**2. The place and importance of -MSH and met-enkephalin in immunomodulation**

Immunomodulatory effects of a combination of -MSH and met-enkephalin involve several mechanisms that support the modern trend in the development of new anti-inflammatory drugs. The method of selective immunomodulation using peptides has only recently been used in immune-mediated diseases and malignant neoplasm. Immunomodulation with antigens and peptides is directed towards achieving a satisfactory long-term remission without the occurrence of toxic side effects typical for immunosuppressive drugs. It is based on two approaches: the first approach is to block the initial activation of antigen recognition of autoreactive T cells, while the second is based on downregulation of specific antigenic

that it is a component of natural immunity (Catania et al, 2000a). The supposed connection between alpha-MSH and immune processes is confirmed by the clinical data on changes of its plasma level under certain pathological conditions (Lipton and Catania, 1997; Lipton et al, 2000). An increased concentration of -MSH has been recorded in patients with an acute myocardial infarction who received thrombolytic therapy, probably as an endogenous antiinflammatory response. This peptide has been identified in the synovial fluid of patients with rheumatoid arthritis (Star et al, 1995) and it is believed that the inflammation and joint destruction in this disease occur due to imbalances of production of proinflammatory and anti-inflammatory cytokines such as -MSH. A higher concentration of -MSH has been found in patients suffering from arthropathy accompanied by heavy inflammation (Lipton et al, 2000). Since -MSH is a natural modulator peptide, its concentration is subject to intensive control to avoid excessive inhibition of the immune response. It is assumed that the increased concentration of peptides leads to downregulation of receptors in cells, which could explain

The other component contained in the drug is met-enkephalin, which belongs to a group of endogenous opioid peptides, widely distributed in the CNS. All opioid peptides contain one or more copies of the simplest opioid peptides: methionine-enkefalin (tyrosine-glycineglycine-phenylalanine-methionine) and leucine-enkephalin (tyrosine-glycine-glycinephenylalanine-leucine) (Janković, 1991). They participate in, and modulate neurotransmission. Opioid peptides also act as growth factors that control proliferation and differentiation of cells and may participate in the process of wound healing, tissue regeneration and immune response. Enkephalins show antidepressant, antianxiety and anticonvulsive effects. They are produced in the adrenal and the hypothalamus. Metenkephalin belongs to a group of enkephalins that are involved in transmitting signals in the nervous system, intestines, endocrine glands, bronchial tree, lymphatic and other tissues (Štambuk et al, 1996). The highest concentrations are present in the core of adrenal,

the biphasic curve of the anti-inflammatory effects -MSH (Catania et al, 2000).

hypothalamus, autonomic ganglia and the gastrointestinal tract (Janković, 1991).

**2. The place and importance of -MSH and met-enkephalin in** 

Todić, 2008).

**immunomodulation** 

Met-enkephalin as an inhibitory peptide modulates the proliferation and migration of cells, as well as the organization of tissues during development, homeostatic cell renewal, wound healing, angiogenesis, and in malignant neoplasms (Zagon et al, 2000). Physiologically, enkephalins have a signalling function in the nervous system, intestine, endocrine glands, bronchial tree, cardiovascular system, lymphatic and other tissues, and participate in the regulation of haematopoiesis (Štambuk et al, 1996). It is assumed that they participate in maintaining homeostasis of the organism exposed to stress (Mulabegović et Rakanović-

Immunomodulatory effects of a combination of -MSH and met-enkephalin involve several mechanisms that support the modern trend in the development of new anti-inflammatory drugs. The method of selective immunomodulation using peptides has only recently been used in immune-mediated diseases and malignant neoplasm. Immunomodulation with antigens and peptides is directed towards achieving a satisfactory long-term remission without the occurrence of toxic side effects typical for immunosuppressive drugs. It is based on two approaches: the first approach is to block the initial activation of antigen recognition of autoreactive T cells, while the second is based on downregulation of specific antigenic inflammatory response of T cells through the activation of regulatory physiological mechanisms (Štambuk et al, 1997).

