. There have been many attempts and ways to use their effects in skin
care, by having included horse‘s milk, protein-rich and antibody-rich colostrum, as well as other milk products as
ingredients into skin care products. Peptides from the thymus gland, the epiphysis, cartilage, from the liver, the
prostate gland, from heart and brain were synthetically reproduced and used in cosmetic creams – in the hope
of improving the metabolism of mature and atrophic skin, boosting the immune response and stopping the aging
of skin. All that was of limited success and essentially just went so far as to increasing skin moisture, superficially
smoothing the skin and producing antioxidant effects.

Later, synthetic peptides were found which stimulated collagen synthesis; they became known as matrikines. Other
oligopeptides, i.e. small peptides with a low mol mass, influence the synapses of the muscles of mimic wrinkles and
folds and can be used in the reduction of wrinkles.

Of major interest are body-own peptides which, in minutest quantities, have hormonal effects. They are called
growth factors. New ones are constantly being discovered and findings and knowledge about their functions are
growing exponentially.

Growth factors are formed, stimulated or distributed by different stimuli. Interactions with their corresponding
receptors trigger cascading signals in and on cells and selectively regulate the activities of genes up or down or
even deactivate them entirely. The following overview shows exemplarily some skin-relevant representatives – a
„snapshot“ so to speak, or a moment in time of an exciting topic.

Fibroblast Growth Factor (FGF)

FGF stimulates vascularization, collagen synthesis, wound contraction, matrix synthesis and epithelization. FGF is
not a single growth factor but rather an entire family meanwhile including more than 20 growth factors. The FGF
receptors end superficially on the outer cell membranes and, after FGF has docked on, they transmit signals into the
cell‘s interior. Receptors of that type are also called trans-membrane receptors; cells are caused to divide, differ-
entiate or even to migrate. FGF-1 is the most prominent representative of this family. It has its impact not only on
the proliferation of fibroblasts (connective tissue cells) which is responsible, inter alia, for collagen synthesis, but
also on endothelial cells lining the inner surface of blood vessels. This has a crucial effect on the new formation of
vessels – the angiogenesis. Wound healing or tissue repair is another vital aspect for the skin.

The Keratinocyte Growth Factor (KGF) is synonymous with FGF-7. This factor controls the epithelization in wound
healing where keratinocytes cover the wound and form the epithelium – in the case of the skin, the epidermis, a
squamous epithelium. Keratinocyte Growth Factor 2 is identical with FGF-10.

Transforming Growth Factor (TGF; Transformierender Wachstumsfaktor)

TGF belongs to the cytokine family. Cytokines also regulate cell functions and stimulate differentiation, growth and

— 17 —
immune reactions. TGF consists, in turn, of several subfamilies. For example, TGF-α is produced by tumor cells while
TGF-β is involved in wound healing.

• TGF-β (Transforming Growth Factor β)

• reduces scarring
• reverses wound healing disorders caused by corticoids
• attracts fibroblasts and promotes their proliferation
• stimulates collagen synthesis
• promotes the secretion of FGF and PDGF by monocytes

• TGF-α (Transforming Growth Factor α)

• stimulates mesenchymal, epithelial and endothelial cells

• TGF-α (Transforming growth factor-α)

• stimulates mesenchymal, epithelial and endothelial cells

Other growth factors of the cytokine type are the interleukins (IL). IL-1 removes damaged cells from connective
tissue by reinforcing inflammatory processes. This group as well is characterized by its high multifunctionality. IL-1α
stimulates collagen synthesis in the skin.

Insulin-like Growth Factor (IGF)

IGF-1 is one representative of this group which is tied into cancer development, cell proliferation and the inhibition
of apoptosis (programmed cell death).

— 18 —
Hepatocyte Growth Factor (HGF)
HGF enhances, inter alia, the synthesis of collagen by fibroblasts and accelerates cell division in wound healing.

Melanocyte Stimulating Hormone (MSH)

MSHs are signal molecules, controlling various biological processes. For example, the α-melanocyte stimulating
hormone (α-MSH) induces the development of pigments.

