U.S. patent application number 12/040669 was filed with the patent office on 2008-09-11 for methods for production of the oxidized glutathione composite with cis-diamminedichloroplatinum and pharmaceutical compositions based thereof regulating metabolism, proliferation, differentiation and apoptotic mechanisms for normal and transformed cells.
This patent application is currently assigned to Novelos Therapeutics, Inc.. Invention is credited to Mark B. Balasovski, Leonid A. Kozhemyakin.
Application Number | 20080220093 12/040669 |
Document ID | / |
Family ID | 26653986 |
Filed Date | 2008-09-11 |
United States Patent
Application |
20080220093 |
Kind Code |
A1 |
Kozhemyakin; Leonid A. ; et
al. |
September 11, 2008 |
METHODS FOR PRODUCTION OF THE OXIDIZED GLUTATHIONE COMPOSITE WITH
CIS-DIAMMINEDICHLOROPLATINUM AND PHARMACEUTICAL COMPOSITIONS BASED
THEREOF REGULATING METABOLISM, PROLIFERATION, DIFFERENTIATION AND
APOPTOTIC MECHANISMS FOR NORMAL AND TRANSFORMED CELLS
Abstract
The present invention relates to a composite for the treatment
of a variety of medical conditions, the composite comprising an
oxidized gluthathione-based compound, which has a disulfide bond,
and a metal material, in particular where the metal is either
platinum or palladium. The oxidized glutathione-based compound and
metal material can be present in a ratio of 3000 to 1 and
preferably 1000 to 1. The oxidized glutathione-based compound can
be oxidized glutathione itself or salts or derivatives. A feature
of the invention is that the composite has a more stabilized
disulfide bond than the oxidized glutathione-based compound itself.
Methods for preparing the composite are provided, such methods
being beneficial in that the composite is provided in high yields
and at high purity. Methods for treating various medical conditions
with the composites of the present invention are also
disclosed.
Inventors: |
Kozhemyakin; Leonid A.; (St.
Petersburg, RU) ; Balasovski; Mark B.; (St.
Petersburg, RU) |
Correspondence
Address: |
WOLF GREENFIELD & SACKS, P.C.
600 ATLANTIC AVENUE
BOSTON
MA
02210-2206
US
|
Assignee: |
Novelos Therapeutics, Inc.
Newton
MA
|
Family ID: |
26653986 |
Appl. No.: |
12/040669 |
Filed: |
February 29, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11646128 |
Dec 27, 2006 |
7371411 |
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12040669 |
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10747855 |
Dec 29, 2003 |
7169412 |
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11646128 |
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10404914 |
Apr 1, 2003 |
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10747855 |
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09842104 |
Apr 30, 2001 |
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10404914 |
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09241232 |
Feb 1, 1999 |
6312734 |
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09842104 |
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09237801 |
Jan 27, 1999 |
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09241232 |
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Current U.S.
Class: |
424/649 |
Current CPC
Class: |
A61K 47/54 20170801;
A61K 47/62 20170801; A61K 33/24 20130101; C07K 5/0215 20130101;
A61K 38/063 20130101; A61K 38/08 20130101; A61P 35/00 20180101;
A61K 33/24 20130101; A61K 2300/00 20130101; A61K 38/063 20130101;
A61K 2300/00 20130101; A61K 38/08 20130101; A61K 2300/00
20130101 |
Class at
Publication: |
424/649 |
International
Class: |
A61K 33/24 20060101
A61K033/24; A61P 35/00 20060101 A61P035/00 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 23, 1998 |
RU |
RU 98-120753 |
Claims
1. A method, comprising: introducing, to a subject in need of
alteration of cellular redox potential, an effective amount of a
composite comprising an oxidized glutathione-based compound and a
metal material wherein the metal material comprises platinum or
palladium, for a period of time sufficient alter a redox state of a
cell within the immune system, thereby altering gene expression of
immunologically significant genes within the subject.
2. The method of claim 1, wherein the oxidized glutathione-based
compound is formed from two monomers, each monomer comprising a
glutamic acid bonded to a cysteine bonded to a glycine, the two
monomers being linked through a disulfide bond bridging the sulfur
atoms of each respective cysteine of each monomer.
3. The method of claim 1, wherein the oxidized glutathione-based
compound is selected from the group consisting of the formula:
##STR00005## and salts of said formula, wherein A, B, D, E, G and H
can be the same or different and each is selected from the group
consisting of an organic unit and salts of the organic unit.
4. The method of claim 1, wherein the metal material is
cis-platin.
5. The method of claim 1, wherein the composite is administered
orally.
6. The method of claim 1, wherein the composite is administered as
a solution form selected from the group consisting of inhalation
solutions, local instillations, eye drops, intranasal
introductions, ointment for epicutaneous applications, intravenous
solutions, injection solutions, and suppositories.
7. A method, comprising: introducing, to a subject in need of an
increase in proliferation and/or differentiation of platelets,
white blood cells, red blood cells, T lymphocytes, neutrophils,
monocytes, macrophages, natural killer cells and/or B lymphocytes,
an effective amount of a composite comprising an oxidized
glutathione-based compound and a metal material wherein the metal
material comprises platinum or palladium.
8. The method of claim 7, wherein the oxidized glutathione-based
compound is formed from two monomers, each monomer comprising a
glutamic acid bonded to a cysteine bonded to a glycine, the two
monomers being linked through a disulfide bond bridging the sulfur
atoms of each respective cysteine of each monomer.
9. The method of claim 7, wherein the oxidized glutathione-based
compound is selected from the group consisting of the formula:
##STR00006## and salts of said formula, wherein A, B, D, E, G and H
can be the same or different and each is selected from the group
consisting of an organic unit and salts of the organic unit.
10. The method of claim 7, wherein the metal material is
cis-platin.
11. The method of claim 7, wherein the composite is administered
orally.
12. The method of claim 7, wherein the composite is administered as
a solution form selected from the group consisting of inhalation
solutions, local instillations, eye drops, intranasal
introductions, ointment for epicutaneous applications, intravenous
solutions, injection solutions, and suppositories.
13. A method, comprising: introducing, to a subject in need of
alteration of a cellular protein, an effective amount of a
composite comprising an oxidized glutathione-based compound and a
metal material wherein the metal material comprises platinum or
palladium.
14. The method of claim 13, wherein the oxidized glutathione-based
compound is formed from two monomers, each monomer comprising a
glutamic acid bonded to a cysteine bonded to a glycine, the two
monomers being linked through a disulfide bond bridging the sulfur
atoms of each respective cysteine of each monomer.
15. The method of claim 13, wherein the oxidized glutathione-based
compound is selected from the group consisting of the formula:
##STR00007## and salts of said formula, wherein A, B, D, E, G and H
can be the same or different and each is selected from the group
consisting of an organic unit and salts of the organic unit.
16. The method of claim 13, wherein the composite is introduced for
a period of time sufficient to stimulate an increase in the
phosphorylation of a cellular protein to obtain a therapeutic
effect.
17. The method of claim 13, wherein the composite is introduced for
a period of time sufficient to stimulate an increase in the
phosphorylation of tyrosine on a cytosolic protein to obtain a
therapeutic effect.
18. The method of claim 13, wherein the metal material is
cis-platin.
19. The method of claim 13, wherein the composite is administered
orally.
20. The method of claim 13, wherein the composite is administered
as a solution form selected from the group consisting of inhalation
solutions, local instillations, eye drops, intranasal
introductions, ointment for epicutaneous applications, intravenous
solutions, injection solutions, and suppositories.
21-34. (canceled)
Description
RELATED APPLICATIONS
[0001] This application is a continuation of Ser. No. 11/646,128,
filed Feb. 27, 2006 (now pending), which is a continuation of U.S.
application Ser. No. 10/747,855, filed Dec. 29, 2003 (now U.S. Pat.
No. 7,169,412), which is a continuation of U.S. application Ser.
No. 10/404,914, filed Apr. 1, 2003 (now abandoned), which is a
continuation of U.S. application Ser. No. 09/842,104, filed Apr.
30, 2001 (now abandoned), which is a continuation of U.S.
application Ser. No. 09/241,232, filed Feb. 1, 1999 (now U.S. Pat.
No. 6,312,734), which is a continuation-in-part of U.S. application
Ser. No. 09/237,801, filed Jan. 27, 1999 (now abandoned), which
claims priority to RU 98-120753, filed Nov. 23, 1998. Each of these
applications is incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The present invention relates to medicine and, more
particularly, to pharmacology, i.e., to methods for producing
medicinal agents based on a composite comprising an oxidized
glutathione composite and a platinum material, in particular
cis-platin, that are intended to be used for preventing and
treating various pathologic syndromes and diseases by way of
differentiated influence on processes of metabolism, proliferation,
differentiation and apoptosis of normal and transformed cells.
BACKGROUND OF THE INVENTION
[0003] Certain issues in modern pharmacological industry involve
counteracting two medical-biological problems: [0004] forming a
resistance (tolerance and pharmacological efficacy decrease) to
medicinal agents including cases due to activation of the MDR-genes
system; and [0005] forming of undesirable resorptive effects
manifested, first of all, by alteration of the immunocompetent cell
system and hemopoiesis; cardio-, hepato-, nephro- and
neurotoxicity.
[0006] A typical cause for these problems is a wide administration
of chemotherapeutic agents that can be quite effective, according
to their physical-chemical properties, but can also be foreign to
an organism at their very nature. Even genetic-engineering
medicines, in spite of human genes (DNA) application as a matrix
for multiplication, use single-celled organisms (Escherichia coli,
yeast cells) that bring in their own, and therefore, xenobiotic
contribution into obtained drugs.
[0007] Theoretical and practical medicinal research are now paying
greater attention to natural key metabolites, i.e., key factors
(low molecular biochemical substances) that are naturally
determined to trigger chain reactions for endogenous production and
modification of many biologically active products for
physiologically important and adequate processes. In some cases
these biochemical substances function as "biochemical gyroscopes"
assigned to restoring the balance of altered equilibrium of basic
metabolic processes, for example, anabolism and catabolism or
proliferation and differentiation. Alteration of the balance of
these vital mechanisms can lead to cell destruction (cytolytic
syndrome) or their transformation into malignant ones, i.e.,
cancer. As a rule, the key regulatory metabolites, i.e., "cellular
hormones", are peptide origin factors (usually not larger than 3-20
amino acids). Obtaining synthetic analogues (biochemical
substances) and, therefore, drugs with predetermined properties is
a desired goal because these drugs are optimal as metabolic therapy
instruments and, in fact, are not foreign to the organism.
[0008] Ideal peptide structures that can function as these
synthetic analogues include sulphur-containing peptides and
derivatives thereof, due to the presence of a thiol group. In
particular, biological effects of the tripeptide "reduced
glutathione" (.gamma.-glutamyl-cysteinyl-glycine; hereinafter--GSH)
are known to be researched extensively. The glutathione tripeptide
dimer, oxidized glutathione (.gamma.-glutamyl-cysteinyl-glycine;
hereinafter--GSSG), where two molecules of the tripeptide with the
aforementioned formula are linked via a covalent bond between
cysteine residues, is also well known.
[0009] Administration of the exogenous synthetic GSSG analogue is
known to induce cytokine and hemopoietic factor synthesis during in
vitro experiments, and to provide the setting of the cytokine
profile to normal values in conditions of cyclophosphamide and
radiation immunodepression models (in vivo experiments) along with
the immunity and hemopoietic system restoration (International
application WO 97/21444, MKI A61 .kappa.38/02, published
19/06/97).
[0010] In their turn, the exogenous GSSG drug forms applied to
severe immunodeficient conditions and suppressed bone marrow
hemopoiesis containing chemical examples relating to clinical
Examples in the International Application on AIDS patients,
patients with oncopathology, aplastic anemia, and other conditions
at treatment courses of different durations, can provide a curative
effect in restoring the organism immune status (including antitumor
immunity indices), immunogenesis and hemopoietic functional
activity (International application WO 97/21444, MKI A61
.kappa.38/02, published 19/06/97).
[0011] Previously, the thiol class of biologically active substance
had applications aimed to provide GSH at increased levels, i.e., to
obtain antioxidant effects. In addition, pharmacological activity
was observed, namely, for the pro-oxidant effect gaining and
forming of a new intracellular redox-balance by introduction of
GSSG that possesses pro-oxidant potential into an organism. This is
the only known case of triggered redox-sensitive mechanisms for
immunologically significant genes, activation of the cellular thiol
metabolism, and therefore, beneficial pharmacological properties
were provided including systemic cell-protective effects and
immunity state regulation depending on initial cell status:
immunodeficiency, i.e., hyporeactivity, immunoautoaggression, i.e.,
hyperreactivity.
[0012] International Application WO 97/21444,MKI A61 .kappa.38/02,
published 19/06/97, is directed to gain a set of technical and
pharmaceutically acceptable solutions effective for the prevention
of the GSSG reduction into GSH and, thus, for extending the
lifetime of GSSG as the oxidized form in biological media.
