New Generation Drugs at Treatment of Viral Hepatitis: Differentiated Effect on Normal and Virus-Infected Cells.
1027-2712/99/0301-059 © 1999 by THESA ExConsilio 1999, 1, 59-65
L. Kozhemyakin, 0.Ketlinskaya, S. Romanova, A. Belokhvostov Military Academy of Medicine, St. Petersburg, Russia.
Hepatitis B and C are the widely spread viral infections, which in their occurrence significantly exceed HIV/AIDS infection.
The hepatitis C virus has the highest chroniogenic potential being the main cause on forming of the chronic hepatic diseases' entire spectrum: chronic hepatitis, cirrhosis, and the primary liver cancer (hepatocellular carcinoma) as well. The basic mechanisms are the ones of "elusion" of the HBV infection and, moreover, the HCV one from the immune surveillance due to the following: (a) the permanent HCV multivarious antigen variability (the so-called "quasispecies"); (b) T-cell energy; (c) the HCV and HBV replication in monocytes and macrophages, that is, in the cells that are non-controlled by immunocytes. Thereupon, there is the basis to think that the cell-mediated immune response playing the central role in regulation of inflammation and liver homeostasis at viral infection as well as for viral persistency and elimination processes is characterized by functional incapacity, first of all, in regard of endogenous production of an adequate and proper "cytokines team". Exactly the cytokines in conditions of the constant "race for speed" between formation of the new quasispecies and the responsive immune reaction are able to modulate the immunity system reply. Therefore, the viral hepatitis therapy methods should imply using of the immunomodulators capable not just to perform fine regulation for the endogenous production of the wide cytokines range but to reproduce the cytokines effects in conditions of blocking (or activity contortion) of the receptors system mediating manifestations of helper and cytotoxic activities.
Nowadays the main methods of the antiviral therapy for hepatitis B and C are interferons and nucleoside analogs. The common interferon therapy program is 3 millions IU 3 times a week during 6 months, that was considered as a golden standard until quite recently, causes the disease remission in 30% of patients.
Therefore, the new approaches to the viral hepatitis treatment are necessary; there is need in the new substances created on the basically new ground in order to make possible answer to the HCV declaration – "Muto ergo sum": There is the medicine of the differentiated action regarding to the normal (non-impaired) and the virus-infected cells.
"Glutoxim" is a chemically synthesized biologically active substance, a hexapeptide with the stabilized disulfide bound (disodiurn salt of bis-(Y-L-glutamyl)-L-cystinyl-bis-glycine) with a formula (C20H32O16N6S2). "Glutoxim" represents a new class of medical remedies—thiopoietins, which have the unique biological activity due to the modulating effect on the intracellular processes of the thiol metabolism, forming the new level of the cell redoxcontour that plays an important role in regulation of the genetic and metabolic processes in cells and tissues.
The basic mechanism of the drug biological and pharmacological efficacy is a regulated escalation of cell redox state. New level of the redox systems and of the key proteins phosphorilating development of signal-transducing systems and transcriptional factors (NFkB and AP-I), first of all, in the immunocompetent cells and in the cells of central immunogenesis organs determines the immunomodulating and systemic cell protective drug activity. "Glutoxim" possesses the bifunctional differentiated effect: (a) capability to stimulate proliferation and differentiation of the normal cells including the bone marrow ones, and, simultaneously, (b) capability to induce selectively the apoptosis mechanisms in the transformed cells (in particular, by the Fas-Ag (CD-95+) expression). Immunophysiologic features of Glutoxim determine: (1) the high drug affinity to the cells of the central immunogenesis organs and the lymphoid tissue system forming the cell protective mechanisms; (2) enhancement of the bone marrow hemopoiesis: processes of erythropoiesis, lymphopoiesis and granulocyto-monocytopoiesis; (3) the phagocytosis system activation including in the acquired immunodeficiency conditions; restoration of the neutrophil, monocyte, lymphocyte level in the peripheral blood and of the functions of the tissue macrophages; (4) the proliferation and differentiation activation of the T-lymphocytes mainly, restoration of the levels of the CD3+ CD4+ CD8+, CD16+/56+ and CD25+cells.