Platelet Derived Growth Factor (PDGF)

PDGF is subdivided into several factors which are released by blood platelets, intervening in wound healing and the
cell division of fibroblasts. They are found especially in the embryonic stage.

• Stimulates collagen synthesis, vascularization and fibroblast division

• Activates macrophages and neutrophils
• Activates beta-TGF
• Attracts stem cells

Granulocyte Macrophage Colony-Stimulating Factor (GM-CSF)

Like the factors G-CSF and M-CSF which are also part of this group, GM-CSF stimulates the development of neutro-
philic granulocytes and macrophages which are both counted among the phagocytes (»killer cells« or scavenger
cells).

Definition of growth factors

Cell biology designates growth factors as proteins which are transmitted as signals from one cell to a second one,
thus transmitting information. They also regulate the most diverse intracellular processes in the sense of »signal
proteins« and play a part especially in the development of multicellular organisms. As a rule, signal transmission is
effected by binding the growth factor to a specific receptor in the cell membrane.

Growth factors – GF for short – are proteins having an impact on cell proliferation and/or the differentiation of
precursor cells (progenitor cells) or stem cells to somatic cells.

— 19 —
Families of growth factors

Six large families of growth factors exist:

• The FGF family (Fibroblast Growth Factor)

• The TGF family (Transforming Growth Factor)
• Hedgehog
• Wingless
• Delta and Serrate
• Ephrine

Growth factors are either secreted – i.e. given off by cells to their environment – or they are membrane-stable.
They act or are effective in that they are detected by a receptor on the surface of the target cell. Only cells bearing
the specific receptor for the respective growth factor (the ligand) can react to the signal. Upon binding to its ligand,
this receptor generates – by conformation change in the cell interior – a signal which results, through further signal
transmissions, in the activation or deactivation of genes. One typical example of the mode of action of growth
factors is the angiogenesis.

Growth factors with different signal inductions

Numerous growth factors with different signal inductions are known today. Examples are as follows:

• Fibroblast Growth Factor (FGF)

• Transforming Growth Factor (TGF)
• Platelet Derived Growth Factor (PDGF)
• Epidermal Growth Factor (EGF)
• Granulocyte-Macrophage Colony Stimulating Factor (GMCSF)
• Vascular Endothelial Growth Factor (VEGF)
• Insulin-like Growth Factors (IGF)
• Hepatocyte Growth Factor (HGF)
• Interleukin-1 beta, -8 (IL-1 beta, IL-8)
• Nerve Growth Factor (NGF)
• Hematopoetic Growth Factors: Erythropoietin and Colony Stimulating Factors, such as G-CSF

— 20 —
— 21 —
 COL AGEN
Collagen stores humidity and promotes
the regeneration of skin cells, it supports
the connective tissue and provides
structure and stability to it.
Collagen - main componenet of the skin
Collagen is the most common protein in the human body and one of the skin’s major components. It is newly
produced every day; it stores humidity and supports the regeneration of skin cells. As long as it functions properly
and well, it supports the connective tissue, giving it structure and stability. Collagen thereby keeps our skin young,
smooth and firm. It consists of rope-like structures, compacting to fibers of high rigidity, thus ensuring elasticity
and suppleness – indispensable for the skin’s vitality. The protein has outstanding tensile strength and minimum
stretch or elongation. Regrettably the body’s own production decreases with advancing age, thus forming fine lines
and wrinkles. Collagen-based cosmetics should counter this, but they only have an effect on the outer layer of skin,
the epidermis.

The skin‘s elasticity begins to decrease already at the age of 25. At the latest as of 30 years of age, smooth, wrin-
kle-free skin is no longer a matter of course. However, whether our skin begins to wrinkles and to what extent will
also depend on other factors – not only on one‘s age. In addition to the skin’s humidity level and the condition of
its surface, the skin’s elasticity is the main factor determining smoothness and tautness of our skin.