Attainment of the biological-pharmacological effects of the
glutathione oxidized form is proven by the biomedical investigation
results obtained in the course of the complex and extensive
preclinical and clinical studies program on synthetic GSSG analog
effectiveness. A strategy for extending the lifetime of oxidized
GSSG includes providing: salts thereof, composite drugs including
GSSG combinations with substances that prolong or enhance the
effect of GSSG or salts thereof, or derivatives as a new composite,
i.e., a mixture of oxidized GSSG and other materials. The
pharmaceutically acceptable GSSG derivative in the form of the
salts thereof, or combinations with extenders of the GSSG existence
in the oxidized form, or GSSG combinations with
enhancers/modifiers, i.e., all technical solutions stabilizing in
the varying degree the GSSG molecule disulfide form were first
demonstrated to be significantly more effective in inducing the
cytokine and hemopoietic factor production in normal conditions and
to a greater degree in pathologic ones.
[0013] GSSG derivative drug forms are characterized with a larger
fraction of the GSSG stabilized in the disulfide form, with maximal
pharmacokinetics in biological media. These forms manifested the
following features:
a) Inducting production of a wider range of cytokine and
hemopoietic factors that can determine the presence of largely
modulating effects rather than only stimulating ones. b)
Reproduction of particular cytokine effects, for instance,
IL-2.
[0014] The events developing in cells (tissues and, therefore,
organs) after interaction with cytokines is well known. These
events are conditioned by the universal cytokine influence on the
main signal-transducing systems and, through the latter, on the
cell genome determining regulating cytokine effects on
proliferation, differentiation, and apoptosis.
[0015] Methods for obtaining the oxidized GSSG form from the
reduced GSH precursor are well known. The labile mercapto-5H-groups
of cysteine in GSH can be oxidized with such soft oxidizers as air
oxygen (R. Douson, D. Elliot, W. Elliot, K. Jones. Biochemist's
manual, Moscow, "Mir", 1991; Tam J. P. et al., Int. J. Pept. Prot.
Res., Vol. 29, p. 421-431, 1987; Ahmed A. K. et al., J. Biol. Chem.
250, p. 8477-8482, 1975). However, the reaction rate is rather low
in this case and desired quantitative yields require very long
periods of time (many days). Catalysis by heavy metals ions and,
especially, by copper can be toxic, and can create significant
problems for obtaining pure pharmaceutical medicines.
[0016] Another oxidation method involves more potent oxidizers such
as, hydrogen peroxide, iodine, potassium ferrocyanide, etc. (Kamber
B. et al., Helv. Chim. Acta., Vol. 63, p. 899-915, 1980; Hope D. B.
et al., J. Biol. Chem., Vol. 237, p. 1563-1566, 1962). These
reactions generally proceed much faster (dozens of minutes to
several hours). A disadvantage, however, is a difficulty in
controlling reaction conditions that can result in significant
contamination of the product with oxidation products, e.g.,
derivatives of the corresponding acids. It is sometimes necessary
to add additional, sometimes rather labor-consuming purification
procedures, that can sharply appreciate the process.
[0017] Yet another oxidation method involves the use of gaseous
substances (nitrogen oxides), sulfoxides and other compounds as
oxidizers. These oxidizers, however, can be toxic. (William A. Kato
et al., Chem. Pharm. Bull., vol. 34 (2), p. 486-495, 1986; A.
Meister et al., Ann. Rev. Biochem., p. 711-718, 1983.)
[0018] All these methods do not necessarily improve the
quantitative yield of the desired product compared to older
methods, and at the same time, can provide additional problems
regarding toxicity and safety, as in case of nitrogen oxides, or
more difficult accessibility of the reagents and their high
cost.
[0019] Another previously known method involves the use of hydrogen
peroxide as an oxidizer (Amber B. et al., Helv. Chim. Acta., Vol.
63, p. 899-915, 1980). The process is performed in the water
solution with pH about 8.0-8.5 using the hydrogen peroxide
equivalent at the room temperature. The reaction time is about 1
hour and the product yield is 90%. The main impurities (up to 10%)
are other oxidation products, which can be removed only by means of
an expensive preparative HPLC separation that can sharply increase
the drug cost.
SUMMARY OF THE INVENTION
[0020] The present invention involves the creation of new
pharmaceutically acceptable compounds with predetermined properties
based on GSSG, i.e., an oxidized glutathione-based compound having
a stabilized disulfide bond. The present invention also provides a
new method for obtaining the oxidized glutathione as a composite
with the stabilized disulfide bond during the product synthesis.
The method involves the production of the composite having the
formula: bis-(.gamma.-L-glutamyl)-L-cysteinyl-bis-glycine disodium
salt with a platinum material such as cis-platin, i.e.,
cis-diamminedichloroplatinum, preferably in the mole ratio 3000:1,
more preferably in a mole ratio of 1000:1.
[0021] According to the invention the composite is characterized as
having a stabilized disulfide bond. Upon introduction into
biological media, consequently, a longer drug half-life time is
provided in the biological media in the disulfide form.
[0022] The general procedure of the present method for the
composite production involves using the reduced glutathione for the
oxidation reaction as a hydrogen peroxide oxidizer combined with a
platinum material, in particular, cis-diamminedichloroplatinum, as
a catalyst. The method allows using lesser amounts of hydrogen
peroxide (for example, 0.9 of an equivalent), resulting in an
elimination of the superoxidation products along with very high
yield for GSSG (more than 98% by the HPLC data). Thus, the product
obtained has high purity and does not require additional
purification.
[0023] One aspect of the present invention provides a composite
comprising an oxidized glutathione-based compound and a metal
material in a ratio between about 3000:1 to about 1:1. The metal
material comprises a metal selected from the group consisting of
platinum and palladium.
[0024] Another aspect of the invention provides a method for
stabilizing a disulfide bond of an oxidized glutathione-based
compound. The method comprises interacting the oxidized
glutathione-based compound with a metal material. The metal
material comprises a metal selected from the group consisting of
platinum and palladium.
[0025] Another aspect of the present invention provides a method of
stimulating endogenous production of cytokines and hemopoietic
factors. The method comprises the steps of introducing to a
mammalian body, in need of stimulation of cytokines or hemopoietic
factors or both, an effective amount of a composite. The composite
comprises an oxidized glutathione-based compound and a metal
material in a ratio of between about 3000:1 to about 1:1. The metal
material comprises a metal selected from the group consisting of
platinum and palladium. The method is carried out for a period of
time to stimulate the endogenous production to obtain a therapeutic
effect.
[0026] Another aspect of the present invention provides a method
for enhancing and prolonging the ability of an oxidized
glutathione-based compound to stimulate endogenous production of
cytokine and hemopoietic factors. The method involves the steps of
interacting the oxidized glutathione-based compound with a metal
material in a ratio of between about 3000:1 to about 1:1. The metal
material comprises a metal selected from the group consisting of
platinum and palladium.
[0027] Another aspect of the present invention provides a method
for treating a subject having a disease. The method comprises
administering to the subject in need of such treatment a composite
comprising an oxidized glutathione-based compound and a metal
material in a ratio of between about 3000:1 to about 1:1 in an
amount effective to stimulate endogenous production of cytokines
and/or hemopoietic factors or both to obtain the therapeutic
effect. The metal compound has a metal selected from the group
consisting of platinum and palladium.
[0028] Other advantages, novel features, and objects of the
invention will become apparent from the following detailed
description of the invention when considered in conjunction with
the accompanying drawings, which are schematic and which are not
intended to be drawn to scale. In the figures, each identical or
nearly identical component that is illustrated in various figures
is represented by a single numeral. For purposes of clarity, not
every component is labeled in every figure, nor is every component
of each embodiment of the invention shown where illustration is not
necessary to allow those of ordinary skill in the art to understand
the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0029] FIG. 1 shows the structure of
bis-(.gamma.-L-glutamyl)-L-cysteinyl-bis-glycine disodium salt with
cis-diamminedichloroplatinum;
[0030] FIG. 2 shows the synthesis scheme of the composite of the
oxidized glutathione disodium salt with
cis-diamminedichloroplatinum;
[0031] FIG. 3 shows the donor-acceptor bond among platinum atom and
two NH.sub.3 groups due to the lone-pair electrons of nitrogen
atoms;
[0032] FIG. 4 shows proposed mechanisms for the GSSG molecule
disulfide bond stabilization due to the ligand exchange
--NH.sub.3-groups on disulfide bonds--and through forming of the
donor-acceptor bond among platinum atom and two sulfur atoms due to
lone-pair electrons of sulfur atoms;
[0033] FIG. 5 shows proposed mechanisms of the general GSSG
molecule stabilization through the mechanism given at FIG. 4 as
well as through exchange of the NH.sub.3 ligands on NH.sub.2 groups
of the glutathione (the NH.sub.2 group convergence and the GS
fragments, correspondingly) forming new "biophysics" of the
GSSG.cndot.Pt composite, with squares (.quadrature.) denoting donor
sites and circles (.largecircle.) denoting acceptor sites;
[0034] FIG. 6 shows the main sites (encircled) for the
GSSG.cndot.Pt molecule chemical modification;
[0035] FIG. 7 shows the structure of
bis-phenylalanyl-GSSG.cndot.Pt;
[0036] FIG. 8 shows the synthesis scheme for
bis-(L-phenylalanyl-.gamma.-L-glutamyl)-L-cysteinyl-bis-glycine
with cis-diamminedichloroplatinum;
[0037] FIG. 9 shows the structure of lithium salt of
bis-(.gamma.-L-glutamyl)-L-cysteinyl-bis-glycine;
[0038] FIG. 10 shows the stage of processing of the source reduced
glutathione with hydrogen peroxide at pH=8 (stage of the synthesis
scheme for the GSSG.cndot.Pt lithium salt);
[0039] FIG. 11 shows the extraction of the free hexapeptide
bis-(.gamma.-L-glutamyl)-L-cysteinyl-bis-glycine (III, stage of the
synthesis scheme for the GSSG.cndot.Pt lithium salt);
[0040] FIG. 12 shows the transfer of the oxidized GSSG (III) form
in the lithium salt of the GSSG.cndot.Pt;
[0041] FIG. 13 shows the DNA degradation character of the normal
cells at the control group (1), after treatment with: GSSG (2),
GSSG.cndot.Pt (3) incubation time--48 hrs;
[0042] FIG. 14 shows the DNA degradation character of the HL-60
cells at the control group (2), after treatment with: GSSG (1),
GSSG.cndot.Pt (3) incubation time--48 hrs;
[0043] FIG. 15(a) shows the structure of
S-thioethylamine-glutathione disulfide;
[0044] FIG. 15(b) shows the structure of bis-[DL-6,8-thioetic
acid].cndot.glutathione disulfide;
[0045] FIG. 15(c) shows the structure of
[.beta.-alanyl-L-histidyl].cndot.glutathione disulfide;
[0046] FIG. 15(d) shows the structure of
[9-.beta.-D-ribofuranosyladenyl].cndot.glutathione disulfide;
[0047] FIG. 15(e) shows the structure of
bis-[L-2-amino-4-[methylthio]butanoic acid].cndot.glutathione
disulfide;
[0048] FIG. 16(a) shows the structure of
bis-[methionyl].cndot.glutathione disulfide;
[0049] FIG. 16(b) shows the structure of
bis-[aspartyl].cndot.glutathione disulfide;
[0050] FIG. 16(c) shows the structure of
bis-[histidyl].cndot.glutathione disulfide;
[0051] FIG. 16(d) shows the structure of
bis-[3-iodine-tyrosyl].cndot.glutathione disulfide;
[0052] FIG. 16(e) shows the structure of
[.gamma.-aminobutanoyl].cndot.glutathione disulfide;
[0053] FIG. 16(f) shows the structure of
bis-[.gamma.-hydroxybutanoyl].cndot.glutathione disulfide;
[0054] FIG. 16(g) shows the structure of
bis-[3,4-dihydroxyphenylalaninyl].cndot.glutathione disulfide;
[0055] FIG. 17(a) shows the structure of bis-nicotinoyl-glutathione
disulfide (bis-[pyridine-3-carbonyl].cndot.glutathione
disulfide);
[0056] FIG. 17(b) shows the structure of
uridine-5'-monophosphatoyl.cndot.glutathione disulfide;
[0057] FIG. 17(c) shows the structure of
inosine-5'-monophosphatoyl.cndot.glutathione disulfide;
[0058] FIG. 17(d) shows the structure of
folliculylsuccinyl.cndot.glutathione disulfide;
[0059] FIG. 17(e) shows the structure of
glycerol-1,3-diphosphatyl.cndot.glutathione disulfide;
[0060] FIG. 18(a) shows the structure of
tetra-dopamine.cndot.glutathione disulfide;
[0061] FIG. 18(b) shows the structure of
theophylline.cndot.glutathione disulfide;
[0062] FIG. 19(a) shows the structure of bis-[carnosyl]-GSSG;
[0063] FIG. 20(a) shows the structure of a non-symmetric mixed
disulfide compound;
[0064] FIG. 20(b) shows the structure of a symmetric mixed
disulfide compound;
[0065] FIG. 20(c) shows the structure of a symmetric and doubly
bridged mixed disulfide compound;
[0066] FIG. 21(a) shows the structure of divanadate salts;
[0067] FIG. 21(b) shows the structure of dihydrofluoride salts;
[0068] FIG. 21(c) shows the structure of dilithium salts;
[0069] FIG. 21(d) shows the structure of dizinc salts;
[0070] FIG. 22 shows a scheme for modifying a GSSG.cndot.Pt
composite having phenylalanyl groups;
[0071] FIG. 23 shows a GSSG pharmokinetic curve for intravenous
introduction;
[0072] FIG. 24 shows a GSSG pharmokinetic curve for intravenous
introduction;
[0073] FIG. 25 shows a chart of immune system response to cancers
and diseases;
[0074] FIG. 26 shows a chart of drug activation of immune systems
to fight cancers and diseases; and
[0075] FIG. 27 shows a chart of cytokines stimulated by
drug-type.
