Glutoxim mechanisms of action.
Glutoxim, a chemically synthesized biologically active compound, is a hexapeptide with a stabilized disulfide bond (bis-(gamma-L-glutamyl)-L-cisteinyl-bis-glycin disodium salt), total formula – (C20H32O16N6S2).
It has been demonstrated that Glutoxim initiates the cytokine system of normal cells of immunopoiesis and hemopoiesis organs, in particular, it regulates endogenous production of a wide range of interleukins and hematopoietic factors (IL-1b, IL-4, IL-6, IL-8, IL-10,IL-12, TNF, IFN, GM-CSF and erythropoietin) and reproduction of IL-2 effects by inducing IL-2Ra and IL-2Kb receptors.
Reproduction of the effects of a number of cytokines by Glutoxim is of exceptional importance, because diseased characterized by marked immune suppression, such as oncological diseases, are primarily associated with impaired reception of cytokine regulatory effects. The above elements of the drug’s specific activity, including those of its immunomodulating activity, have been experimentally confirmed in studies of apoptosis regulating mechanisms, in radiation and chemically (cyclophosphane) induced immune deficiency as well as in clinical studies in cancer patients.
Clinical study results make it possible to assume that the basic mechanism of the drug’s general biological activity consists in regulated escalation of the redox state of cells and modification of “critical cysteines” of signal-transmitting systems’ key proteins. The new level of cellular redox contour as well as that of phosphorylation dynamics (cAMP/cGMP ratio) and of nFkB and AP-1 transcription factor activity leads to a chain reaction of genetically determined biochemical mechanisms constituting the functional response of cells to the drug.
Thus, in terms of its molecular mechanisms of action the drug can be classified as regulator of redox-sensitive expression of genes, primarily of immunologically relevant ones, which include interleukine-2 (IL-2) alpha chain, tumour necrosis factor alpha (TNFa), alpha and gamma interferons (IFNa,г), c-fos, Bax and Bcl-2 genes.
Besides, there are experimental and clinical data suggesting that the drug:
- induces differentiation of pre-T lymphocytes (Thy-1 marker) of the bone marrow;
- can activate proliferation and differentiation of normal hemopoietic tissue CD34+ cells and trigger apoptosis-inducing mechanisms in actively proliferating cells in leukemia (as determined by flow cytometry using monoclonal antibodies to Fas-antigen);
The above mechanisms of action account for functional integrity of immunophysiological effects, which include:
- high tropism of the drug to the cells of central immunity organs and of the lymphoid tissue system associated with formation of cytoprotective mechanisms;
- enhancement of erythropoiesis, lymphopoiesis and granulocyte-monocytopoiesis;
- activation of the phagocytosis system, including that in acquired immune deficiency, restoration of neutrophil, monocyte, lymphocyte and platelet counts in peripheral blood;
- predominant activation of T-lymphocyte proliferation and differentiation, also in the situation of radiational and chemical immunosuppression and AIDS, restoration of CD3+, CD4+, CD8+, CD16+/56+ and CD25+ cell counts.
On the whole, Glutoxim can be classified as an immunomodulator possessing multicytokine-activating and hemopoietic activity.
Absorption Bioavailability of the drug following intramuscular,
intravenous or subcutaneous injection exceeds 90%. Whether Glutoxim is
administered intravenously, intramuscularly or subcutaneously, its dose/blood
plasma concentration ratio is linear. Plasma concentration of the drug reaches
its maximum 5 minutes after intravenous administration or 7-15 minutes after
intramuscular or subcutaneous injection.
Distribution following intravenous administration of 5 mg of
Glutoxim to healthy volunteers, the drug distribution volume in equilibrium
state constituted 0.220-0.680 l/kg (0.386 l/kg on the average), suggesting that
the drug is primarily localized in additional compartments (excluding blood
plasma), in the intra- or extracellular space or is selectively accumulated in
Metabolism Glutoxim is rapidly captured by various organs. Its
uptake is highest in the liver, kidneys and immuno- and hemopoiesis organs and
lowest in the adipose tissue. After the drug is reduced by natural cellular
metabolic systems (mostly through enzymatic reduction in the presence of
glutathionereductase or non-enzymatically through sulfhydryl groups oxidation),
its metabolites, such as reduced glutathione, can undergo intracellular
degradation to its constituent amino acids or eliminated as mercaptopurine
acids. In the former case the free amino acids can be used as protein
construction material or participate in cellular energy metabolism. In case of
mercaptopurine acid formation (mostly through conjugation in the presence of
glutathionetransferase), the metabolite is eliminated into the blood and
further via kidneys.
Glutoxim is available as 1% or 3% solution in 1 ml ampoules containing 10 or 30 mg of the active ingredient, respectively, or in 2 ml ampoules containing 20 or 60 mg of the active ingredient, respectively.
Experimental single administration of 1000 times the therapeutic dose of the drug as well as chronic (6 months) administration of 100 times the therapeutic dose demonstrated absolute safety of the drug. Glutoxim did not cause negative changes in the major biochemical and physiological systems of the body. The width of Glutoxim therapeutic action is of equal importance.
