Physiology and Pathology of Cytokine: Commercial Production and Medical Use

2.1. Production and medical use of IFN-α

Interferon production process includes the fermentation process, purification and the formation of the final product. Fermentation is conducted in specially designed vessels made of stainless steel. This process involves transfer and expression of genes in E. coli and the subsequent intracytoplasmic protein production of the inserted gene. Thus, recombinant proteins constitute intracytoplasmic accumulation of inclusion bodies. Homogenization E. coli and centrifugation, followed by a chromatographic purification process, need to be done in order to prepare inclusion bodies. The crystallization method is used for the final purification of interferons. The crystallized product is then dissolved in a phosphate buffer which contains glycine and human albumin (carriers of an active substance). Finally, lyophilization and packaging of the product is done.

There are several significant advantages achieved by the modification of interferons. Recombinant interferon modification is achieved by the process of “pegylation.” This process was first described by Frank Davis and Abraham Abuchowski and his associates in 1977 [8]. In contrast to the modification process of drug formulation (using colloidal systems or osmotic pumps), the process of interferon pegylation involves covalently binding polyethylene glycol (PEG) polymer chain to the amino group of interferons, which significantly improves its pharmacokinetic and pharmacodynamic characteristics [9]. Many existing pharmacological limitations of interferons are overcome by the process pegylation. Native PEG is an inert, nontoxic polymer with two terminal hydroxyl groups. Chemically active form of PEG molecule is obtained by modifying the terminal hydroxyl group (substitution process of a hydroxyl group with reactive (functional) one). The incubation of purified interferon with methoxypolyethylene glycol (chemically active form of PEG) spontaneously forms covalent bonds [10]. There are different configurations of PEG molecules today, including linear and branched structures of various molecular weights. Formation of stable (covalent) bonds in the process of pegylation of interferon is necessary for a long-term preservation of incurred pharmacological changes. Resultant molecules of interferon are predominantly monopegylated with small impurities of di-, and non-pegylated molecules. Sodium phosphate, saccharose and polysorbate are used as carriers of the active substance for the pegylated product in lyophilized form.

After subcutaneous administration IFN-α is rapidly absorbed and the peak of serum concentration is reached 7–12 hours after which its concentration decreases rapidly. As half-life of IFN-α is 3–8 hours, its serum concentration falls below detection limit within 24 hours after administration [11]. Since the administration of IFN-α is conducted 3 times a week, the desired serum concentration is not maintained in most intervals between applications.

Different linear and branched structures of PEG molecules are used for the pegylation of IFN-α, so that the resulting preparations exhibit different pharmacokinetic and pharmacodynamic characteristics which are improved in comparison to standard IFN-α. PEG group protects IFN-α from enzymatic degradation, prolongs the absorption period at the administration site of subcutaneous injection and reduces its clearance from the body, so the period to reach maximum concentration is significantly prolonged (80 hours). Taking into consideration the above mentioned, it is clear that IFN-α concentration in blood is higher and more constant for a much longer period of time when compared to standard IFN-α. These pharmacokinetic properties enable once-a-week subcutaneous administration of pegylated IFN-α to achieve and maintain the concentration in blood that provides the desired effectiveness (desired effects are time and concentration dependent). Pegylation of IFN-α greatly improves its pharmacodynamic characteristics, and testing shows 100 times stronger antiviral activity and 20 times stronger antitumor activity. Nowadays, IFN-α is used in the treatment of hepatitis B and C, chronic myeloid leukemia, malignant melanoma, non-Hodgkin’s lymphoma, Kaposi’s sarcoma, and other diseases (Table 2).

2.2. Production and medical use of IFN-β

There are several preparations made of IFN-β used for therapeutic purposes. Some of them were made by recombinant DNA technology in E. coli, but nowadays CHO cell line is mostly used in their production [12]. Recombinant human IFN-β produced in CHO cell line (rhIFN-β-1a) is glycosylated and it has the same amino acid sequence as natural IFN-β [13, 14]. Although E. coli does not generate glycosylation of IFN-β [15], this deficiency does not affect the efficiency of its therapeutic applications. IFN-β is presently used in the treatment of multiple sclerosis. However, in IFN-β glycans play an important role in the protein stabilization and thus enhance its biological activity [16] (Table 3).

2.3. Production and medical use of IFN-γ

IFN-γ preparations are made by recombinant DNA technology in bacteria E. coli. Despite the fact that recombinant human IFN-γ is not glycosylated [17], its biological activity is not affected. It is used for the treatment of chronic granulomatous disease (Table 4).

2.4. Side effects of interferon application

Administration of interferons can cause many side effects [18].

Application of IFN-α causes increased temperature, chills and headache. These reactions are often manifested a few weeks after the application and paracetamol is simultaneously applied to alleviate them. Severe cases develop anorexia, insomnia, cardiovascular complications and autoimmune reactions, which requires immediate termination of its application.

Application of IFN-β causes increased temperature, chills and headache. Severe cases develop hypersensitivity reactions, depression and menstrual disorders.

Application of IFN-γ causes increased temperature, chills and headache. Severe cases develop heart failure, metabolic disorders and disorientation.

