New
Generation Drugs at Treatment of Viral Hepatitis: Differentiated Effect on
1027-2712/99/0301-059 © 1999 by THESA ExConsilio 1999, 1, 59-65
L.
Kozhemyakin, 0.Ketlinskaya, S. Romanova, A. Belokhvostov Military
Introduction
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 (
Results
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 15%.
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
performed once.
Discussion
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 stable
remission.
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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For more references:
Dr. Giorgio Castello
Corso Torino, 32/6
16129 – Genova (Italy)
Tel: +39.010.58.94.95
Mobil
phone: +39.335.628.34.24
e-mail:
castello@tiopoietine.info