The collagen fibers in our skin act like rubber bands and as a whole provide a sort of support corset for the cells.
When we laugh or frown, the supporting fibers made of protein elements ensure that our skin returns to its initial
state after movement of our muscles. Additionally, they can store water, thus ensuring that our skin looks full and
smooth – at least in younger years. Because in our mid-twenties already, our body reduces the production of colla-
gen, with a simultaneous increase in the degradation of elastic fibers – especially due to environmental effects.
Beginning with the menopause, the altered hormonal balance additionally increases the degradation process. And
the result: The skin is getting thinner and more limp or flabbier. This change is especially visible in wrinkles and
facial contours, with ever lower resistance to gravity.

1 Definition

The family of collagens is a heterogeneous group of proteins, making up about one quarter of total protein volume
in the human body. Collagen is the most important fibrous component of skin, bones, tendons, cartilage, blood
vessels and teeth. So far, 25 collagen polypeptides have been described, forming over 28 different types of collagen
in the extracellular matrix.

— 23 —
2 Structure

Collagen molecules have a high amount of proline and glycine, in addition to a number of hydroxylated amino acids
such as hydroxyproline and hydroxylysine, which allow for cross-linking of proteins and the formation of a stable
collagen matrix. Collagen fibers consist of three alpha-helical protein chains, with respectively three of these mole-
cules intertwining in the form of a super helix or a triple helix – thus forming fibers. Depending on its specific type,
each collagen helix can consist of between several hundred up to several thousands of amino acids.

3 Physiology

In the organism, collagen is mainly found in connective tissue where – depending on its condition – it can make up
the bulk of the extracellular matrix. The different properties of the various classes of collagen are caused by the
numerous characteristics of connective tissue and supporting tissue. The tensile strength of ligaments and tendons,
the flexibility of bones or the pressure resistance of joint cartilage is due, to a large extent, to the collagen preva-
lent in the tissue.

4 Pathophysiology

The synthesis of collagen depends on ascorbic acid. Vitamin C deficiency results in scurvy symptoms. A multitude
of genetic disorders is linked to defects of collagen or associated proteins (for example Ehlers-Danlos syndrome,
osteogenesis imperfecta, Stickler syndrome, Alport syndrome).

— 24 —
5 Classification

Numerous types of collagen have been discovered so far; yet, their function is partly still unclear.

• Collagen I (fiber-forming in collagen fibers): skin, tendons, bones, dentin, fibrous cartilage, cornea
• Collagen II (fiber-forming): hyaline cartilage, fibrous cartilage, elastic cartilage, vitreous body
• Collagen III (fiber-forming in reticuline fibers): skin, skeletal muscles, blood vessels
• Collagen IV (reticular): basement membrane (in particular, lamina densa)
• Collagen V (fiber-forming): fetal tissue, placenta, interstitial connective tissue
• Collagen VI (fiber-associated, spherical): connective tissue
• Collagen VII (anchoring fibrils): anchoring epithelia on basement membrane and stroma
• Collagen VIII (reticular): Descemet’s membrane
• Collagen IX (fiber-associated): cartilage, vitreous body
• Collagen X (reticular): cartilage area of growth
• Collagen XI (fiber-forming): cartilage
• Collagen XII (fiber-associated): embryonic skin, embryonic tendons
• Collagen XIII: bones, cartilage, skin, striated muscles
• Collagen XIV: in particular, in the nerves’ intraneural connective tissue, in epimysium and perimysium, as well as
in the interior of muscles following a denervation
• Collagen XV: Basement membrane of muscles, in kidneys following fibrosis
• Collagen XVI: in internal organs, the eyes and to some extent in muscle tissue
• Collagen XVII (trans-membranous): associated with hemidesmosomes of squamous epithelial cells of the
epidermis
• Collagen XVIII: unknown
• Collagen XIX: (fiber-associated): fetal skin, fetal tendons

— 25 —
Detection of anti-inflammatory cytokines
with different incubation time

native blood 0 hours 1 hours 3 hours

* The blood was transferred to the medical

device and immediately centrifuged.