3.1. Production and medical use of interleukin 1 (IL-1)

IL-1 is a proinflammatory cytokine which stimulates the synthesis of substances involved in the induction of inflammation. Activated mononuclear phagocytes are the main cellular source of IL-1. When secreted in small quantities, IL-1 acts as a paracrine mediator of local inflammation, while in larger amounts the endocrine effect can induce body temperature increase, synthesis of acute-phase proteins in the liver and the production of neutrophils and platelets in the bone marrow. In diseases with the elevated level of IL-1 it is important to decrease IL-1 level due to its effect in the induction of inflammation [20]. It has been shown that preparations which reduce the level of IL-1 are therapeutically useful when administered alone or, more preferably, in the combination with low doses of other therapeutic agents. In accordance with the above, the reduction of IL-1 level can be achieved by the administration of:

1. Anti-IL-1 antibody

2. The IL-1 receptor antagonist (IL-1Ra)

3.2. Production and medical use of interleukin 2 (IL-2)

As it was the case with most other cytokines, the use of IL-2 for medical purposes was initially impractical because of small production quantities. Some transformed cell lines, particularly cell line Jurkat (T cell leukemia), produced IL-2 in larger quantities [21]. The largest amounts of IL-2 used in initial studies were obtained from this source. The production of significant amounts of IL-2 is possible by the development of recombinant DNA technology. Today, complementary DNA (cDNA) to the IL-2 gene is expressed in many cell lines, while E. coli was used at the beginning (absence of glycosylation of the recombinant product does not alter biological activity of IL-2).

Basiliximab (anti-IL-2Rα (CD25); chimeric monoclonal antibody) is produced in a cell line Sp2/0 and is administered intravenously to patients after kidney transplantation. Basiliximab and cyclosporine are administered in the treatment of kidney transplant [22]. It is important to note that studies have shown that basiliximab slows cyclosporine elimination from the body (it is believed that cytochrome P450 plays an important role in this process).

Daclizumab (anti-IL-2Rα (CD25); humanized monoclonal antibody) was administered intravenously to patients after kidney transplantation. It was withdrawn from the market of the European Union and the United States of America in 2009.

Recombinant IL-2.

Proleukin (Aldesleukin) is indicated for the treatment of adults with metastatic renal cell carcinoma (year of first approval-1992) and metastatic melanoma (year of first approval-1998). Proleukin helps an increased production of several different components of the immune system found in the blood, including T lymphocytes and natural killer cells. Proleukin should be restricted to patients with healthy heart and lung function. Proleukin can cause capillary leak syndrome and the treatment is associated with a reduced neutrophil chemotaxis and with an increased risk of disseminated infection, including sepsis.

3.3. Production and medical use of interleukin 6 (IL-6)

IL-6 has pro- and anti-inflammatory properties and plays a crucial role during the transition from innate to acquired immunity. It has the ability to stimulate neutrophil production, promote expansion and activation of T cells, the differentiation of B cells, and the regulation of the acute-phase response [23]. Proteolytic shedding of IL-6Rα from invading neutrophils subsequently drives IL-6 trans-signaling in resident tissue cells, leading to a switch from neutrophil to monocyte recruitment by suppressing mainly neutrophil-attracting (CXCL1, CXCL8 and CX3CL1) and enhancing mainly monocyte-attracting chemokines (CCL2, CCL8, CXCL5 and CXCL6), and cellular adhesion controlled by the lymph node–homing receptor CD62L, and modulates expression of adhesion molecules ICAM-1 and VCAM-1. Besides its role in attracting monocytes, IL-6 trans-signaling has been shown to skew monocyte differentiation toward macrophages by upregulating M-CSF receptor expression. The finding that IL-6 induces neutrophil apoptosis contributes to the resolution of acute neutrophil infiltration. Increased IL-6 level is often a better predictor of disease activity in the context of infection, autoimmunity or cancer than C-reactive protein [24, 25]. Consistent with the early description of IL-6 as a lymphocyte-stimulating factor, IL-6 deficiency leads to impaired innate and adaptive immunity to viral, parasitic and bacterial infection. Indeed, children with inhibitory autoantibodies to IL-6 develop recurrent staphylococcal cellulitis and subcutaneous abscesses. While IL-6 has a protective role in many infections, the same activity can be key to the maintenance of chronic inflammation that includes rheumatoid arthritis and multicentric Castleman’s disease. The reduction of IL-6 level (Table 5) as the treatment of chronic inflammatory diseases can be achieved by the administration of:

1. Anti-IL-6 antibody

2. Anti-IL-6 receptor antibody

3.4. Production and medical use of tumor necrosis factor (TNF)

Tumor necrosis factor (TNF) was originally described as a circulating protein which causes tumor necrosis (discovered in 1975 as endotoxin-induced glycoprotein which causes necrosis of sarcoma in mice). Human TNF was first produced in 1985 by recombinant DNA technology in E. coli [26]. Activated mononuclear phagocytes are the most important cellular source of TNF. TNF exerts a variety of effects on vascular endothelial cells and leukocytes. In response to TNF endothelial cells enhance the expression of different combinations of leukocyte adhesion molecules (including E-selectin, ICAM-1 and VCAM-1), which in combination with the secretion of chemokines (IL-8, including, MCP-1 and IP-10) from peripheral blood leukocytes and endothelial cells induces chemotaxis of different populations of leukocytes to the site of inflammation apart from the recognition of the antigen. In addition, TNF stimulates microbicidal activity of neutrophils and monocytes in order to eliminate the cause easier. Secreted in small amounts, TNF acts locally (autocrine and paracrine), primarily on vascular endothelial cells and leukocytes. The effect of TNF on the endothelium and leukocytes is most likely crucial for a successful local inflammatory response to microorganisms. TNF is produced in large quantities in severe infections. It enters the bloodstream and affects distant cites (endocrine), causing pathological and clinical effects on the body. Systemic effects caused due to excessive TNF production or exogenous administration in high doses can induce increased body temperature, synthesis of acute-phase proteins in the liver, production of neutrophils and platelets in the bone marrow. TNF is presently considered a key mediator of inflammatory response which has a wide range of effects in inflammation, infection and response to tumors. In this respect, several approaches can be useful, and this includes the administration of:

1. Anti-TNF antibody

2. TNF receptor (TNFR)

3. TNF