DETAILED DESCRIPTION
[0076] The present invention discloses a number of oxidized
glutathione-based compounds having a stabilized disulfide bond and
in particular, a composite comprising the compound of this
invention with a metal material. Methods for the preparation of a
composite and for using the composite to treat a variety of
diseases are also disclosed.
[0077] One aspect of the present invention provides a composite
comprising an oxidized glutathione-based compound and a metal
material. Generally, a "composite" can refer to a mixture of
different chemical species. The "mixture" can be a physical mixture
or a chemical mixture, i.e., having a chemical interaction
involving either a chemical bond or an electrostatic interaction.
In one embodiment, the mixture can be prepared by dissolving and/or
suspending the different chemical species in a solution and
precipitating out or filtering out a resulting solid. In another
embodiment, the mixture can be a homogeneous solution comprising
the different chemical species.
[0078] An "oxidized glutathione-based compound" refers to any
compound having a basic dimer structure where each unit of the
dimer comprises a glutamic acid group or salt or derivative bonded
to a cysteine group or salt or derivative bonded to a glycine group
or salt or derivative, and each unit is correspondingly bonded to
each other by the cysteine sulfur atoms to form a sulfur-sulfur
bond (disulfide bond). A "derivative" can be prepared by reacting
at least one reactive group of the oxidized glutathione-based
compound or precursor with another chemical species. An example of
an oxidized glutathione-based compound is GSSG itself. Other
examples are provided below.
[0079] In one embodiment, the oxidized glutathione-based compound
has the general formula:
##STR00001##
[0080] A, B, D, E, G and H can each be selected from the group
consisting of an organic unit and salts of the organic unit.
Preferably, the "organic unit" allows the glutathione-based
compound to remain soluble in biological media and in addition, the
organic unit should not impart toxicity to the oxidized
glutathione-based compound in an applied dosage. It is understood
that A, B, D, E, G and H can be the same or different. Preferably,
groups A-H can each include a unit selected from the group
consisting of amine groups, carboxyl groups, and amides. For
example, A-H can represent amino acids or derivatives bonded via an
amide bond. Alternatively, any two of A-H can be linked to each
other by at least one covalent bond. Thus, A-H can be part of a
cyclic structure.
[0081] In one embodiment, the composite comprises a large excess of
the oxidized glutathione-based compound relative to the metal
material, preferably in a ratio of between about 3000:1 and 1:1,
more preferably in a ratio of between about 1000:1 and 1:1, more
preferably in a ratio of between about 1000:1 and 10:1, even more
preferably in a ratio of between about 1000:1 and 100:1. In another
embodiment, the composite comprises equal amounts of the oxidized
glutathione-based compound and the metal material, i.e., a ratio of
about 1:1.
[0082] In one embodiment, the metal material comprises a metal
selected from the group consisting of platinum and palladium.
Preferably, the metal is platinum. Ideally, the metal material, in
combination with the oxidized glutathione-based compound, renders
the composite soluble in biological media. Small portions of the
metal material can be insoluble, as long as the insoluble portion
does not result in any toxic or hazardous effects to the biological
system. A platinum material can be selected from the group
consisting of a platinum salt, a coordination compound and an
organometallic compound. Preferably, the platinum material is a
platinum coordination compound such as cis-platin
(cis-Pt(NH.sub.3).sub.2Cl.sub.2 or
cis-diamminedichloroplatinum--FIG. 3).
[0083] In one embodiment, the oxidized glutathione-based compound
is oxidized glutathione itself (GSSG) and salts thereof, where both
A and E are --CO.sub.2H, both B and D are --NH.sub.2 and both G and
H are --CO.sub.2M, M being a counterion. The counterion can be a
proton, an organic-based ion such as tetralkyl-amononium, an
alkaline metal, an alkaline earth metal, or a transition metal. It
is understood that in aqueous media, any of A-H can comprise an
ionized group, e.g., A and E can be --CO.sub.2.sup.-, and B and D
can be --NH.sub.2.sup.+ and the ionized groups are neutralized by
an appropriate counterion.
[0084] In a preferred embodiment, the present invention relates to
the production of a new biologically active compound, i.e., a
composite comprising an oxidized glutathione-based compound and
cis-diamminedichloroplatinum. A convenient short-form notation will
be used herein. For example, a composite comprising GSSG itself and
cis-platin will be denoted as GSSG.cndot.Pt. Derivatives will be
denoted by the newly appended chemical group, e.g.,
bis-[histidyl]-GSSG. While not wishing to be bound by any theory, a
proposed structural formula can be found in FIG. 1. (Gross-formula:
C.sub.20H.sub.30N.sub.6O.sub.12Na.sub.2S.sub.2.[Pt(NH.sub.3).sub.2Cl.sub.-
2]; molecular weight: 656.59 on
C.sub.20H.sub.30N.sub.6O.sub.12Na.sub.2S.sub.2 with Pt content
0.022-0.042%.)
[0085] The composite can be prepared by various methods. For
example, the composite can result from the addition of a metal
material to glutathione in the presence of an oxidant.
Alternatively, the composite can result by the addition of a metal
material to the oxidized glutathione-based compound.
[0086] Thus, another aspect of the invention provides a method for
stabilizing a disulfide bond of an oxidized glutathione-based
compound. "Stabilizing a disulfide bond" refers to a process for
maintaining a bond between two sulfur atoms and preventing facile
reversion of the oxidized glutathione-based compound (e.g., GSSG)
back to the reduced form (e.g., GSH). Alternatively, stabilizing
the disulfide bond can result in an increased lifetime of the
oxidized glutathione-based compound. In the presence of reductants,
the disulfide bond can cleave resulting in formation of the reduced
form of the glutathione-based compound, which is an undesired
reaction. By maintaining the glutathione-based compound in an
oxidized form for a greater amount of time, the compound can be
pharmaceutically effective for a correspondingly longer period of
time in biological media.
[0087] In one embodiment, the disulfide bond can be stabilized by
interacting the oxidized glutathione-based compound with a metal
material. As discussed previously, the metal material is preferably
a platinum or palladium material, such as cis-platin. In one
embodiment, the platinum material is present in an amount of
between about 0.0003 equivalent to about 1 equivalent relevant to
the oxidized glutathione-based compound, preferably in a ratio of
between about 0.001 equivalent to about 1 equivalent relevant to
the oxidized glutathione-based compound.
[0088] In one embodiment, "interacting the oxidized
glutathione-based compound with a metal material" comprises
providing a glutathione-based compound and reacting this compound
with an oxidant and a platinum material. A "glutathione-based
compound" refers to any compound having a structure comprising a
glutamic acid/salt/derivative bonded to a cysteine/salt/derivative
bonded to a glycine/salt/derivative. Examples of glutathione-based
compound include glutathione itself or any derivative, where a
derivative can be prepared by reacting a reactive group with
another chemical species. The resulting product will be an oxidized
glutathione-based compound having a stabilized disulfide bond.
Thus, in this embodiment, a glutathione-based compound is in a
reduced form, such as GSH, and the reaction with a oxidant involves
oxidizing the glutathione-based compound to produce a sulfur-sulfur
bond. The oxidant can be any species which can cleave a S--H bond
of a glutathione-based compound to produce a hydrogen atom and a
compound having a sulfur-based radical which ultimately can react
with another sulfur-based radical to provide the sulfur-sulfur
bond. Various oxidants that can perform this S--H bond cleavage are
well known in the art. In a preferred embodiment, the oxidant is
selected from the group consisting of oxygen and hydrogen
peroxide.
[0089] In this method, reacting a glutathione-based compound with
an oxidant and the platinum material comprises an oxidation
reaction. Relative amounts of the reactants are preferably about 1
equivalent of the glutathione-based compound with less than about 1
equivalent of the oxidant such as hydrogen peroxide, and more
preferably, with about 0.9 equivalent of the hydrogen peroxide. In
another embodiment, the oxidation reaction comprises reacting about
1 equivalent of the glutathione-based compound with between about
0.0003 equivalent and about 1 equivalent of the platinum material,
preferably between about 0.001 equivalent and about 1 equivalent of
platinum material, more preferably between about 0.001 equivalent
and about 0.1 equivalent, and even more preferably between about
0.001 equivalent and 0.01 equivalent, in the presence of less than
1 equivalent of the oxidant. In another embodiment, about 1
equivalent of the glutathione-based compound is reacted with about
1 equivalent of the platinum material in the presence of less than
1 equivalent of the oxidant.
[0090] In one embodiment, the method involves oxidizing the
glutathione-based compound with about 0.9 equivalent of hydrogen
peroxide and about 0.001 equivalent of cis-platin. One advantageous
feature of this method is an increased rate of oxidation of the
glutathione-based compound. Another advantageous feature of this
method is that the yield of the resulting composite is increased to
an amount greater than about 98% and this increased yield is
accompanied by an increased purity. The purification of this
composite is simplified to a significant degree in that liquid
chromatography can be performed to obtain a purity of the composite
of greater than 99%, which complies with pharmaceutical standards.
Prior art methods have achieved a purity of only 75-93% of oxidized
glutathione, depending on the method.
[0091] In a preferred embodiment, the composite is synthesized in
one step by oxidizing the reduced glutathione in the presence of
cis-diamminedichloroplatinum, which may function as an oxidation
reaction catalyst. The reaction conditions can be regulated
accurately by using less than 1 equivalent of hydrogen peroxide.
Consequently, formation of superoxidation products can be reduced,
resulting in a near quantitative yield of the product. Thus, the
one-step composite synthesis provides significant technological
simplification and production of the composite GSSG.cndot.Pt with
the stabilized disulfide bond.
[0092] In a preferred embodiment, the reaction is performed in a
solution involving reduced glutathione as a monosodium salt and
adding about 0.9 equivalent of the hydrogen peroxide and about
0.001 equivalent of cis-diamminedichloroplatinum at room
temperature with stirring. The oxidation reaction typically
proceeds in about 1.5-2 hours. Control for the completeness of the
oxidation process can be conducted by an HPLC assay. The process is
completed by the reaction solution lyophilic drying to produce the
composite consisting of oxidized glutathione and
cis-diamminedichloroplatinum in a mole ratio of 1000:1 (confirmed
by spectral analysis on platinum and sodium). The peptide
constituent of the obtained composite according to the data of an
amino acid assay, a NMR (.sup.1H) spectrum, retention time by HPLC
corresponds to GSSG. The admixtures content do not exceed 2%, and
the product yield as a disodium salt is 96-98% calculating for the
dry composite.
[0093] A composite synthesis scheme can be found on FIG. 2.
[0094] While not wishing to be bound by any theory, the increased
stabilization of the disulfide bond can be the result of an
interaction of the sulfur atoms with the platinum material (see
also FIG. 4).
##STR00002##
[0095] In an interaction between the platinum material and the
oxidized glutathione (GSSG) molecule there is a possibility for
ligand exchange, i.e., instead of the NH.sub.3 groups, two sulphur
atoms possessing two pairs of the lone-pair electrons can be
involved in donor/acceptor bonds with the platinum atom. One can
also consider the possibility for the addition in regard to the
aforesaid stabilization of the disulfide bond due to the
convergence of the NH.sub.2 groups of the oxidized
glutathione-based compound and stabilization of the general GSSG
conformation (FIG. 5).
[0096] It is an advantageous feature of the present invention that
obtaining derivatives of GSSG can produce a compound having
different biological/chemical properties and/or activity. Thus,
depending on the desired use of a drug comprising a composite
including the oxidized glutathione-based compound, it is possible
to obtain a particular drug for treatment of a particular disease.
In addition, new chemical modifications of the oxidized
glutathione-based compound, such as aminogroup acylation (for
instance, bis-phenylalanyl-glutathione, and etc.), can result in a
significant decrease in the risk of secondary reactions due to
disulfide bond destruction. Reactions such as S-alkylation,
oxidation to the corresponding acids, etc., can cause particular
hardships in the working process and, in the case given, can be
minimized or excluded.
[0097] The synthetic method of the present invention and
physical-chemical modifications thereof directly in the course of
the synthesis (the disulfide bond and general molecular
conformation stabilization; the new reactive functional sites
appearance) can result in obtaining the biologically active
composite, such as the platinum material and GSSG, having new
biophysical, chemical and biochemical parameters as dictated by the
various structural-functional properties.
[0098] In one embodiment, the oxidized glutathione-based compound
can be a derivative of glutathione. The glutathione can be
derivatized after preparation of the composite, or it can be
derivatized prior to preparation of the composite, i.e., the GSH
can be derivatized prior to oxidation. FIG. 15 depict various
examples of derivatives of glutathione-based compounds, i.e.,
oxidized glutathione-based compound. FIG. 15a shows the structure
of S-thioethylamine.cndot.glutathione disulfide. FIG. 15b shows a
structure of bis-[DL-6,8-thioetic acid].cndot.glutathione
disulfide. FIG. 15c shows a structure of
[1-alanyl-L-histidyl].cndot.glutathione disulfide. FIG. 15d shows a
structure of [9-.beta.-D-ribofuranosyladenyl].cndot.glutathione
disulfide. FIG. 15e shows a structure of
bis-[L-2-amino-4-[methylthio]butanoic acid].cndot.glutathione
disulfide.