Until recently it was thought that the immune/inflammatory reaction develops exclusively in the periphery. Today it is known that the nervous, endocrine and immune systems are interconnected, and that they communicate by signals that are transmitted through neuropeptides. In this way, central neurogenic influences can either enhance or modulate the peripheral response, so by analogy the treatment of inflammatory reactions could be improved by the influence of the proinflammatory signals in the central nervous system. Thus, Catania et al. (1999) suggest that one of these strategies could be based on the action of -MSH.

In the past several years, particular attention has been given to the anti-inflammatory effects of this peptide, as a result of the introduction of -MSH and related peptides in clinical practice for the treatment of inflammatory diseases (Getting, 2006). Undoubtedly, -MSH, administered systemically or locally, expresses strong anti-inflammatory effects. Antiinflammatory effects of this peptide are mediated by direct effects on cells of the immune system, as well as by indirect effects that are achieved by changing the function of nonresident immune cells of peripheral tissues (Luger and Brzoska, 2007).

Three basic mechanisms of -MSH anti-inflammatory action can be distinguished: direct effects through melanocortin receptors on cells in the periphery (monocytes/macrophages and neutrophils); effects on glial cells; and descendent anti-inflammatory effects through melanocortin receptors on neurons (Lipton et al, 2000). These mechanisms of action correspond to the concept of neuroimmunomodulation in which nerves, endocrine and immune systems are independent networks that mutually communicate through soluble mediators such as cytokines and neuropeptides.

Multiple anti-inflammatory effects that -MSH has have been identified in various *in vitro* systems of cell cultures. Studies of anti-inflammatory effects of -MSH on cellular level were primarily concentrated on the issue whether and to what degree -MSH suppresses the production of pro-inflammatory cytokines. In this respect, several studies have shown that treatment with -MSH results in significant downregulation of pro-inflammatory cytokines such as IL-1, IL-2, IL-4, IL-6, IL-13 and TNF-, as well as chemokines such as IL8, Gro and interferon (IFN) (Brzoska et al, 2008). Furthermore, chemotaxis induced by IL8 in the cells of human neutrophils and monocytes has been suppressed under the influence of -MSH (Manna et al, 2006), which indicates that the function of these types of phagocytic cells during the inflammatory response is blocked by the peptide via multiple effector pathways.

Contrary to the inhibitory effects of -MSH on the production and activity of proinflammatory mediators, it has been found that -MSH induces IL-10, a cytokine with a potent anti-inflammatory activity. Specifically, in monocytes of human peripheral blood and cultivated human monocytes, -MSH increases the production and expression of IL-10 (Bhardway et al, 1996). Since IL-10 reduces the production of pro-inflammatory cytokines in macrophages, its upregulation could result in anti-inflammatory influences.

Studies on different cell types of human skin, including pigment cells, fibroblastic cells and dermal microvascular endothelial cells, as well as rat mast cells, showed that -MSH is able to suppress the expression of several intracellular adhesion molecules (interstitial adhesion molecule-1 - ICAM-1 and P-selectin) induced by proinflammatory stimuli such as IFN-, LPS and TNF-Böhm et al, 2005; Hill et al, 2006; Sarkar et al, 2003). Finally, the inhibition of adhesion and transmigration of inflammatory cells may contribute to the anti-

Enkorten – A Potential Drug for the Treatment of Rheumatoid Arthritis 159

therefore its inhibition results in the alteration of mediators' production and cell functions. NF-B participates in the regulation of hundreds of genes, including those for cytokines, chemokines, factors for haematopoietic system growth, antiapoptotic factors and the inducible synthesis of nitric oxide (iNOS). Therefore, the discovery that -MSH inhibits activation of NF-B provides an explanation for numerous effects of this peptide on the