Not a single case of drug withdrawal due to intolerance has been observed during comprehensive clinical studies. Glotoxim has proven to be safe in terms of all criteria used for drug safety evaluation.
Glutoxim effect on major intracellular regulation systems, including the Ras-signal cascade
Glutoxim is the first differentially acting drug which on the one hand acts favourably on normal cells while on the other hand initiating elimination of genetically defective cells (tumour cells or cells affected by viruses) from the body. Genetically defective cells are eliminated due to restoration of their capacity for apoptosis (programmed cell death). In particular, the feect of Glutoxim on genetically intact cells promotes activation of the intracellular proteinkinase cascade, proliferation and restoration of cell susceptibility to humoral factors and mobilizes redox complex enzymes involved in glutathione metabolism (Kozhemyakin et al., 1999). The effect on genetically impaired cells manifests itself in their reaction to humoral factors of apoptosis induction as well as on redox-dependent cell division and apoptosis regulation factors. On molecular level Glutoxim, being a structural analog of oxidized glutathione, activates gluthationereductase, gluthathionetransferase and glutathioneperoxidase, which in their turn activate intracellular reactions of thiolic metabolism and conjugate the processes the synthesis of sulfur- and phosphorous-containing compounds required for normal functioning of intracellular regulatory systems. It is known that cells use an active ATP-dependent system for capturing oxidized glutathione, while the uptake of its reduced from is minimal. Stabilization of oxidized glutathione disulfide bond multiplies its pharmacological effects compared to those of oxidized glutathione.
Experimental and clinical results of the use of Glutoxim have revealed its anti-tumour activity realized through depressing redox potential in transformed cells. It has been demonstrated that redox potential depression can induce apoptosis both due to increased p53 protein half-life and by influencing the cascade of Ras-signal pathway phosphoproteinkinases. In vitro studies of the effect of Glutoxim on the number of HL60 cells demonstrate that 48 hour incubation in the presence of the drug in the concentration of 100 ug/ml resulted in the death of practically all malignant cells despite defective p53 gene. Addition of Glutoxim to culture medium containing transformed fibroblasts induces apoptosis points to involvement of the Ras-signal cascade in this process. However, while 2 days after of exposure to Glutoxim (C8 cells) in case of cells with enhanced production of Ras-protein and a normally functioning p53 protein death due to apoptosis is almost 100%, in case of defective p53 gene only half the cells died under the same conditions. Thus, apoptosis induction by Glutoxim involves the proteinkinase Ras-signal cascade and follows both p53-dependent and p53-independent pathway. If both the pathways are intact, the effect of Glutoxim is greater than if only one of them functions.
Glutoxim acts on the Ras-signal pathway of an intracellular cascade of phosphorylation of proteins which ultimately trigger cell proliferation. The Ras-signal pathway is characterized by a dual effect of its activation. In normal cells proliferation and differentiation are activated while in malignantly degenerated cells or in cells with impaired GTP-GDP ratio (GTP depletion) capacity for apoptosis increases. This is due to the fact that in the end of the pathway the cascade of Ras-dependent phosphokinase reactions branches out. In normal cells it is the proliferation branch components that are active, while in genetically impaired cells the potentially active branch is that of cell self-destruction, which accounts for the dual effect of the Ras-signal pathway activation by Glutoxim.
Ras-protein activation requires detachment of its C-terminal tripeptide, which takes place due to formation of a high-energy bond between sulfur atoms in the tripeptide cysteine molecule and of phosphorous atoms in the DTP molecule. Acting on this process, Glutoxim contributes to normalization of Ras-protein processing. Only after the cysteine-containing tripeptide is detached does the Ras-protein adequately attach to the cytoplasmic membrane.
Besides, the fact that the Ras-signal cascade factors include a number of other GPP-binding proteins containing “critical cysteines” does not exclude its interaction with Glutoxim. The multicomponent effect of Glutoxim on normalizing the Ras-dependent cascade of intracellular reactions proves its preventive role in situations of high oncological risk.
Many cytokines act by activating the Ras-signal pathway. Ensuring functional stability of the Ras-signal pathway, Glutoxim contributes to adequate immunocorrection.
Thus, the effect of Glutoxim on key processes of vital functions of cells in a body affected by a tumour promotes both restoration of specific anti-tumour immunity and increased apoptosis of malignant cells. Glutoxim improves the condition of genetically normal cells and initiates elimination of genetically defective ones. It induces a wide range of cellular reactions, including those on genetic level, thus enhancing organism resistance to extreme chemical, physical and biological impacts. The versatility of intracellular regulatory effects of Glutoxim determines its value in treating acute and chronic diseases characterized by hypoxia, cytolysis and impairment of cell proliferation and differentiation ratio. Preventive use of the drug as a modifier of cellular response to aggressive exogenous and endogenous influences appears equally advisable. Effect of Glutoxim on cell proliferation, differentiation and programmed death via the Ras-signal system. RAS – Ras-protein; P53 – p53-protein; P21 – p21-protein; 1,2,3 – Rho, Rac, Mos guanyl proteins and other proteins influencing the activity of Ras-signal pathway including Raf, MEK, MAPK/ERK, PI 3 phosphokinases.
For more references:
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