[0099] In one embodiment, the oxidized glutathione-based compound
has an acylated primary glutamic acid amino group. This variant is
most suitable for acylation by N-protected activated amino acid
derivatives, where after the stage of temporary protection of the
cysteine mercapto-groups followed by condensation (activated ester
method), and removal of the N- and S-protective groups and
oxidation by the hydrogen peroxide with addition of
cis-diamminedichloroplatinum results in the GSSG.cndot.Pt composite
modified by amino-acids at the glutamic acid aminogroups. In this
embodiment the oxidized glutathione-based compound can be selected
from the group consisting of bis-[methionyl].cndot.glutathione
disulfide (FIG. 16a), bis-[aspartyl].cndot.glutathione disulfide
(FIG. 16b), bis-[histidyl].cndot.glutathione disulfide (FIG. 16c),
bis-[3-iodine-tyrosyl].cndot.glutathione disulfide (FIG. 16d),
[.gamma.-aminobutanoyl].cndot.glutathione disulfide (FIG. 16e),
bis-[.gamma.-hydroxybutanoyl].cndot.glutathione disulfide (FIG.
16f), bis-[DL-6,8-thioetic acid].cndot.glutathione disulfide (FIG.
15b), and bis-[3,4-dihydroxyphenylalaninyl].cndot.glutathione
disulfide (FIG. 16g).
[0100] When the composite includes phenylalanyl groups, the
compound is bis-phenylalanyl-GSSG.cndot.Pt (see FIG. 7). According
to this basic scheme (as shown in FIG. 22), all other GSSG.cndot.Pt
modifications based on the aminogroups acylation by the derivatives
of the protected aminoacids, oxy-acids, carbonic acids and
derivatives thereof are synthesized. Slight changes of the methods
are possible due to a specific nature of modifying molecule.
Protecting groups for the initial GSH compound include Bam
(N-hydroxymethylbenzamide). The amino acid (AA) can be protected
with groups such as BOC (butyl carbamate) or O-Su (N-oxysuccinimide
ester; see FIG. 8 for specific details).
[0101] When the composite is bis-methionyl-GSSG.cndot.Pt, i.e.,
(Met).sub.2GSSG.cndot.Pt, incorporation of a methionine group may
involve BOC-protective group deblocking after the condensation
stage.
[0102] When the composite is bis-aspartyl-GSSG.cndot.Pt, an
aspartic acid group can be introduced according to the general
aforementioned scheme. A temporary protective group for the
.beta.-carboxyl group is preferably removed, if possible, with the
BOC-protective group simultaneously if needed. A .beta.-tert-butyl
ester of N-BOC-aspartic acid, i.e., BOC-Asp(OBu.sup.t)-OSu, can be
applied at the condensation stage. The protective groups can be
removed by trifluoroacetic acid (TFA).
[0103] When the composite is bis-histidyl-GSSG.cndot.Pt, a
histidine imidazole ring can be protected via a
di-tert-butyl-oxycarbonyl histidine derivative, i.e.,
BOC-His(BOC)--OSu, which can be used at the condensation stage. As
in previous cases, the protective groups can be removed by
trifluoroacetic acid.
[0104] When the composite is bis-3-iodine-tyrosyl-GSSG.cndot.Pt, a
possible protecting group is an acid-labile protective group such
as tert-butyl ester. The tyrosine derivative used at the
concentration stage can be BOC-Tyr(OBu.sup.t)--OSu.
[0105] When the composite is GABA-GSSG.cndot.Pt,
(.gamma.-aminobutanoyl-GSSG.cndot.Pt), an acid-labile protective
tert-butyloxycarbonyl (BOC) group can be used at the condensation
stage.
[0106] When the composite is GOBA-GSSG.cndot.Pt,
(.gamma.-hydroxybutanoyl-GSSG.cndot.Pt), an acid-labile group
tert-butyl ester (which can be removed by a trifluoroacetic acid
solution) can be used to protect a GOBA hydroxyl group. A
derivative, used at the condensation stage, can be
GABA(OBu.sup.t)--OSu.
[0107] When the composite is bis-lipoyl-GSSG.cndot.Pt, it is
believed that side functional groups of lipoic acid do not require
special protection. At the condensation stage it is possible to
apply an activated (hydroxysuccinimide) ester of lipoic acid. There
may be no need for a TFA treatment.
[0108] When the composite is
bis-3,4-dihydrooxyphenylalanyl-GSSG.cndot.Pt
(bis-DOPA-GSSG.cndot.Pt), to introduce DOPA molecule, it may be
necessary to previously protect two hydroxyl groups of
3,4-dihydroxyphenylalanine by tert-butyl esters and to protect the
aminogroup by a BOC-protective group. For condensation with a
composite precursor, an activated ester can be obtained
(hydroxysuccinimide or pentafluorophenyl one) that is used in
excessive mole amount. Removal of the protecting groups can occur
simultaneously with a trifluoroacetic acid solution.
[0109] When the composite is bis-[carnosyl]-GSSG.cndot.Pt
(bis-.alpha.-alanyl-L-histidyl-GSSG.cndot.Pt), carnosine can be
protected as a di-BOC derivative of the .beta.-alanine aminogroup
and histidine imidazole group before condensation. Then
condensation and following deblocking can be performed as
previously described. A structure of bis-[carnosyl]-GSSG can be
found in FIG. 19a.
[0110] In another embodiment, the oxidized glutathione-based
compound has an amide or phosphoramide bond to a unit selected from
the group consisting of heterocyclic carbonic acids and
nucleotides. In this embodiment, examples of the oxidized
glutathione-based compound include bis-nicotinoyl-glutathione
disulfide (bis-[pyridine-3-carbonyl].cndot.glutathione disulfide)
(FIG. 17a), uridine-5'-monophosphatoyl.cndot.glutathione disulfide
(FIG. 17b), inosine-5'-monophosphatoyl.cndot.glutathione disulfide
(FIG. 17c), folliculylsuccinyl.cndot.glutathione disulfide (FIG.
17d) and glycerol-1,3-diphosphatyl.cndot.glutathione disulfide
(FIG. 17e).
[0111] When the composite is bis-nicotinoyl-GSSG.cndot.Pt
(bis-pyridine-3-carbonoyl-GSSG.cndot.Pt), a nicotinic acid
containing no side functional groups can be introduced into
condensation with a composite precursor without obtaining protected
derivatives as corresponding activated esters such as
hydroxysuccinimide or pentafluorophenyl. TFA treatment for the
removal of protecting groups may not be required.
[0112] When the composite is
uridine-5'-monophosphatoyl-GSSG.cndot.Pt (UMP-5'-GSSG.cndot.Pt),
uridine-5'-monophosphate in presence of
N,N-dicyclohexyl-carbodiimide can form phosphoamide links in
reactions with amides (Chambers R. W., J. Am. Chem. Soc., 80, 3752,
1958). The composite precursor can have protected carboxyl groups,
such as tetratrimethylsilyl derivatives to be used as an
aminocomponent. Deblocking can proceed in mild water-alcohol
systems.
[0113] When the composite is
ionosine-5'-monophosphatoyl-GSSG.cndot.Pt, IMP-5'-GSSG.cndot.Pt,
the synthetic scheme would most likely be similar to that of the
previous derivative (UMP-5'-GSSG).
[0114] When the composite is folliculylsuccinyl-GSSG.cndot.Pt, a
link between GSSG.cndot.Pt and estrone can be made by amide and
ester bonds through a succinyl residue. Estrone can be transformed
into an activated derivative by reaction with succinanhydride with
following condensation by N,N-dicyclohexylcarbodiimide with a
protected or blocked composite precursor and with
tetra-trimethylsilyl derivatives as well. Deblocking can be
performed in a water-alcohol system.
[0115] When the composite is
glycerol-1,3-diphosphatyl-GSSG.cndot.Pt, the modification can
proceed by a carbodiimide method using a composite precursor
protected, as a tetra-trimethylsilyl derivative as an amino
component, the synthesis is similar to that in the synthesis of
phosphoamide derivatives (See Examples 13 and 14).
[0116] In another embodiment, the oxidized glutathione-based
compound can be selected from the group consisting of
tetra-dopamine.cndot.glutathione disulfide (FIG. 18a) and
theophylline.cndot.glutathione disulfide (FIG. 18b). The formation
of amide links can occur between composite carboxyl groups and
amides. The reactivity of all four carboxyl groups is very similar
and, therefore, a mixture of products can result.
[0117] When the composite is tetra-dopamine-GSSG.cndot.Pt, a
3,4-di-tert-butyl ester of dopamine can be used as an
aminocomponent and di-tert-butyloxycarbonyl derivatives the
composite can be used as a carboxyl component. Condensation can
proceed by a N,N-dicyclohexyl-carbodiimide, and removal of the
protective groups can be performed with a trifluoroacetic acid
solution.
[0118] When the composite is GSSG.cndot.Pt-theophylline,
theophylline can be used as an aminocomponent, a composite
precursor can be used for a carboxyl component as a
di-tert-butyloxicarbonyl derivative. Condensation can be performed
with an "F" complex. The removal of protective group can be
performed with a trifluoroacetic acid solution.
[0119] In another embodiment, the oxidized glutathione-based
compound can include mixed disulfides. Possible combined structures
can involve mixed disulfide formation (symmetric and
non-symmetric). (See FIGS. 20a-20c)
[0120] One structure (FIG. 20b) can be formed via mutual oxidation
of mercaptogroups starting materials. There may be no need for
additional protective groups and condensation methods.
[0121] Another compound (FIG. 20a) can be obtained by formation of
an amide bond between the cyteamine aminogroup and one of the
composite precursor carboxyl groups. It may be necessary to
introduce N-protective groups and to activate the composite
precursor carboxyl groups. Due to the presence of four carboxyl
groups it may be necessary to manipulate the stoichiometry and/or
perform chromatographic separation of the resulting products.
[0122] The synthesis conditions are different from the structure
20b by presence of the additional aminocomponent equivalent. At the
chromatographic purification the structure 20b is used a
witness.
[0123] It is obtained from the structure 20c through formation of
an additional disulfide bond at the mercapto-group reaction. During
the chromatographic separation of products, it may be necessary to
have the structure 20c as a witness.
[0124] In another embodiment, the oxidized glutathione-based
compound can be a salt selected from the group consisting of alkali
metal salts, alkaline earth metal salts, and transition metal
salts. Examples of such salts include divanadate salts (FIG. 21a),
dihydrofluoride salts (FIG. 21b), dilithium salts (FIG. 21c),
didopammonium salts, and dizinc salts (FIG. 21d).
[0125] The salts can be obtained through addition of the
corresponding amount of the salt-forming components, a base or an
acid. Examples of salts with aminogroups includes a divanadate of
GSSG.cndot.Pt((HVO.sub.3).sub.2-GSSG.cndot.Pt) or a dihydrofluoride
of GSSG.cndot.Pt ((HF).sub.2.GSSG.cndot.Pt). Examples of salts with
carboxyl groups include a dilithium salt GSSG.cndot.Pt (see Example
2), a GSSG.cndot.Pt didopammonium salt or a GSSG.cndot.Pt zinc
salt.
[0126] In another aspect of the invention, a drug comprising the
composite is obtained according to the previously described method,
such as the hexapeptide
bis-(.gamma.-L-glutamyl)-L-cysteinyl-bis-glycine disodium salt and
cis-diamminedichloroplatin (cis-platin). Thus, the present
invention provides a new class of medicinal substances
"thiopoietins" that can be introduced into biological media,
resulting in a new level of metabolism and cellular genetic
activity.
[0127] An advantageous feature of the composite of the present
invention is the biological activity, in particular: [0128]
Stimulation/modulation of the endogenous production for a
significantly large range of the cytokine, growth and hemopoietic
factors in conditions of radiation and chemical immunosuppression
(IL-1.alpha. and .beta., IL-2, IL-3, IL-4, IL-6, IL-8, IL-10 and
IL-12, TNF-.alpha., IFN-.alpha. and IFN-.gamma., erythropoietin,
colony-stimulating factors) (See Examples 5-7); [0129] Reproduction
of some cytokine effects (IL-2, IL-12, IFN-.alpha. and IFN-.gamma.)
due to the mechanism induction for the redox-sensitive expression
of the immunologically significant genes and the key protein
"critical" cysteine modification for the cellular
signal-transducing systems (See Example 8); [0130] Restoration of
the depressed bone-marrow haemopoiesis including erythrocyte,
leukocyte and platelet counts as well as levels of CD3.sup.+,
CD4.sup.+, CD8.sup.+, CD16/56.sup.+, CD19/20.sup.+, CD25.sup.+,
CD34.sup.+, CD95.sup.+ (See Examples 9-13) at patients receiving
radiation and high-dose combined chemotherapy; [0131] In conditions
of an active antibacterial, antiviral and antitumor chemotherapy,
hepatotropic effects as well as diminution of the cardio-, hephro-
and neurotoxicity signs; [0132] The differentiated impact with
regard to normal cells (including the ones being under functional
stress) and transformed ones, namely, the metabolism, proliferation
and differentiation stimulation at the normal cells/tissues and,
simultaneously, the apoptosis mechanism induction only in the
tumor- and/or virus-transformed cells. The data appears to support
the induction of p53-dependent and p53-independent, Bcl-2-intensive
apoptosis pathway/types occurring (See Examples 14-16).