Anti-inflammatory effects of -MSH *in vitro* have also been identified *in vivo* in various animal models. These have confirmed a number of effects of melanocortin on the production of mediators of inflammation. The anti-inflammatory activity of -MSH in animal models of arthritis is of special interest. Repeated intraperitoneal application of the peptide twice daily resulted in a significant reduction of clinical and histological signs of adjuvant-induced experimental arthritis as compared to control animals. The efficiency of -MSH was similar to that of prednisolone, but -MSH did not cause significant weight loss (Ceriani et al, 1994). Some data indicate that the melanocortin receptor MC-3R may be a relevant target for the

During the early phase of inflammation (associated with tissue destruction and activation of nociceptors) mediators of inflammation, including cytokines, chemokines, bradykinin, prostaglandins act as mediators of hyperalgesia. At the same time, the expression of peripheral opioid receptors on sensory nerve endings increases during inflammation (Rittner et al, 2003), which leads to the release of pain mediators, such as opioid peptides, somatostatin, endocannabinoids and certain cytokines. Endogenous opioid peptides, leucine and met-enkephalin act presynaptically when they inhibit the release of neurotransmitters, and postsynpatically when they suppress the activity of the postsynaptic neurone, and block

In addition to the central nervous system, met-enkephalin influences the function of the immune system (Vujić-Redžić, 2000). It is released from polymorph nuclear cells during inflammation and periods of stress (Rittner et al, 2006). In inflammation, there is a migration of leukocytes that contain opioid peptides, and also the upregulation of opioid receptors of afferent fibres. This is supported by the fact that IL-1 is a specific inductor of expression of kappa opioid receptors (Puehler et al, 2006). Interleukin-1, together with TNF-α, is the main proinflammatory cytokine released from macrophages and many other cells, which initiates

In *in vitro* studies, it was found that T lymphocytes have opioid receptors on their surface (Wybran et al, 1985), and according to Plotnikoff and associates (1991), the effects of enkephalin are precisely related to the immune response mediated by T cells. In addition, the corticotropin-releasing hormone, similarly to the pituitary gland, appears dosedependent locally, through delta opioid receptors, and leads to the release of opioid peptides from leukocytes and the induction of endogenous analgesia (Menzebach et al, 2003;

Met-enkephalin manifests central, bimodal and dose dependent immunomodulatory effects. A bi-phasic effect was registered in *in vivo* studies on rats. High doses have suppressed, while low doses potentiated a humoral immune response (Janković and Marić, 1994). The studies *in vivo* of the immunomodulatory effect of met-enkephaline on rats showed a suppressive action of high doses of met-enkaphalin on inflammatory reactions, such as the systemic anaphylactic shock (Janković and Marić, 1987), Arthus phenomenon and postponed skin reactions to protein antigens (Marić and Janković, 1987), the rejection of

production of mediators.

treatment of arthritis.

Cabot et al, 2001).

the transmission of ascending pain signals (Rang, 2005).

the release of other cytokines (Rajora et al, 1997).

inflammatory potential of -MSH (Böhm et al, 2005; Scholzen et al, 2003). Moreover, it was found that -MSH inhibits the maturation of dendritic cells and downregulates the expression of co-stimulatory molecules on antigen-presenting dendritic cells, such as CD86, CD40 and CD54 (Luger, 2003; Capsoni et al, 2007).

One of the mechanisms that could explain the anti-inflammatory action of -MSH is the suppression of pro-inflammatory non-cytokine mediators under the influence of this peptide. The inhibitory effect of -MSH on the synthesis of prostaglandin has long been known. This peptide suppressed the synthesis of PGE in the fibroblast of foetal human lung stimulated with IL-1 (Cannon et al, 1986). The effect of -MSH on the synthesis of PGE is cell specific. Confirmation of this is the fact that under the influence of -MSH, TNFinduced production of PGE2 in FM55 melanoma cells was blocked, but not in HaCaT keratinocytes (Nicolaou et al, 2004).