— 26 —
Determination of the concentration of the
cytokines il-1α, il-1β, il-6, tnf-α and il-1ra
after various incubation times
Aim

The detected ingredients are intended to underline the efficacy of the autologous serum, the quantity of which is
multiplied by the Sanakin® technology. The attending physician is therefore in a position to effectively treat his or
her patients for osteoarthritis and inflammation with serum produced from their own body.

The aim is to prove that after 1–3 hours of incubation, there is an increased number of anti-inflammatory cytokines
present in the serum, while at the same time the number of inflammatory cytokines is not excessive.

Methodology

Withdrawal of 40 ml of native blood from each of 4 patients.

Native blood without incubation ­­­— not in the medical product, native blood 0 hours and after incubation lasting 1
hour and 3 hours each in the medical product.

Result

It was possible to show that the serum produced after 1–3 hours of incubation does not contain a high concentration
of inflammatory cytokines. The quantity of anti-inflammatory cytokine IL-1Ra was increased 3-fold after 1 hour and
4.5-fold after 3 hours.

Graphical Analysis and Explanation

• The interleukin-1 receptor antagonist (IL-1Ra) occurs in the body as a natural inhibitor of interleukin-1 (IL-1) and
plays an essential role in the regulation of inflammatory processes mediated by interleukin-1 (IL-1).

• Its action is based on the fact that it slows down and stops the effect of interleukin-1 (IL-1) by docking on to the
interleukin receptor of the target cell instead of interleukin-1 (IL-1) and thereby preventing the docking of inter-
leukin-1 (IL-1).

— 27 —
 • The polymorphisms in pro-inflammatory IL-1 α
induce increased synthesis. Their induced presence
results in an inflammatory response.
• In the graph it can be seen that the pro-inflam-
matory IL-1α does not increase for any incubation
times (1 and 3 hours). This means that no pro-in-
flammatory IL-1 α is produced by the incubation.

native blood 0 hours 1 hours 3 hours

• IL-1β is a highly effective cytokine which belongs to

the inflammation mediators.
• IL-1β acts like Interleukin-1α.
• IL-1β triggers the release of interleukin-6.
• This graph also shows that the pro-inflammatory
interleukin does not increase during both incuba-
tion periods.

native blood 0 hours 1 hours 3 hours

• IL-6 is one of the interleukins which regulate the

inflammatory response of the organism.
• The concentration of IL-6 in the plasma of healthy
individuals is approximately 1 pg/ml.
• The graph clearly illustrates that IL-6 ranges
between the value of 1.5 and 2 pg/ml. The interleu-
kin can therefore be considered non-critical.

native blood 0 hours 1 hours 3 hours

• TNF-α is a multifunctional cytokine of the immune

system which is involved in local and systemic
inflammation.
• In the joints of patients with chronic polyarthritis,
TNF-α plays a significant role in the process of
inflammatory joint destruction.
• The standard value for TNF-α is between 0 and 25
pg/ml, so that the measured values are within the
native blood 0 hours 1 hours 3 hours normal range (see graph).

* The blood was transferred to the medical device

and immediately centrifuged.

— 28 —
Observational study of 100 patients
from November 2011 to October 2014

Cases analysed

43 men, 59.28 years ∅ 5 injections (1 x / week)

57 women, 60.81 years ∅ ∅ Degree of osteoarthritis III (Kellgren)
∅ 60.15 years 3 h incubation