[0133] The method for the production of the composite makes
possible synthesis of different composites as a basis for design of
drugs possessing a range of new features, namely: [0134] Increased
biochemical drug stability, i.e., "non-assailability" by the GSSG
metabolism enzymes (i.e., far less accessible for these enzyme
action), first of all, by NADP.cndot.H.sup.+-dependent glutathione
reductase that basically increases the drug half-life time in the
biological media exactly in the disulfide form; [0135] New
biophysical component with high level of the donor-acceptor
potential; [0136] Presence of new reactive sites within the said
molecule and, therefore, entirely new capacity for chemical
modification.
[0137] The composite properties allow it to function as a unique
cellular "gyroscope" that in conditions of the extreme external
environmental factors (physical, chemical and biological ones)
provides restoration of a balance: [0138] Within the cytokine
profile, i.e., the cytokines regulating proliferation mainly; and
the cytokines regulating mainly differentiation of the
immunocompetent cells; [0139] Ratio of the cellular redox potential
including donor/acceptor balance of the electron dynamics due to
restoration of the thiol-disulfide metabolism; NAD/NAD.cndot.H and
NADP/NADP.cndot.H ratios; [0140] cAMP/cGMP ratio, changes of the
extra- and intracellular ionized calcium; [0141] Relationship of
the transcriptional differentiation factors (NF.kappa.B) and
proliferation factors (AP-1); relationship of the functional
activity manifestations of p53, p21 and ras-proteins, therefore,
balance of cellular proliferation, differentiation and apoptosis
taking into account basically different exhibitions of these
effects in the normal and transformed cells.
[0142] In the applied invention, including examples of the
preferred embodiments, the following terminology accepted is
used.
[0143] "Subject in need thereof" as used in this application herein
comprises a mammal, e.g., man, domestic animals and livestock
including cats, dogs, cattle and horses, having one or more
manifestations of a disease in which stimulation of endogenous
cytokine or hemopoietic factor (or both) production as well as an
apoptosis mechanism regulation would be considered beneficial by
those skilled in the art with an up-to-date biomedical
knowledge.
[0144] "Medicinal drug" as used in this application includes any
drug form containing the composite of the present invention, e.g.,
GSSG.cndot.Pt and derivatives, which has a therapeutic effect on
neoplastic, infectious, hematologic, immunologic, neurodegenerative
or other diseases.
[0145] "Therapeutic effect" indicates any effect in man and other
mammals which is beneficial, including curative, preventative,
allowing maintenance at a beneficial level, or being in any way
advantageous in regard to a body of man and other mammals.
[0146] "Pharmaceutically acceptable salt" as used in this
application comprises any composite derivative in the form of a
salt that is acceptable for use in the body without unwanted
detrimental effect on the body, and including, for example, sodium,
lithium, zinc or vanadium cations, or sodium, lithium, zinc or
vanadium salt respectively.
[0147] "Pharmaceutically acceptable composition" as used in this
application involves the composite, or derivative thereof, as a
pharmaceutically acceptable substance and may include, in addition,
a group of active metabolites or other chemical compounds. For
example, the composite or derivative can include or can be
covalently bound to phenylalanine or to cystamine.
[0148] "Metabolism" as used in this application involves the
totality of all biochemical reactions taking place within the
living organism responsible for vital function maintenance in the
said organism (Robert C. Bohinski "Modern Concepts in
Biochemistry", 4th edition, 1987).
[0149] "Proliferation" as used in this application involves
reproduction or multiplication of similar forms (cells) due to
constituting (cellular) elements (Stedman's Medical Dictionary,
26th edition, Moscow, 1995, p. 519; Miller-Keane "Encyclopedia
& Dictionary of Medicine, Nursing, & Allied Health:, 5th
edition, W.B. Saunders Company, 1992, p. 1221).
[0150] "Differentiation" as used in this application involves cell
changes including acquisition or possession of features
distinguishing from an original with the cell conversion from
relatively simple functions to more complex, specialized functions
as is in morphological and/or functional heterogeneity incident to
the given cellular type through the tissue-specific gene expression
(A. Horst "Molecular Basis for Disease Pathogenesis", Moscow, 1982,
p. 125; Brian W J Mahy "A Dictionary of Virology", 2-e Academic
Press, 1997, c. 87; Stedman's Medical Dictionary, 26th edition,
Moscow, 1995, p. 179; and Miller-Keane "Encyclopedia &
Dictionary of Medicine, Nursing, & Allied Health", 5th edition,
W.B Saunders Company, 1992, p. 424).
[0151] "Apoptosis" as used in this application involves
morphologically distinguishable forms of genetically programmed,
physiological cell death initiated by extra- or intracellular
signals when there are activated enzymes (e.g., the caspases group)
causing destruction (e.g., fragmentation) of nuclear DNA through
intranucleosomic cuts and morphologically manifested by (1) cell
shrinkage; (2) condensation, margination, and fragmentation of
chromatin; and (3) retention of cytoplasmic organelle structure,
but loss of positional interrelationships; further apoptotic cells
or apoptotic bodies formed out of them are engulfed
(incurphagocytosis) (Harrison's Principles of Internal Medicine,
14th edition, p. 511, 1998; Apoptosis: a role in neoplasia, C. D.
Gregory, 1996; and The Molecular Biology of Apoptosis, D. L. Vaux,
A. Strasser; Proc. Natl. Acad. Sci. USA 93 (1996)).
[0152] "Cytokines" as used in this application comprises
peptide-origin regulatory compounds produced by the different cell
types playing a key role in the immune response development,
hemopoiesis and different disease pathogenesis, performing their
effect through gene activation, participating in regulation for all
immune system elements (proliferation and differentiation of immune
competent cell precursors; antigen representation,
antigen-sensitized lymphocyte proliferation, B-lymphocyte
differentiation into antibody-producing cells, T-lymphocyte
differentiation into functionally different T-lymphocytes;
functions of macrophages, neutrophils, eosinophils, mast cells and
basophils), as well as controlling growth, differentiation,
apoptotic processes and functional activity for different tissue
cells (including fibroblasts, chondrocytes, keratinocytes,
endotheliocytes, nerve tissue cells and cardiomyocytes) (Harrison's
Principles of Internal Medicine, 14th edition, p. 511 1998;
Apoptosis: a role in neoplasia, C. D. Gregory, 1996).
[0153] As used herein, the terms "neoplastic and infectious
disease", "hemopoiesis and immunity depression of various origin",
and "other diseases" mean any neoplastic or infectious disease, any
conditions caused or accompanied by the erythroid or myeloid
suppression, or a reduction in quantitative or functional immunity
parameters, as well as any other disease or pathological condition,
in which stimulation/modulation of the aforesaid cytokine and/or
hemopoietic factor endogenous production as well as apoptosis
mechanism induction would be considered advantageous by those
skilled in the art. Thus, modulating the cytokine and hemopoietic
factor endogenous production for a person in need thereof by using
the composite with the stabilized disulfide bond (e.g.,
GSSG.cndot.cis-platin), which being introduced parenterally also
has a new feature of influencing the cytokine profile allowing
regulation of the normal cell metabolism, proliferation and
differentiation processes.
[0154] One embodiment of the present invention provides a method
for stimulating endogenous production of cytokines and hemopoietic
factors. The method includes the step of introducing to a mammalian
body in need of such stimulation an effective amount of a composite
comprising the oxidized glutathione-based compound and a metal
material for a period of time to stimulate the endogenous
production to obtain a therapeutic effect. As described previously,
the ratio of the oxidized glutathione-based compound to the metal
material is between about 3000:1 to about 1:1 where the metal
material comprises a metal selected from the group consisting of
platinum and palladium.
[0155] Therapeutic effect includes alleviation, prevention or
curing of an unwanted body condition and can comprise a process
selected from the group consisting of regulating proliferation in
normal cells, regulating differentiation in normal cells, and
inducing apoptosis of transformed cells where the transformed cells
can include diseased cells. The therapeutic effect includes
preventative, alleviation and curing effects in various
diseases.
[0156] "Disease" refers to any unwanted condition of the body
including, but not limited to, selected oncological diseases,
infectious diseases, immunological diseases, ischemic diseases,
neurodegenerative diseases, metabolic diseases, endocrinal
diseases, and any other unwanted medical condition.
[0157] The composite can be administered by various methods: orally
or as a solution form selected from the group consisting of
inhalation solutions, local instillations, eye drops, intranasal
introductions, an ointment for epicutaneous applications,
intravenous solutions, injection solutions, and suppositories.
Preferably, the glutathione is introduced parenterally or
topically. The method is carried out by introducing the composite,
derivatives or salts thereof to enhance the regulatory influence on
stimulating of the cytokine and hemopoietic factor endogenous
production; or to induce apoptosis mechanisms in transformed
tissues, which provides regulation of metabolism, proliferation and
differentiation in tissues and achieving a corresponding
therapeutic effect.
[0158] In one embodiment, the composite is administered in a dosage
of between about 0.1 mg/kg to about 1.0 mg/kg by body weight. In
another embodiment, the composite is administered in a dosage of
between about 1 mg/m.sup.2 to about 100 mg/m.sup.2 by body surface.
In another embodiment, the drugs can be applied one or more times a
day, by one or more day pulses or continuous administration until a
desired therapeutic effect is achieved.
[0159] In a preferred embodiment, the GSSG.cndot.Pt material
pharmaceutically acceptable derivatives are introduced to the body
at a dose from 0.01 to 1.0 mg of GSSG.cndot.Pt material per kg of
body weight for the GSSG.cndot.Pt material or salt thereof, or at a
dose from 1 to 100 mg per 1 m.sup.2 of body surface and in case
when applied epicutaneously/through instillations at a dose from 1
to 100 mg per 1 m.sup.2 of body surface as well, at least, once
during each 24 hour period. Also the drug can be continuously
injected or otherwise introduced to the body to have a 24 hour
total dosage from 0.01 to 1.0 mg per kg of body weight for
GSSG.cndot.Pt base and salts thereof, and from 1 to 100 mg per 1
m.sup.2 of body surface during each 24 hour period. Preferably,
administration and introduction to the body should be carried out
until a desired stimulating effect on the cytokine and hemopoietic
factor production or the apoptosis induction and, thus, the
cellular metabolism, proliferation and differentiation regulation
and corresponding therapeutic effect is obtained.
[0160] Where the composite is administered as a solution,
preferably the solution has a concentration of between about 1% to
about 10% of the composite. Preferably, the pharmaceutically
acceptable derivatives of the GSSG.cndot.Pt material for parenteral
use is in a pharmaceutically acceptable solvent as, for example, an
aqueous solution including water, glucose solution, isotonic
solutions of sodium chloride, buffered salt solutions. Other
physiological solvents or carriers can be used. Where the composite
is administered as an injectable form, preferably the injectable
form comprises the composite in a solution in a concentration of
between about 0.01% to about 3.0%.
[0161] For topical application including application for different
body cavities, organic solvents or carriers may be used in the form
of ointments, pastes, creams or suppositories.
[0162] The examples of the invention embodiments given below
demonstrate feasibility of the invention practical use and confirm
its effectiveness, and also expediency of using these medicinal
drugs as an injectable solution containing 0.01% to 3% of
GSSG.cndot.Pt base or the salts thereof with the dosage range from
0.1 to 1.0 mg/kg by body weight or from 1 to 100 mg/m.sup.2 of body
surface. In cases when the GSSG.cndot.Pt drugs are administered
like inhalation solutions, local instillations, eye drops,
intranasal introduction, or an ointment for epicutaneous
applications, or suppositories, the recommended concentration range
is from 1% to 10% of GSSG.cndot.Pt base or the salts thereof.
[0163] The active principle, the composite of the hexapeptide with
the stabilized disulfide bond--GSSG.cndot.Pt--capable of
stimulating/beneficial modulating the endogenous cytokine and
hemopoietic factor production as well as inducing of transformed
cells apoptosis, may be obtained by original, developed by the
authors the peptide synthesis technique provided herein. Thereby,
the obtained hexapeptide composite (GSSG.cndot.Pt) with purpose for
subsequent usage in animals and humans is applied as a
pharmaceutically acceptable GSSG.cndot.Pt derivative in an
injectable drug form prepared by dissolving of the bulk substance
in sterile water for injections or in any pharmaceutically
acceptable solvent with the resultant concentration 0.01-3.0%. For
an in vitro use in experimental settings, GSSG.cndot.Pt or the
derivatives thereof may be dissolved in solvents acceptable for
performance of corresponding experiments such as culture media,
isotonic saline solutions, glucose solutions and the like.
[0164] Injectable medicinal forms of the GSSG.cndot.Pt, salts and
compositions thereof have been tested in animal studies and as well
in wide clinical studies and pilot trials on sick persons. The drug
form for human and animal use should be prepared under sterile and
pyrogen-free conditions while exerting every effort to prevent
chemical or bacterial contamination of the medicinal form.