Immunobiochemical features provide: (I) drug stimulating effect on the cascade mechanisms of the phosphate modifications of the main proteins of the signal-transducing systems; (2) initiation of the cytokine system including the endogenous production of IL-I, IL-6, TNF, IFN-a and y, and expression of receptors to IL-2.
Glutoxim might be used as a metabolic immunomodulator providing basically new quality for the immunological support of antibacterial, antiviral and antitumor antibiotic- and chemotherapy.
Being analog of the key cell metabolite the "'Glutoxim" active principle has high bioavailability and, therefore, being introduced into biological media it rapidly translocates into target cells initiating cascade mechanism of biochemical reactions of regulation of metabolism, cell proliferation, and differentiation, forming wide spectrum of the immunomodulating and system cell protective effects of drug. In the ground of the HBV and HCV replicative activity inhibition mechanisms there is the differentiated impact on unaffected hepatocytes and resident macrophages (metabolism, proliferation and differentiation stimulation) and on virus-infected cells (induction of apoptosis mechanisms including FasAg-CD95+ expression on fibroblasts). "Glutoxim" corrects the cell-mediated immune response, which plays a leading role in the hepatocellular necrosis development, processes of virus persistency and elimination as well as the fibrogenesis processes to prevent cirrhosis" that determines etiopathogenic therapy efficacy.
Materials and Methods
Patient's description. We have examined 75 patients of 19 to 39 years old having chronic viral hepatitis C (CHCV) and prolonged forms of acute viral hepatitis B (AHBV). All patients were examined and treated in the Specialized Hospital of the Viral Infections (St. Petersburg, Russia) from August till December, 1997. Criteria of the drug administration at CHCV were presence of RNA HCV in the blood serum, increased levels of ALT (GPT) and bilirubin. This group included 32 persons, 26 males and 6 females. The drug administration criteria at AHBV were presence of the marked cytolytic syndrome, which was uncontrolled until the disease 20th day against the background of the HBsAg persistency and the DNA HBV manifestation in the blood serum. This group comprised 43 people: 13 females and 30 males.
The patients in both groups were administered with one course of the therapy with "Glutoxim". The treatment course was conducted during 24 days. "Glutoxim" was introduced intramuscularly and intravenously every day, the daily dose 5-10 mg.
Evaluation scale. There were evaluated the general blood analysis,
biochemical blood analysis (ALT (mmol h-11-1), bilirubin (mmol 1-1)), serologic
blood analysis (HbsAg (ng m1 1-1), PCR HBV and HCV, anti-HBs, antiHBcor IgM,
antiHBcor IgG, HCV IgG (core, ns)), immunologic parameters and cytokines (IL-lв, IL-2, IL-6, TNF-б and IFN- б). The evaluation
of this indices was performed in the following terms: before the treatment,
after treatment completion and one month after the treatment completion.
Laboratory methods. The blood serum was tested on the presence of HCV-RNA and HBV-DNA by the PCR with the diagnostic reagent "Amplicor" (La Roche). The "Orgenics" test-systems (Israel) were used to detect the serologic markers HBsAg, anti-HBs, antiHBcor IgM, antiHBcor IgG, HCV IgG (core, ns). The cytotoxic test with using of the monoclonal antibodies produced by the Immunology Institute of the Russian Medical Sciences Academy was applied to reveal the membrane markers of the lymphocytes subsets. The solid phase enzyme immunoassay was applied to define the spectrum of cytokines (IL-lв, IL-2, IL-6, TNF- б and IFN- б) where the test-systems manufactured by the company "Proteinoviy contur" (St. Petersburg, Russia) were used.