Initial pain Final Pain Positive change

∅ 69,86 VAS prae. ∅ 17,11 VAS post. ∅ 75,51 %

20 Shoulders
75,75 %

17 Spines
74,43 %
13 Thumbs
82,60 %

45 Knees
73,99 %
3 Achillodynia
80,55 %
2 Large toe
joints
66,67 %

— 29 —
 ANNEX
Scientific papers — further reading
Scientific papers
1. Dinarello CA: Interleukin-1. Rev Infect Dis 1984; 6:51–95.
2. Arend WP, Joslin FG, Massoni RJ: Effects of immune complexes on production by human monocytes of
interleukin 1 or an interleukin 1 inhibitor. J. Immunol 1985; 134:3868–75.
3. Dinarello CA, Thompson RC: Blocking IL-1: interleukin 1 receptor antagonist in vivo and in vitro. Immunol
Today 1991;12: 404–10.
4. Arend WP, Smith MFJ, Janson RW, Joslin FG: IL-1 receptor antagonist and IL-1 beta production in human
monocytes are regulated differently. J. Immunol 1991; 147: 1530–6.
5. Arend WP, Leung DY: IgG induction of IL-1 receptor antagonist production by human monocytes. Immunol
Rev 139 (1994) 71–78.
6. Caron JP, Fernandes JC, Martel-Pelletier J, Tardif G, Mineau F, Geng C et al: Chondroprotective effect of
intraarticular injections of interleukin-1 receptor antagonist in experimental osteoarthritis. Suppression of
collagenase-1 expression. Arthritis Rheum 39(1996)1535–1544.
7. Evans CH, Ghivizzani SC, Herndon JH, Wasko MC, Reinecke J, Wehling P et al: Clinical trials in the gene
therapy of arthritis. Clin Orthop 379 (2000) Suppl, S300–307.
8. Frisbie DD, Ghivizzani SC, Robbins PD, Evans CH, Mcliwraith: Treatment of experimental equine
osteoarthritis by in vivo delivery of the equine interleukin-1 receptor antagonist gene. Gene therapy 9
(2002)12–20
9. Goldring MB: Osteoarthritis and cartilage: the role of cytokines. Curr Rheumatol Rep 2 (2000) 459–465.
10. Goldring MB: Anticytokine therapy for osteoarthritis. Expert Opin Biol Ther 1(2001) 817–829.
11. Heilmann HH, Lindenhayn K, Walther HU: Synovial volume of healthy and arthrotic human kneejoints. Z
Orthop Ihre Grenzgeb 134 (1996) 144–148.
12. Moss SG, Schweitzer ME, Jacobson JA, Brossmann J, Lombardi JV, Dellose SM et al: Hip joint fluid:
detection and distribution at MR imaging and US with cadaveric correlation. Radiology 208 (1998) 43–48.
13. Neidel J, Schulze M, Sova L, Lindschau J: Practical significance of cytokine determination in joint fluid in
patients with arthroses or rheumatoid arthritis. Z Orthop Ihre Grenzgeb 134 (1996) 381–385.
14. Pelletier JP, Caron JP, Evans CH, Robbins PD, Georgescu HI, Jovanovic D et al: In vivo suppression of early
experimental osteoarthritis by interleukin1 receptor antagonist using gene therapy. Arthritis Rheum 40
(1997) 1012–1019.
15. Firestein GS, Alvaro-Gracia JM, Maki R.: Quantitative analysis of cytokine gene expression in rheumatoid
arthritis. Published erratum appears in J. Immunol 1990 Aug. 1; 145(3): 10371.
J. Immunol 1990; 144: 3347–53.
16. Granowitz EV, Clark BD, Mancilla J, Dinarello CA: Interleukin-1 receptor antagonist competitively inhibits
the binding of interleukin-1 to the type II interleukin-1 receptor. J. Biol Chem 1991; 266: 14147–50
17. Arend WP et al.: Interleukin-1 receptor antagonist: role in biology. Annu Rev Immunol 1998;
16: 27–55
18. Gabay C, Arend WP (1998): Treatment of rheumatoid arthritis with IL-1 inhibitors. Semin Immunopathol
20(1-2): 229–246
19. Van den Berg WB et al. (1991): In vivo evidence for a key< role of IL-1 in cartilage destruction in
experimental arthritis. Agents Actions 32 (suppl): 159–163
20. Towle CA et al. (1997): Detection of interleukin-1 in the cartilage of patients with osteoarthritis: a possible
autocrine/paracrine role in pathogenesis. Osteoarthr Cartil 5(5): 293–300

— 31 —
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