[0165] Using the maximum achievable concentration of the
GSSG.cndot.Pt material sodium salt solution (10.0%, 100 mg/ml) in
water for injections (or in normal saline), and using the maximum
tolerable volumes administered to mice intra-peritoneally (IP, 2.0
ml), intravenously (IV, 0.5 ml), and intramuscularly (1M, 0.05 ml),
there have been reached the GSSG.cndot.Pt dosage levels of 5000
mg/kg (IP), 1350 mg/kg (IV), and 135 mg/kg (IM), i.e. 1000, 270,
and 27 times, respectively, have been obtained in mice, exceeding
the maximum recommended human dose. In none of the cases either
animal deaths or any toxic signs were observed, proving, in fact,
the GSSG.cndot.Pt material in injectable drug form are essentially
non-toxic.
[0166] Through chemical modifications of the basic GSSG.cndot.Pt
molecule, it is possible to create new drugs where, in molecules
along with the basic structure that have already demonstrated high
medical-biological activity based on the oxidized glutathione
composite with cis-diamminedichloroplatinum, there are fragments of
other covalently-bound biochemically active molecules. Usage of the
covalently bound combinations allows enhancing of a range of
important drug characteristics such as stability and standardized
properties of composition. Foundation for the chemical
modifications is the GSSG.cndot.Pt hexapeptide core with two
primary glutamic acid aminogroups, cysteine disulfide bond and
carboxyl groups of glycine and .alpha.-carboxyl groups of glutamic
acid (FIG. 6).
[0167] Purposely elected covalently-bound fragments of
biochemically significant molecules can considerably improve
medical-biological features of the basic GSSG.cndot.Pt material
composite making them more selective for each particular
therapeutic purpose, resulting in a sharp increase in a desirable
treatment course. It might be conditioned with an additive effect
of fragments in the biochemical activity mechanisms, sharp
improvement of the basic molecule transport to a target-cell or a
target-molecule, larger affinity for a receptor, necessary
redistribution of oxidative-reductive (redox) potential and a range
of other factors as well as combination thereof.
[0168] In one embodiment, the drug including the composite of the
present invention can be useful for the treatment of a variety of
oncological diseases. Included in these oncological diseases are
solid tumors including those selected from the group consisting of
lung cancer, melanoma, cerebral tumors, colorectal cancer, breast
cancer, prostate cancer and ovarian cancer. The drug of the present
invention can be effective for hematologic tumors (non-solid
tumors) including acute lymphoblastic leukosis and acute
myeloblastic leukosis.
[0169] For lung cancer treatment, application of GSSG.cndot.Pt is
preferred for the first or fourth stages of the disease as a
monoregime. The monthly course includes dosages from 10 to 100
mg/m.sup.2 of the body surface of intravenous (IV) and
intramuscular (IM) drug introduction. In the second or third stage,
GSSG.cndot.Pt can be applied in combination with common
chemotherapy by means of IV, subcutaneous and local (intrapleural)
introduction of the drugs. The GSSG.cndot.Pt dosage is preferably
from 30 to 50 mg/m.sup.2 of the body surface.
[0170] For melanoma treatment, bis-[-iodine-tyrosyl]-GSSG.cndot.Pt
can be particularly effective. The monthly course can comprise IV
and IM drug introductions every day, with a dosage of about 30 to
100 mg/m.sup.2 of the body surface. Maintenance therapy can occur
over a period of up to six months-one year. It comprises
subcutaneous (locally, close to the impaired zones) and IV drug
introductions once a week, with a dosage of about 10-30 mg/m.sup.2
of the body surface.
[0171] For treatment of cerebral tumors,
bis-[dopamine]-GSSG.cndot.Pt is preferred and may be applied by
means of IV injections and catheterization of the carotid artery.
The dosage is preferably about 10 to 100 mg/m.sup.2 of the body
surface. The IV introduction can last three weeks, once every other
day. For introduction through the carotid artery, application
preferably occurs twice every day during six to seven days. The
repeat course can be administered two weeks later, over a period of
21 days as well and comprise IV injections mainly.
[0172] For treatment of colorectal cancer, i.e., adenocarcinoma of
stomach and pancreas, bis-[cystamine]-GSSG.cndot.Pt is preferred
and can be applied by means of IV and subcutaneous injections with
a dosage of about 10 to 30 mg/m.sup.2 of the body surface. Two to
three week administration can be used as a pre-surgery preparation
and post-surgery patient management. In case of inoperable stomach
cancer, applying the drug through a fibergastroscope is
recommended, introducing the drug around the cancer infiltrate once
every week with the single drug dose of 30-100 mg in this case.
Preferably, the solution volume does not exceed 3 mL.
[0173] For breast cancer treatment, cystamine-GSSG.cndot.Pt is
preferred by means of IV and subcutaneous injections with a dosage
of about 10 to 100 mg/m.sup.2 of the body surface as a pre-surgery
preparation and post-surgery patient management. Maintenance
therapy can take up to one year and can comprise application of
cystamine-GSSG.cndot.Pt along with ordinary chemotherapy by courses
lasting up to two weeks. There can be one-month intervals between
courses.
[0174] For prostate cancer treatment, application of the zinc salt
of GSSG.cndot.Pt is preferred through IV injections and through the
drug introduction into lymphatic spaces with a dosage of about 10
to 100 mg/m.sup.2 of the body surface. The approximate treatment
course duration can occur over a period of three to seven
weeks.
[0175] For ovarian cancer treatment, application of
theophylline-GSSG.cndot.Pt is preferred as an anticancer medicine
and as a remedy that restores the cancer susceptibility to
cis-platin. There can be IV and endolumbal routes for the drug
introduction with a dosage of about 10 to 100 mg/m.sup.2 of the
body surface over a period of three to seven weeks. It is possible
to combine this treatment with antiestrogen therapy.
[0176] For treatment of acute lymphoblastic leukosis, lithium salt
of GSSG.cndot.Pt is preferably applied intravenously as a month
course with a dosage of about 10 to 50 mg/m.sup.2 of the body
surface along with ordinary chemotherapy.
[0177] For treatment of acute myeloblastic leukosis, the lithium
salt of GSSG.cndot.Pt is preferably applied in combination with
cystamine-GSSG.cndot.Pt through IV introduction with a dosage of
about 10 to 100 mg/m.sup.2 of the body surface. The duration of
treatment can occur over a period of about three weeks. The
maintenance treatment can be repeated after three-month periods
during one year with a treatment course duration of about 14-17
days.
[0178] Infectious diseases that can be effectively treated with the
drug of the present invention include tuberculosis, viral hepatitis
B and C and mixed infections (HBV and HCV), herpes, meningitis
(sepsis), peritonitis, acute pancreatis and supporative
post-surgery sequalae.
[0179] The treatment of tuberculosis can involve a disseminated
process with destructive pathologic changes (cavities +) and
bacterial discharge (BK +). Preferably,
bis-[histidyl]-GSSG.cndot.Pt is applied by means of IV and IM
injections during one month with a dosage of about 3-10 mg/kg of
the body weight. For the following two months, the dosage can be
1-5 mg/kg of the body weight. The drugs can be introduced
intravenously twice a week or intramuscularly every other day.
[0180] For the treatment of viral hepatitis B and C and
mixed-infections (HBV and HCV), GSSG.cndot.Pt and
inosine-5-monophosphatyl-GSSG.cndot.Pt (IMP-5-GSSG.cndot.Pt) is
preferably applied by means of IV and IM injections with daily
dosage of 30 and 40-50 mg, respectively. In the case of hepatitis B
(HBV), the treatment course duration can be up to one month. In the
case of hepatitis C(HCV) and mixed infections, the treatment course
duration is preferably not less than three months with 10-12 day
intervals after each month of the treatment.
[0181] For the treatment of herpes, the course therapy preferably
involves GSSG.cndot.Pt and IMP-5-GSSG.cndot.Pt in a manner similar
to that for hepatitis C(HCV) patients.
[0182] For the treatment of meningitis, sepsis,
tetra-dopamine-GSSG.cndot.Pt is preferably applied by means of IV
and IM injections with a daily dosage of about 3-10 mg/kg of the
body weight. In meningitis patients, applying intralumbal
injections is recommended in dosage of 30-70 mg once every three to
four days until the patient's clinical state, blood and liquor
indices restore to normal.
[0183] For the treatment of peritonitis, GSSG.cndot.Pt and
tetra-dopamine-GSSG.cndot.Pt is preferably applied by means of IV
and IM injections with a daily dosage of about 30-70 mg over a
period of 10-14 days until there is full restoration to norm of the
patient's clinical state and objective indices (blood and urine
analyses; "liver" biochemistry).
[0184] For the treatment of acute pancreatitis, GSSG.cndot.Pt and
IMP-5-GSSG.cndot.Pt is preferably applied by means of IV and
subcutaneous (along a left costal arch) injections with a daily
dosage of about 3-10 mg/kg of the body weight, preferably every day
during the first week and then three times a week during the
following 14-17 days. This treatment can result in the restoration
of the patient's clinical state to normal.
[0185] For the treatment of supporative post-surgery sequalae,
preferably GSSG.cndot.Pt and IMP-5-GSSG.cndot.Pt is applied in a
manner similar to that for peritonitis.
[0186] The drug of the present invention can be effective for
various immunological diseases. Such diseases include
immunosuppressive diseases such as AIDS and immunosuppressions of
infectious diseases of radiation or toxic origin. Autoimmune
diseases include glomerulonephritis, rheumatoid arthritis,
collagenosis, systemic lupus erythematosus, and diabetes types I
and II. Other immunological diseases include atopic forms of
allergic conditions which include allergic rhinitis, atopic
dermatipis, bronchial asthma and urticaria.
[0187] For the treatment of AIDS, GSSG.cndot.Pt and
uridine-[5-monophosphatyl]-GSSG.cndot.Pt (UMP-5-GSSG.cndot.Pt) are
preferably applied by means of IV and IM injections alternated
every other day with a daily dosage of about 1-3 mg/kg of the body
weight during 30 days. The treatment course total duration can be
up to six months with two to three week intervals after each month
of the therapy application. This treatment can be combined with
common antiviral therapy. In the case of such a combination, the
antiviral remedies can be used as "impulse", i.e., short-term
courses (7-10 days for each course). In case of the AIDS-associated
encephalopathy, using GSSG.cndot.Pt and UMP-5-GSSG.cndot.Pt is
recommended in single doses of about 30 to 70 mg, respectively,
once every week, during one month.
[0188] For treatment of immunosuppressions of infectious diseases
of radiation or toxic (chemical) origin, GSSG.cndot.Pt and
UMP-5-GSSG.cndot.Pt is preferably applied by means of IV and IM
injections with a dosage of about 1-3 mg/kg of the body weight,
every day, during 10-12 days until hemopoiesis is corrected to
normal and the immune system is restored.
[0189] For the treatment of glomerulonephritis, GSSG.cndot.Pt and
lithium salt of GSSG.cndot.Pt is preferably applied by means of IV
and IM injections with a daily dosage of about 10-30 mg, 1-2 times
every day during first two weeks. Subsequent treatments preferably
involve applying IM injections only with a dosage of about 30 mg,
once every day during one month. The full course duration can be up
to three months with two week intervals after each month.
[0190] For the treatment of rheumatoid arthritis, GSSG.cndot.Pt and
lithium salt of GSSG.cndot.Pt is preferably applied by means of IV
and subcutaneous injections close to impaired joints with a dosage
of about 10 mg, two times every day during three weeks. Preferably,
subsequent treatments involve applying subcutaneous injections
only, one injection two to three times a week during three
months.
[0191] For the treatment of collagenosis, the treatment course
preferably involves GSSG.cndot.Pt and lithium salt of GSSG.cndot.Pt
in a manner similar to that of rheumatoid arthritis. The treatment
can also involve the addition of no greater than 500 mg of vitamin
C per day.
[0192] For the treatment of systemic lupus erythematosus, the
treatment course preferably involves GSSG.cndot.Pt and lithium salt
of GSSG.cndot.Pt in a manner similar to that of collagenosis.
[0193] For the treatment of atopic forms of allergic conditions
(allergic rhinitis, atopic dermatitis, bronchial asthma,
urticaria), GSSG.cndot.Pt and dihydrofluoride-GSSG.cndot.Pt
[(HF).sub.2.GSSG.cndot.Pt] is preferably applied by means of 1M,
subcutaneous injections and nasal drops with a dosage of about
0.1-1 mg/kg of the body weight, two times every day during three
weeks. No less than two or three treatment courses is preferred,
usually in spring and late fall.
[0194] For the treatment of diabetes-type I, vanadium salt of
GSSG.cndot.Pt (divanadate-GSSG.cndot.Pt) is preferably applied by
means of IV and IM injections with a dosage of about 1-2 mg/kg of
the body weight, along with bis-[nicotinoyl]-GSSG.cndot.Pt with a
dosage of about 1-3 mg/kg of the body weight. Preferably, three to
four week treatment courses are performed every three months.
[0195] For the treatment of diabetes-type II,
bis-[lipoyl]-GSSG.cndot.Pt is preferably applied by means of IV and
IM injections with a dosage of about 3-7 mg/kg of the body weight,
along with bis-[nicotinoyl]-GSSG.cndot.Pt, dosage-1-3 mg/kg of the
body weight (mainly within the dropper composition based on a 0.5%
solution of glutamic acid). The treatment courses can occur over a
period of one month, two to three times a year.