Biochemical and serological changes of indices. In the group of patients with AHBV there were 43 patients who all received a monotherapy course by "Glutoxim". All patients were examined three times: before, after the treatment course and I month after the treatment (without administration of any kind of therapy) (Fig. 1).
Before the treatment the ALT level in this group of patients was 10.5±2.1, after the treatment completion 1.2±0.01, and one month after the treatment 0.68± 0.009 mmol h-11-1 (norm is 0.1-1.0 mmol h-11-1). The bilirubin level in the group of patients with AHBV at the control observation terms was 29.36±3.2, 15.0±1.2, and 13.0±1.6 mmol h-11-1 (norm is 8.5-20.5 mmol h-11-1).
At the HBsAg examination we obtained the following dynamics of the index: before the treatment it was 146.1+4.3, after the treatment 23.85±2.3, and after one observation month 0 ng ml-1.
It should be noted that the general state of the patient and clinical symptoms due to the high cytolytic syndrome exhibited positive changes as well and it correlated with the biochemical and serological levels of indices. The patient's state improvement was manifested by disappearing of icterus, pains under the right costal arch, nausea, with the significant diminution of weakness. In the group of patients with CHCV there were 32 persons who received single course of "Glutoxim" monotherapy and were examined at the similar terms. Before the treatment the ALT level was 3.24±0.2, after the treatment completion 0.66±0.07, and one month after the treatment 1.4±0,02 mmo1 h-11-1. The bilirubin level at CHCV on the control observation terms was within the normal limits and it was 18.0±2.2, 16.5±1.8, and 22.3±2.1mmol h-11-1 respectively.
PCR evaluation results. In the group of patients with AHBV PCR was
positive in 100% cases. After the performed treatment the viral activity was
determined at 40% of cases, after the monthly observation PCR was positive only
In the group of patients with CHCV before the treatment PCR was positive in 100% cases, after the treatment completion the viral replicative activity was determined only in 1 patient that was 7,7%, and after the monthly observation (without any therapy) it reversed in 30% of cases.
Changes of immunological indices. In two examined groups the
following immunological indices were assessed: CD2+, CD3+, CD4+, CD8+, CD16+,
CD72+, CD4+CD8+, CD4+/CD8+HLA-DR, CIC, NST-test, migrating activity of
lymphocytes (spontaneous and induced by concavalin A). Evaluation was made at
the first treatment day and after the therapy course by "Glutoxim".
Citokines level development. The antiinflammatory
cytokines levels were assessed in the blood serum of the patients with CHCV.
The solid phase enzyme immunoassay was applied. 32 persons in main group
(patients with CHCV) and 20 persons in the control group (healthy volunteers)
were examined. In the main group patients were examined before and after the
monotherapy with "Glutoxim". In the control group examination was
The Glutoxim monotherapy course including 24 parenteral injections
showed in the patients with AHBV the normalization of biochemical indices (ALT,
bilirubin) after the treatment course and stabilization of the this state after
the observation month without any additional treatment. Assessment of the
serological indices revealed considerable lowering of the HBsAg level (from
146.1±4.3 to 23.85±2.3 ng ml-1), and after the month observation absence of the
HbsAg persistency in the blood serum of these patients. Hepatitis B virus
replicative activity after the treatment course was significantly decreased and
it was determined only in 40% of the patients, and after the month observation
in 15%. "Glutoxim" application at the CHCV treatment provided ALT
normalization and stabilization after the application at the treatment CHCV to
month observation. The bilirubin level stayed without change before the
treatment and in the control terms. PCR index in creased in 100% of the
patients before the treatment and after the treatment only one patient
manifested the viral replicative activity, however, after the month observation
without any kind of therapy PCR reversed in 30% of patients. This result allows
us to make the conclusion on sufficiency of the single "Glutoxim"
monotherapy course for the treatment of the patients with AHBV and necessity to
develop methodology of the drug application at the treatment CHCV to gain
The cytolytic syndrome is controlled at the treatment of the viral hepatitis B and C through the drug hepatotropic effects and the losing the toxic influence on the hepatocytes. Absence of the viral replication and elimination of the HBsAg persistency confirm the main ideological direction of the "Glutoxim" application: activation of the apoptotic mechanisms of the virus infected hepatocytes to cause the virus elimination from tissues and cytokine-replacing immunocorrection to enhance antiviral immunity (Fig. 3).