The induction of the inducible form of the enzyme nitric oxide synthase (iNOS) and the release of gaseous vasodilator nitric oxide (NO) after the stimulation of cells with various pro-inflammatory stressors, i.e. LPS, -IFN and -amiloid, can also be suppressed under the influence of -MSH (Tsatmali et al, 2000; Gupta et al, 2000; Caruso et al, 2007; Jung et al, 2007). The increased expression of NOS, and nitration of cellular proteins in the blood and synovial fluid of a patient with rheumatoid arthritis has been registered. Immunomodulatory effects of -MSH realized through NO may be more important in rodent than in human cells, since human monocytes are known to contain only marginal amounts of this gaseous mediator.

Recent studies have shown that -MSH inhibits the production of superoxide radicals in neutrophils of rats treated with LPS or forbolester (Oktar et al, 2004). Similarly, -MSH reduced the amount of oxidative burst in HL-60 cells, a cell line of human monocytes (Manna et al, 2006). Although there is no evidence to suggest that -MSH itself is a real radical quencher, these findings are in accordance with other reports that indicate an effect of this peptide on cellular redox balance and the process of apoptosis as well.

Regarding the release of histamine by mast cells, various effects of -MSH have been described which are likely related to species-specific differences, the type of mast cells and experimental conditions. In mast cells obtained from rat bone marrow, -MSH inhibited the antigen-induced histamine release together with the suppression of other pro-inflammatory cytokines (Adachi et al, 1999).

There are only a few studies that have researched the effect of -MSH on the function of lymphocytes, probably due to the fact that the total expression of melanocortin receptors in several lymphocyte lines is low or undetectable (Andersen et al, 2005). It is known that - MSH induces subpopulation of regulatory T lymphocytes. Regulatory T cells induced with -MSH are characterized by the expression of CD25, CD4 and CTLA4 and production of increased levels of TGF-2 (Namba et al, 2002). In the human system, -MSH also expresses modulator effects on T cells. Cooper and associates (2005) found that -MSH suppresses the proliferation of human T lymphocytes stimulated by streptokinase/streptodornase. This inhibitory effect of -MSH was independent of the genotype MC-1R, which is highly polymorphous with more than 35 genetic variations.

Numerous effects of -MSH on the production of inflammation mediators were a mystery to researchers until it was found that this peptide inhibits the activation of the nuclear factor kappa B (NF-B) (Mandrika et al, 2001; Hassoun et al, 2002). This essential nuclear factor induces the transcription of many molecules involved in the inflammatory process, and

inflammatory potential of -MSH (Böhm et al, 2005; Scholzen et al, 2003). Moreover, it was found that -MSH inhibits the maturation of dendritic cells and downregulates the expression of co-stimulatory molecules on antigen-presenting dendritic cells, such as CD86,

One of the mechanisms that could explain the anti-inflammatory action of -MSH is the suppression of pro-inflammatory non-cytokine mediators under the influence of this peptide. The inhibitory effect of -MSH on the synthesis of prostaglandin has long been known. This peptide suppressed the synthesis of PGE in the fibroblast of foetal human lung stimulated with IL-1 (Cannon et al, 1986). The effect of -MSH on the synthesis of PGE is cell specific. Confirmation of this is the fact that under the influence of -MSH, TNFinduced production of PGE2 in FM55 melanoma cells was blocked, but not in HaCaT

The induction of the inducible form of the enzyme nitric oxide synthase (iNOS) and the release of gaseous vasodilator nitric oxide (NO) after the stimulation of cells with various pro-inflammatory stressors, i.e. LPS, -IFN and -amiloid, can also be suppressed under the influence of -MSH (Tsatmali et al, 2000; Gupta et al, 2000; Caruso et al, 2007; Jung et al, 2007). The increased expression of NOS, and nitration of cellular proteins in the blood and synovial fluid of a patient with rheumatoid arthritis has been registered. Immunomodulatory effects of -MSH realized through NO may be more important in rodent than in human cells, since human monocytes are known to contain only marginal