[0196] The drug of the present invention can be effective for the
treatment of ischemic diseases such as ischemic cerebral conditions
including post-insult conditions (e.g., infantile cerebral
paralysis) and ischemic heart diseases such as those manifested
mainly as a syndrome of conduction impairment and arrythmias
(tachyarrythmia, bradyarrythmia, and impairment of ventricular
conduction due to blockage of the His bundle or branches) and
diseases manifested mainly as a syndrome of functional myocardial
failure (cardiomyopathies of different origin, metabolic myocardio
dysfunctions, and post-infarction conditions).
[0197] For the treatment of ischemic cerebral conditions including
post-insult ones (e.g., infantile cerebral paralysis),
bis-[phenylalanyl]-GSSG.cndot.Pt is preferably applied by means of
IV and IM injections with a dosage of about 1-7 mg/kg of the body
weight, as treatment courses last three to four weeks and intervals
after each course up to one month. The total treatment duration can
take up to two years.
[0198] For the treatment of ischemic heart disease (IHD) manifested
mainly as a syndrome of conduction impairment and arrhythmias
(tachyrhythmia, bradyrhythmia, impairment of ventricular conduction
due to block of the His's bundle or branches),
bis-[carnosyl]-GSSG.cndot.Pt
(bis-.beta.-alanyl-L-histidyl-GSSG.cndot.Pt) is preferably applied
by means of IV and IM injections with a dosage of about 2-5 mg/kg
of the body weight as treatment courses lasting three to four
weeks.
[0199] For the treatment of IHD manifested mainly as a syndrome of
functional myocardial failure (cardiomyopathies of different
origin; metabolic myocardial dysfunctions, post-infarction
conditions), glycerol-[1,3-diphosphatyl]-GSSG.cndot.Pt is
preferably applied by means of IV and IM injections with a dosage
of about 3-7 mg/kg of the body weight, mainly within the dropper
composition containing a 5% glucose solution. The treatment courses
duration can occur over a period of two to three weeks, two to
three times a year.
[0200] The drug of the present invention can be effective for the
treatment of neurodegenerative diseases such as Alzheimer's
disease, hereditiary (Huntington's) chorea, amyotrophic lateral
sclerosis, neuro-AIDS and demyelinating diseases such as multiple
sclerosis. Neurodegenerative disease can also include
neurobehavioral diseases such as diseases caused by drug (narcotic)
abstinence and behavioral diseases created with psychotropic and
nootropic drugs, such as cerebral hypoxia (post-ischemic
conditions), manic-depressive psychosis and schizephrenia.
[0201] For the treatment of neurodegenerative diseases, such as
Alzheimer disease, hereditary (Huntington's) chorea, amyotrophic
lateral sclerosis, neuro-AIDS,
bis-[3,4-dihydroxyphenylalanyl]-GSSG.cndot.Pt is preferably applied
by means of IV, subcutaneous (along cervical-thoracic spine) and IM
injections with a dosage of about 1-5 mg/kg of the body weight, as
treatment courses lasting up to one month during one year.
Intervals between courses can be two or three weeks.
[0202] For the treatment of demyelinating diseases, such as
multiple sclerosis, bis-[3,4-dihydroxyphenylalanyl]-GSSG.cndot.Pt
is preferably applied by means of IV and subcutaneous (along the
spine and endolumbal route) injections with a dosage of about 1-10
mg/kg of the body weight, as treatment courses lasting up to one
month. The total treatment duration can last up to one to two
years. The drug can be introduced through endolumbal route one to
two times a month.
[0203] Psychotropic and nootropic drugs can be used for treatment
of neurodegenerative diseases such as cerebral hypoxia
(post-ischemic conditions), y-hydroxy-[butanoyl]-GSSG.cndot.Pt can
be applied by means of IV and IM injections with a dosage of about
1-4 mg/kg of the body weight as two to four week treatment courses.
Introduction of the drug endolumbally is recommended once a week,
with a dosage of about 1 mg/kg of the body weight.
[0204] For the treatment of manic-depressive psychosis, such as
schizophrenia, .gamma.-amino-[butanoyl]-GSSG.cndot.Pt is preferably
applied by means of IV and IM injections with a dosage of about 3-5
mg/kg of the body weight as four to six week treatment courses two
to three times a year.
[0205] The drugs of the present invention can be effective in
diminishing the contents of pre-.beta.- and .beta.-lipoproteins in
blood for the treatment of atherosclerosis and other metabolic
diseases. Bis-[nicotinoyl]-GSSG.cndot.Pt
(bis-pyridine-3-carbanoyl-GSSG.cndot.Pt) can be applied by means of
IV and IM injections with a dosage of about 1-3 mg/kg of the body
weight as treatment courses lasting two to three weeks three times
a year.
[0206] The drugs of the present invention can be effective for the
treatment of endocrinal diseases such as
hypothalamic-hypophysial-ovarian associated functional link
impairments that can produce hormonal abnormalities and cause
sterility. Folliculyl-[succinyl]-GSSG.cndot.Pt can be applied by
means of IM and subcutaneous injections with a dosage of about 3-10
mg/kg of the body weight, as monthly treatment courses three to
four times a year.
[0207] Other preferable derivatives include the GSSG.cndot.Pt
material derivatives in the form of its sodium, lithium, potassium,
calcium, zinc, molybdenum, vanadium and other salts, as well as the
GSSG.cndot.Pt derivatives obtained through covalent binding to
phenylalanine, or to methionine and some other aminoacids including
D and L forms of the aminoacids herein; or to cystamine, lipoic
acid, or to inosine.
[0208] In one embodiment, manifestation of the immunological,
biochemical and molecular-biological effects of the GSSG.cndot.Pt
therapeutical impact can be obtained in the case when a combination
comprising 50% of GSSG.cndot.Pt with all aminoacids in L-form and
50% of GSSG.cndot.Pt with two chemically equal aminoacids being
represented in D-form and others being represented in L-form is
used.
[0209] The present invention presents the advantageous feature that
the drug comprising the composite or derivatives thereof has a
regulating effect on the endogenous cytokine production processes
and, thus, on the proliferation and differentiation processes of
the T- and B-lymphocyte subpopulations (CD.sup.+-cells). Drug
induction can result in production of a wide cytokine and
hemopoietic factor range and CD.sup.+-lymphocytes. Therefore, in
this range, from the point of view of cytokine interaction, there
are both agonist-cytokines and antagonist-cytokines regarding the
effects they stimulate (e.g., "relationship" of IL-1.alpha. and
.beta. and IL-4). In connection with that, depending on the initial
patient's immunogenesis system state, hyper- or hypoactivity, the
drugs of the present invention can restore a disturbed balance in
the system.
[0210] The given provision is illustrated in Examples 9-13 which
show that patients with depressed immunity (oncological patients
receiving radiation or combined chemotherapy) the cytokine
synthesis induction (IL-1.alpha. and .beta., IL-2, IL-3, IL-4,
IL-6, IL-10 and IL-12, IFN-.alpha. and IFN-.gamma.) can be
accompanied by restoration of CD3.sup.+, CD4.sup.+, CD8.sup.+,
CD16/56.sup.+, CD25.sup.+, CD34.sup.+ counts; and patients with the
immunoautoagression signs--at the clones of cytotoxic lymphocytes
or fibroblasts in case of viral hepatitis C the Fas-Ag (CD95.sup.+)
is expressed that promotes the apoptosis mechanism induction and
elimination of the virus-transformed and/or "aggressive" cells.
[0211] Another advantageous feature of the present invention
involves the finding of the composite impact on the isolated human
lymphocytes 10 minutes (a peak is observed at the 30.sup.th minute
(the maximal level of phosphorilating of the cytosol proteins
obtained from the lymphocytes)) after parenteral introduction of
GSSG.cndot.Pt material, a significant increase of the
phosphorylating level on tyrosine for lymphocyte cytosole proteins
that is an integrative characteristic for the cellular
signal-transducing system activity. These changes in state for key
factors of cAMP, cGMP, inositol-phosphate-dependent signal systems
owing to the GSSG.cndot.Pt material influence (See Example 8) calls
forth the redox-sensitive gene expression, first of all, for the
immunologically significant genes responsible for the cytokine and
hemopoietic factor synthesis. Therefore, the GSSG.cndot.Pt material
application in the treatment purposes not only stimulates the
cytokine and hemopoietic factor endogenous production but also
provides reproduction of the biochemical and physiological cytokine
effects, in particular, in the case of sensitivity loss of
receptors to cytokines that is observed at oncological and
retroviral pathology.
[0212] In the tumor- and/or virus-transformed cells the apoptosis
mechanisms are induced through the GSSG.cndot.Pt material
multicytokine-activating impact, its influence on p53-dependent and
p53-independent apoptosis induction mechanisms as well as through
changing of the donor/acceptor .pi.-electron balance in malignant
(cancer) cells (see Examples 14-16).
[0213] Depending on the initial patient's biological status
including his immunity condition: immunodeficiency, i.e.,
hyporeactivity; or immunoautoaggression, i.e., hyperreactivity;
presence of the tumor- or virus-transformed cells--the composite
and/or pharmaceutically acceptable derivatives thereof are able to
act as the endogenous cytokine production stimulators/modifiers
and/or as the apoptosis mechanism inducers, respectively.
[0214] These compounds and the drug forms thereof obtained which
include the GSSG.cndot.Pt material composite are applied as
medicinal drugs capable in the therapeutic purposes depending on
the initial subject's biological status of the subject in need
thereof to stimulate/modulate the wide range cytokine and
hemopoietic factor endogenous production and/or to reproduce the
cytokine effects as well as to perform the differentiated effect
regarding the normal (the metabolism, proliferation and
differentiation regulation) and the transformed cells (the
apoptosis mechanism induction). "Transformed cells" refers to
tumor- and/or virus-transformed cells.
[0215] Performed experimental and clinical investigations show that
therapeutic effects of the drugs obtained from the GSSG.cndot.Pt
material and derivatives thereof are based on the
multicytokineactivating action and capacity to reproduce cytokine
and hemopoietic factor effects. At the same time, the data was
obtained indicating the GSSG.cndot.Pt material has a direct
antitumor effect, especially regarding GSSG.cndot.Pt material salts
administered in pharmaceutically acceptable drug forms. Moreover,
the GSSG.cndot.Pt material effect has revealed to be different for
normal and tumor cells. The research with use of the normal and
tumor cells demonstrated that the GSSG.cndot.Pt material or the
GSSG.cndot.Pt material in pharmaceutically acceptable compositions
initiated tumor cell death through apoptosis mechanism. In case of
normal cells, they did not undergo destruction (See Examples
14-16).
[0216] Also high effectiveness of the applied medicinal drugs based
on the GSSG.cndot.Pt material in regard to apoptosis mechanism
induction in the virus-transformed cells, for example, in case of
viral hepatitis B and C, should be noted as exemplified in Examples
11-13.
[0217] Therapeutic effects of GSSG.cndot.Pt material and
pharmaceutically acceptable derivatives thereof, particularly,
salts thereof for the treating of oncological, infectious (viral)
diseases can be explained as a stimulation of the wide-ranged
endogenous cytokine production with a unique ability to activate
apoptotic death of the transformed cells exclusively. Moreover, the
majority of the GSSG.cndot.Pt therapeutical effects and the
pharmaceutically acceptable derivatives thereof in the experimental
and clinical conditions can be applied to be connected with
revealed properties of the GSSG.cndot.Pt material and the drug
forms thereof to stimulate/beneficially modulate the endogenous
cytokine production or to reproduce their effects in regard to
stimulation of the normal cell proliferation and differentiation
and, at the same time, to activate apoptotic death of the
transformed cells exclusively.
[0218] Another advantageous feature of the drugs of the present
invention is a correcting influence of the GSSG.cndot.Pt material
and the salts thereof on the metabolic abnormalities, particularly,
on the impairment of carbohydrate metabolism at diabetes, type II.
In this case (See Example 7) restoration of the normal cAMP/cGMP
ratio as well as the thiol-disulfide ratio in tissues due to the
GSSG.cndot.Pt material impact (vanadium salt thereof) provided
stable setting to normal values for the glucose content in the
patient blood, which is a considerable therapeutic effect.
[0219] Resuming the results of performed experimental, preclinical
and clinical studies of the medicinal remedies group developed on
the GSSG.cndot.Pt material one should emphasize that the parenteral
(intravenous, intramuscular, subcutaneous, instillations into
urinary bladder or per rectum, etc.) introduction of the indicated
medicinal remedies provides: a) stimulation/beneficial modulation
of the endogenous production of IL-1.alpha. and .beta., IL-2, IL-3,
IL-4, IL-6, IL-8, IL-10 and IL-12, TNF-.alpha., IFN-.alpha. and
IFN-.gamma., erythropoietin, G-CSF, M-CSF and GM-CSF; and,
thereupon, wide range of biochemical and immunological effects; b)
reproduction of the effects of the said cytokines and hemopoietic
factors in case of cytokine receptor desensitization; as well
as--c) induction of apoptosis mechanism in tumor- or
virus-transformed cells only, calling forth in the organism of the
subject in need thereof the corresponding therapeutic effect.