AHBV treating provided increase of CD8+ that indicates activation of the anti viral cellular immunity. Decrease of CD4+ and NST can be explained by diminution of activity of the macrophages and production of the active oxygen forms, lowering of IFN-y level and, respectively, lowering of the viral load. Decrease of the CD4+/CD8+ index manifests diminution of an inflammatory process; decrease of the CD4+CD8+ cells amount is explained with enhancement of differentiation of the T-cells, and CIC lowering reveals the virus quantity diminution in blood and absence of the viral replicative activity. At CHCV after the treatment CD8+ lowering lessened the liver tissue affection, CD72+ lowering indicates the viral replication decrease and, respectively, the humoral immunity lowering. HLADR diminution correlated with the CD72+ level and the viral replication absence. CD16+ lowering could be explained by the decrease in amount of the infected cells. The obtained data indicates positive development of the immunological indices at the AHBV and CHCV treatment by the drug's of the "Glutoxim" series and correlated with serological, biochemical indices and PCR results that prevents and ceases the immunoautoaggression signs especially in regard to the hepatitis C virus.
Development of the anti-inflammatory cytokine levels at the CHCV treatment reveals considerable increase of the studied indices before the treatment beginning and their decrease after the treatment. It is related to the following indices: IL-2, 4 and 10, TNF-a, IFN-y that correlated with the inflammatory reaction diminution after the "Glutoxim" treatment course, normalization biochemical indices and absence of the virus C hepatitis replicative activity.
The Glutoxim clinical effectiveness might be determined by direct activation of the Ras-signal pathway proteins. This pathway includes phosphokinases that coordinate and complete the post-receptor signal process. To activate the Ras-signal pathway an interaction of two proteins, Ras-protein and phosphokinase MEK is necessary. Glutoxim might be assumed to increase rate of formation of the Ras-protein functionally active complex.
Then in the chain of the initiated Ras-signal pathway there is a sequential activation (Fig. 3) of Rab, MEK, MAPK/ERK phosphokinases. Response of normal and genetically impaired, virus infected cells on the Ras-signal pathway is different. In the normal cells activation of the Ras-signal pathway by Glutoxim stimulates a proliferative process through induction of the phosphatidilinositol-3 kinase PI3 and NFKB protein. On the contrary, in the virus- infected cells Glutoxim cause the cell cycle arrest in the G 1 phase and apoptosis induction through P21 and P53 proteins. The Glutoxim capability of optimizing the cellular redox-contour and thiol-disulfide metabolism provides enhanced resistance of hepatocytes and resident macrophages against infection and vice versa in the virus infected cells the immunobiochemical reactions initiated by Glutoxim neutralize induced by hepatitis B and C viruses processes of blocking of apoptosis mechanisms in the virus infected cells through synthesis of the specialized Bcl-2-inhibiting proteins.
In its turn, Glutoxim action on Kupffer's cells in conditions of viral infection restores regulatory capabilities of the Ras-signal pathway in conditions of desensitization and (or) shedding of the receptor part to cytokines.
Complex of the Glutoxim molecular genetic effects cuase apoptosis of the virus infected cells and have a hepatoprotective impact on the normal cells. Considering the clinical data obtained and molecular mechanisms of activity we suggest that the theoretical platform we have worked out on the differentiated impact of the drugs on the virus infected and normal cells should be the basis for the viral hepatitis treatment.
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