Recent studies have shown that -MSH inhibits the production of superoxide radicals in neutrophils of rats treated with LPS or forbolester (Oktar et al, 2004). Similarly, -MSH reduced the amount of oxidative burst in HL-60 cells, a cell line of human monocytes (Manna et al, 2006). Although there is no evidence to suggest that -MSH itself is a real radical quencher, these findings are in accordance with other reports that indicate an effect

Regarding the release of histamine by mast cells, various effects of -MSH have been described which are likely related to species-specific differences, the type of mast cells and experimental conditions. In mast cells obtained from rat bone marrow, -MSH inhibited the antigen-induced histamine release together with the suppression of other pro-inflammatory

There are only a few studies that have researched the effect of -MSH on the function of lymphocytes, probably due to the fact that the total expression of melanocortin receptors in several lymphocyte lines is low or undetectable (Andersen et al, 2005). It is known that - MSH induces subpopulation of regulatory T lymphocytes. Regulatory T cells induced with -MSH are characterized by the expression of CD25, CD4 and CTLA4 and production of increased levels of TGF-2 (Namba et al, 2002). In the human system, -MSH also expresses modulator effects on T cells. Cooper and associates (2005) found that -MSH suppresses the proliferation of human T lymphocytes stimulated by streptokinase/streptodornase. This inhibitory effect of -MSH was independent of the genotype MC-1R, which is highly

Numerous effects of -MSH on the production of inflammation mediators were a mystery to researchers until it was found that this peptide inhibits the activation of the nuclear factor kappa B (NF-B) (Mandrika et al, 2001; Hassoun et al, 2002). This essential nuclear factor induces the transcription of many molecules involved in the inflammatory process, and

of this peptide on cellular redox balance and the process of apoptosis as well.

CD40 and CD54 (Luger, 2003; Capsoni et al, 2007).

keratinocytes (Nicolaou et al, 2004).

amounts of this gaseous mediator.

cytokines (Adachi et al, 1999).

polymorphous with more than 35 genetic variations.

therefore its inhibition results in the alteration of mediators' production and cell functions. NF-B participates in the regulation of hundreds of genes, including those for cytokines, chemokines, factors for haematopoietic system growth, antiapoptotic factors and the inducible synthesis of nitric oxide (iNOS). Therefore, the discovery that -MSH inhibits activation of NF-B provides an explanation for numerous effects of this peptide on the production of mediators.

Anti-inflammatory effects of -MSH *in vitro* have also been identified *in vivo* in various animal models. These have confirmed a number of effects of melanocortin on the production of mediators of inflammation. The anti-inflammatory activity of -MSH in animal models of arthritis is of special interest. Repeated intraperitoneal application of the peptide twice daily resulted in a significant reduction of clinical and histological signs of adjuvant-induced experimental arthritis as compared to control animals. The efficiency of -MSH was similar to that of prednisolone, but -MSH did not cause significant weight loss (Ceriani et al, 1994). Some data indicate that the melanocortin receptor MC-3R may be a relevant target for the treatment of arthritis.

During the early phase of inflammation (associated with tissue destruction and activation of nociceptors) mediators of inflammation, including cytokines, chemokines, bradykinin, prostaglandins act as mediators of hyperalgesia. At the same time, the expression of peripheral opioid receptors on sensory nerve endings increases during inflammation (Rittner et al, 2003), which leads to the release of pain mediators, such as opioid peptides, somatostatin, endocannabinoids and certain cytokines. Endogenous opioid peptides, leucine and met-enkephalin act presynaptically when they inhibit the release of neurotransmitters, and postsynpatically when they suppress the activity of the postsynaptic neurone, and block the transmission of ascending pain signals (Rang, 2005).

In addition to the central nervous system, met-enkephalin influences the function of the immune system (Vujić-Redžić, 2000). It is released from polymorph nuclear cells during inflammation and periods of stress (Rittner et al, 2006). In inflammation, there is a migration of leukocytes that contain opioid peptides, and also the upregulation of opioid receptors of afferent fibres. This is supported by the fact that IL-1 is a specific inductor of expression of kappa opioid receptors (Puehler et al, 2006). Interleukin-1, together with TNF-α, is the main proinflammatory cytokine released from macrophages and many other cells, which initiates the release of other cytokines (Rajora et al, 1997).

In *in vitro* studies, it was found that T lymphocytes have opioid receptors on their surface (Wybran et al, 1985), and according to Plotnikoff and associates (1991), the effects of enkephalin are precisely related to the immune response mediated by T cells. In addition, the corticotropin-releasing hormone, similarly to the pituitary gland, appears dosedependent locally, through delta opioid receptors, and leads to the release of opioid peptides from leukocytes and the induction of endogenous analgesia (Menzebach et al, 2003; Cabot et al, 2001).

Met-enkephalin manifests central, bimodal and dose dependent immunomodulatory effects. A bi-phasic effect was registered in *in vivo* studies on rats. High doses have suppressed, while low doses potentiated a humoral immune response (Janković and Marić, 1994). The studies *in vivo* of the immunomodulatory effect of met-enkephaline on rats showed a suppressive action of high doses of met-enkaphalin on inflammatory reactions, such as the systemic anaphylactic shock (Janković and Marić, 1987), Arthus phenomenon and postponed skin reactions to protein antigens (Marić and Janković, 1987), the rejection of

Enkorten – A Potential Drug for the Treatment of Rheumatoid Arthritis 161

in the body reduces the likelihood of toxicity and tolerance, and allows for better control of

Enkorten toxicity testing was conducted according to the Instructions and Rules of Good

Objectives of preclinical toxicological studies were to determine the degree of toxicity of the investigated substance; to identify the target organs of toxicity and to determine the reversibility of the toxic effects; to detect the toxic effects that may help in the identification of the parameters for subsequent clinical trials and to determine the

Preclinical toxicological studies were carried out in rats and rabbits. The experimental animals were Wistar albino rats bred in the breeding quarters in Harlan, Italy, and the rabbit strain was HY plus giant PS 19 gene, from France. Environmental conditions for animals in the vivarium were maintained constant with daily temperature and humidity monitoring. Semisynthetic food for laboratory animals *ad libitum* and the tap water that meets the Sarajevo city water-supply criteria for drinking water were used during study. Animals were randomly divided in groups according to dose levels. Three dose levels were determined using the method of multiplication of the anticipated maximum human therapeutic dose of 10 mg of met-enkephalin (substance a) and 2 mg of alpha-MSH (substance b): the dose equivalent to the anticipated human therapeutic dose (0.071 mg /kg **a** + 0.014 mg/kg **b**); 5 times higher dose (0.355 mg/kg **a** + 0.07 mg/kg **b**); 10 times higher dose (0.71 mg/kg **a** + 0.14 mg/kg **b**). Enkorten was applied as a constant volume dose of 0.0005 ml/g according to the following dosage regime: during the first month - three times a week; during the second month - twice a week, and during the third month - once a week.

Treatment was followed by a 10-day observation period. The appearance of toxic signs was monitored on a daily basis. Weighing of individual animals was performed before dosing, once a week, and before the animals were sacrificed. Daily food and water consumption per

In subchronic and chronic studies, haematological and biochemical analyses were performed of the fasting blood samples taken from 12 animals from each of the experimental groups and from 24 animals from the control group, before the animals were sacrificed. The following haematological analyses were performed: hematocrit, haemoglobin concentration, red blood cell count, red blood cell morphology, total and differential white blood cell count, platelets. The following biochemical analyses were performed: Calcium

aspartate aminotransferase (AST) , Alanine aminotransferase (ALT), alkaline phosphatase (AP), Gamma-glutamyl transpeptidase (GGT), blood urea nitrogen (BUN), total proteins,

Necropsy was performed only on the sacrificed animals (no lethality observed). Upon completion of the experiment, animals were sacrificed and both external and internal examinations of the sacrificed animals were performed. The following organs: brain, heart, lungs, liver, spleen, thymus, kidneys and adrenal glands, as well as samples of other

), Phosphorus (PO24-), glucose, Aspartal

Laboratory Practice (GLP) and Harmonized Tripartite Guideline issued in 1998.

The control group was treated by 0.9% of physiological NaCl solution.

albumin, globulin, total bilirubin, creatinine, lipids, and cholinesterase.

macroscopically changed tissues were subjected to histopathological analysis.

cage, as well as the stool mass were recorded on a weekly basis.

(Ca+2), Potassium (K+), Sodium (Na+), Chloride (Cl -

the pharmacological effects (Catania et al, 2004).

**3. Preclinical toxicological studies** 

maximum tolerated dose.

allograph (Janković, 1991), arthritis (Janković, 1991) and the experimentally induced allergic encephalomyelitis (Veljić et al, 1991).

In studies conducted at the Charité University in Berlin on the effect of intraarticular application of opioids in comparison to the intraarticular application of glucocorticoids in patients with the chronic inflammatory arthritis accompanied by pain and functional impairment, according to the established hypothesis, local application of opioids led to a significant reduction of pain and inflammation, which can be explained by the activation of peripheral opioid receptors (that have been identified on peripheral sensory nerve endings), and subsequent decreases in neural excitability, transmission of nociceptive impulses, and reduced release of proinflammatory neurotransmitters (Stein et al, 2001; Mousa et al, 2001).

In summary, preclinical studies indicate that the activation of melanocortin receptors could be a new strategy to control inflammation (Catania et al, 2004). Similarly to any new therapeutic approaches, this strategy may have both advantages and disadvantages in comparison to the currently available medications. The main advantage of melanocortin in the treatment of inflammation is that its anti-inflammatory activity is not restricted to a specific mediator or a chemical process. In fact, as a consequence of reduced activation of the nuclear factor NF-B, the collective reduction of all important molecules involved in the inflammatory process is evident.

Another positive aspect is the fact that the treatment with melanocortin peptides never reverses the inflammatory response, but modulates it. It is well known that the inflammatory response is a crucial reaction of the host that contributes to the elimination of pathogens and harmful molecules. Cytokines, which are a major component of the inflammatory process, also have a significant function in regulating the recovery of tissues, haematopoiesis and immune responses. Any agent that completely inhibits their production or action would have a detrimental impact on the host defence mechanism. Melanocortin peptides modulate the increased production of cytokines during infection or inflammation, but do not prevent their release. Besides, they do not affect the production of mediators of inflammation when being on complete rest. So, for example, alpha-1-13 corticotrophin modulates the febrile response caused by pyrogens, while on the other side, it does not cause any changes to the non-febrile temperature of the body.

The main advantage of the melanocortin in relation to the currently used anti-inflammatory drugs, particularly corticosteroids, is that neuropeptides do not reduce the microbicidal activity of neutrophils, but instead increase it. This feature could be very important in the treatment of inflammation in immunecompromised persons.

The lack of selectivity could be a potential problem when it comes to the use of natural melanocortin peptides. Today, it is clear that the melanocortins affect many body functions, including the regulation of food consumption, sexual behaviour and pigmentation. Systemic injection of non-selective peptide could, therefore, cause adverse effects by stimulating all subtypes of receptors. However, the design and synthesis of new melanocortin analogues with a selective affinity for specific receptors could greatly facilitate the achievement of target effects. Knowledge of amino acid substitution that reduces binding to all receptors can also help prevent unwanted receptor activation. (Catania et al, 2004). Another potential problem lies in the rapid splitting, or degradation of peptide molecules in the circulation and other body fluids, and natural melanocortins are no exception.

The relatively short half-life could be a problem if it is necessary to maintain the concentration in the blood. On the other hand, the fact that the peptides are not accumulated in the body reduces the likelihood of toxicity and tolerance, and allows for better control of the pharmacological effects (Catania et al, 2004).