[0220] The beneficial effects of the drug of the present invention
can be illustrated in FIGS. 25-27. FIG. 25 delineates the humoral
and cellular responses to the drugs comprising the composites of
the present invention. FIG. 26 depicts the components of the immune
system activated by the drugs of the present invention, the
components including platelets, white blood cells, red blood cells,
cytokines, erythropoietines, T-lymphocytes, neutrophils, monocytes,
macrophages, natural killer cells, and .beta.-lymphocytes. FIG. 27
depicts the types of cytokines stimulated by the drugs of the
present invention, the cytokines including IL-1, IL-2, IL-4, IL-6,
IL-8, IL-10, IL-12, alpha and gamma interferons, TNF-alpha and
GM-CSF. Thus, the novel method for obtaining the composite
comprising the GSSG.cndot.Pt material the new medicinal substance
(thiopoietins) class is applied and proved, in that therapeutical
effects are determined by firstly found and previously unknown
properties of performing stimulation of cytokine and hemopoietic
factor endogenous production and their effect reproduction,
thereby, performing stimulation and/or modulation of the normal
cell proliferation and differentiation as well as inducing
apoptosis mechanisms in virus- and tumor-transformed cells.
[0221] The presence of a chemical interaction between the disulfide
bond of the oxidized glutathione-based compound and the platinum
material may play a biophysical role in the electron balance of
biological systems. While not wishing to be bound by any theory,
the chemical interaction can be loosely thought of as a
donor/acceptor pair where the lone electron pairs on the sulfur
atoms can potentially interact with an electron-deficient material,
such as the platinum material. Again, while not wishing to be
limited by any mechanism, if cellular activity and cellular
physical state are determined, at least in part, by donor/acceptor
interactions throughout a biological system, then a balance among
electron donors and acceptors having equal biopotentials can be a
life parameter. Such balance alteration can be used for regulation
of different cell functions and physical properties (Szent-Gyorgyi
A., Proc. Natl. Acad. Sci. U.S., 58,2012 (1967); Introduction to a
Submolecular Biology, Academic Press, New York (1960)). Sources of
mobile electrons can include .pi.-electrons such as lone-pair
electrons of nitrogen, oxygen, and sulfur. There are few acceptor
groups (e.g., --C.dbd.O-groups) in a normal cell which can be
balanced via donor/acceptor dynamics of donor electrons such as
.pi.-electrons.
[0222] Continuing along with this non-limiting theory, the
malignant cell can be characterized as having dramatic disturbances
of the donor/acceptor balance towards an excess of donors
electrons. Acceptor molecules are almost absent in cancer cells. A
possible solution to this imbalance in this situation is the
presence of molecules that possess donor/acceptor features within
the same molecule, such as a GSSG.cndot.Pt material. Introducing
GSSG.cndot.Pt into biological media can cause a restoration of the
electronic balance in the biological media. This restoration can
involve a catalysis by the platinum atom in a reaction involving
the formation of an active oxygen form, e.g., superoxide anion
radicals, singlet oxygen.
[0223] Another non-limiting theory involving cellular electronic
balance is an interaction of GSSG.cndot.Pt material with cell GSH
generating oxidative-reductive, i.e. donor/acceptor pair. If there
is a high GSH level in cells that is characteristic for
tumor-transformed cells with a high proliferative impulse, the
pro-oxidative, i.e. oxidative, properties of GSSG.cndot.Pt material
exhibit in the most evident way. In the case the oxidative stress
forms in the tumor cells only causing alteration of the tumor cells
mitochondria functions forming intracellular signal for the
apoptosis mechanisms induction.
[0224] For the normal but "tired", "exhausted" cells, there may be
an oxidative-reductive potential optimization, bioenergetic supply
for the metabolic transformations, redox-sensitive adequate
expression of the genome functional sites, in particular, the
immunologically significant genes and transcription factors.
[0225] For the transformed cells GSSG.cndot.Pt, there may be an
incompatibility with vital functions involving chain transfer
reaction of .pi.-electrons, disturbance of the mitochondrial
oxidative-reductive reactions of electron/protons transfer
reactions along the respiratory chain and dislocation of the
NADP.cndot.H.sup.+/NADP.cndot.H ratio, i.e., forming the
intracellular signal for the apoptosis mechanism induction.
[0226] The new GSSG.cndot.Pt pharmacokinetics (comparing to GSSG by
itself) in blood and tissues (organs) being introduced into
biological media indicates that the GSSG.cndot.Pt molecule is much
less available for the GSSG metabolism enzymes and, first of all,
for the NADP.cndot.H.sup.+-dependent reductase, the main enzyme for
the GSSG into GSH reduction. Thereupon, the GSSG.cndot.Pt half-life
time in the disulfide form in biological media increases
significantly (See Examples 3 and 4).
[0227] Basically novel pharmacokinetics of the hexapeptide with the
stabilized disulfide bond (GSSG.cndot.Pt) compared to the
structural analog, i.e., the oxidized glutathione (GSSG), provided
an optimal manifestation for the newly determined
biological-pharmacological effects.
EXAMPLES OF THE INVENTION EMBODIMENTS
[0228] SYNTHESIS METHOD. 170 g (0.55 mole) of the reduced
glutathione are suspended in 200 ml of water and, along with
stirring, 139 ml (0.55 mole) of the 4N NaOH solution and 170 ml of
0.05% cis-diamminedichloroplatinum cis-[Pt(NH.sub.3).sub.2Cl.sub.2]
water solution are added.
[0229] The obtained transparent, slightly yellow solution is cooled
to 18-20.degree. C. and 283 ml of the 3% hydrogen peroxide solution
(H.sub.2O.sub.2 solution) is added in little portions over a time
period of five minutes with such a speed rate that the reaction
mixture temperature will not exceed 22-25.degree. C.
[0230] Thirty minutes after addition of the hydrogen peroxide
solution (H.sub.2O.sub.2) the pH is measured, and then the 4N
caustic soda solution is added drop-by-drop to reach pH=5.6-5.8
along with simultaneous temperature control, which should be within
22-25.degree. C. Then the cooling is taken away and stirring
continued at indoor (room) .degree. C. temperature for 30 minutes
more.
[0231] Control of the oxidation reaction completeness is performed
by the HPLC assay. A liquid chromatograph for HPLC, type Beckman,
Sol. Module 126, Det. 168, with a column Luna Phenomenex ODS
4.6.times.250 mm, or an identical one, is prepared. To prepare the
HPLC mobile phase 20 cm.sup.3 of acetonitrile and 1 cm.sup.3 of
freshly distilled trifluoroacetic acid is introduced into a
1000-cm.sup.3 graduated flask, and the volume is increased up to
the mark by the deionized water. The solution is mixed and degassed
by shaking in vacuum.
[0232] Thirty minutes after addition of the entire hydrogen
peroxide solution amount one will check the oxidation reaction
completeness by means of the highly productive liquid
chromatography (HPLC). Thereto with a microsyringe one will take 10
.mu.l of the reaction mixture and dissolve them in 1 ml of the
mobile phase (0.1% trifluoroacetic acid:acetonitril, 98:2). 20
.mu.l of the obtained solution is introduced into the chromatograph
Beckman 126 Solvent Module, Diod Array Detector Module 168, the
column Luna Phenomenex ODS 4.6.times.250 mm, or the identical.
Elution is performed in isocratic regime, 30 min., in the system
0.1% trifluoroacetic acid:acetonitrile, 98:2; the flow-speed rate 1
ml/min, detection at 220 nm, scanning 190-600 nm.
[0233] The retention time in the aforesaid conditions is 5.0.+-.0.5
min for reduced glutathione, 11.0.+-.0.5 min for oxidized
glutathione.
[0234] In case if, according to the HPLC data after the standard
chromatogram integration, the oxidized glutathione content is less
than 97%, the stirring is continued in the same regime 30 minutes
more and the HPLC control is repeated.
[0235] In case when the result is equal or exceeds 97%, the
reaction is considered as completed and one will pass to the
reaction solution filtration. Thereto, there is used a filter
having pore size not larger than 0.7 .mu.m.
[0236] Weight loss at drying will not exceed 5% at drying to the
constant weight at 100.degree. C. in vacuum (1 mm Hg) above
CaCl.sub.2 and P205.
[0237] The main material content in the ready product by the HPLC
data will not be lower than 98%.
[0238] Thus, the oxidized glutathione as a composite with
cis-diamminedichloroplatinum is obtained.
[0239] Appearance: white odorless powder.
[0240] Solubility: soluble in water, 0.9% isotonic solution of
sodium chloride for injections; insoluble in 95% alcohol,
chloroform, ether and other organic dissolvents.
[0241] Solution transparency and color: 0.05 g of the drug solution
in 10 ml of water is transparent and colorless solution.
PH of 1% solution: 5.0-6.0 potentiometrically, the device is
pH/mV/.degree. C. meter Cole Parmer, model 59003-15 or
identical.
[0242] Authenticity:
a) amino-acid analysis (6 n HCl, 110.degree. C., 20 hrs.),
glycine-2.0.+-.15%; glutamic acid--2.0.+-.15%; cysteine-2.0.+-.40%;
amino-acid analyzer AAA T-339 M Prague or identical.
[0243] b) HPLC--at the outlet time it corresponds to the standard
of bis-(y-L-glutamyl)-L-cysteinyl-bis-glycine disodium salt.
[0244] Chromatography conditions: device--BECKMAN "Gold Nouveau
Chromatography Data System" Version 6.0, Diod Array Detector Module
126 or identical.
[0245] Assay--20 .mu.l of 0.1% drug solution in the mobile phase,
chromatography on the column ULTRASPERE ODS 250.times.4.6 mm with
the converted C.sub.18 phase in the isocratic conditions
acetonitrile-0.1% trifluorideacetic acid (2:98); flow rate 1
ml/min., detecting at 220 nm, scanning 190-600 nm.
[0246] Purity (main substance content):
[0247] a) at HPLC not less than 98%:
[0248] b) at the amino-acid analysis: not less than 85% (analysis
according to Section "Authenticity", Item "a" with an exact
weight).
[0249] c) Sodium (Na) content according to the emission spectral
method is 7.0.+-.0.5%.
[0250] d) Platinum (Pt) content according to the mass spectrometric
analysis is 0.032.+-.0.01%
Method for Element Content Determination:
[0251] The exact assay weight (about 50 mg) is dissolved in 50 ml
of bidistilled water and the solution is used for the analysis.
[0252] The platinum content is determined quantifiably by the
method of mass spectrometric analysis with inductively bound plasma
at the device of the PQe model made by VG Elemental, England. The
analysis relative precision is 5%.
[0253] The other element content is determined quantifiably by the
method of the atomic-emission spectroscopy with inductive bound
plasma on the device of the model TRACE 61E made by Thermo Jarell
Ash, USA. The analysis relative precision is 5%.
Element Content, .mu.g/g:
TABLE-US-00001 [0254] Silver (Ag) <1.0 (less than 0.0001%)
Aluminum (Al) 2.0 Arsenic (As) <1.0 Barium (Ba) <0.50
Beryllium (Be) <0.05 Calcium (Ca) 7.0 Cadmium (Cd) <0.05
Cobalt (Co) <0.5 Chromium (Cr) 1.7 Copper (Cu) <0.5 Iron (Fe)
<1.0 Potassium (K) <2.5 Selenium (Se) <2.0 Magnesium (Mg)
<2.5 Manganese (Mn) <0.2 Molybdenum (Mn) <0.2 Nickel (Ni)
<0.5 Lead (Pb) <0.40 Strontium (Sr) 1.9 Titanium (Ti) <0.5
Vanadium (V) <0.5 Zinc (Zn) 0.65 Antimony (Sb) <0.5
Example 1
Synthesis of
bis-(L-phenylalanyl-.gamma.-L-glutamyl)-L-cysteinyl-bis-glycine
disodium salt
(I.) General Drug Characteristics.
[0255] 1. Name:
bis-(L-phenylalanyl-y-L-glutamyl)-L-cysteinyl-bis-glycine disodium
salt, composite with cis-diamminedichlorplatinum.
[0256] 2. Structural formula--see FIG. 8.
[0257] 3. Gross-formula:
C.sub.38H.sub.48N.sub.8O.sub.14Na.sub.2S.sub.2.[Pt(NH.sub.3).sub.2Cl.sub.-
2]
[0258] 4. Molecular weight: 950,94 on
C.sub.38H.sub.48N.sub.8O.sub.14Na.sub.2S.sub.2 with Pt content
0.033%.
[0259] 5. Appearance: white odorless powder.
[0260] 6. Solubility: soluble in water, 0.9% isotonic solution of
sodium chloride for injections; insoluble in 95% alcohol,
chloroform, ether and other organic dissolvents.
[0261] 7. Solution transparency and color: 0.05 g of the drug
solution in 10 ml of water is transparent and colorless.
[0262] 8. pH of 0.1% solution: 5.75 (potentiometry).
[0263] 9. Authenticity: [0264] a) amino-acid analysis (6 n HCl,
110.degree. C., 20 hrs.), (error margin 20%, for cysteine--35%), in
correspondence: glycine--2.00; glutamic acid--1.92; cysteine--1.81;
phenylalanine--2.04. [0265] b) NMR (.sup.1H)-spectroscopy,
according to--"BRUKER" AM 500, 500 MHz, D.sub.2O.
TABLE-US-00002 [0265] .delta. Amino-acid 7.20 --C.sub.ar--H Phe
4.70 --C.sub..alpha.H-- Cys 3.75 ##STR00003## Glu 3.27 --CH.sub.2--
Gly 2.95 --CH.sub.2-- Cys 2.52 --CH.sub.2-- Glu 2.15 --CH.sub.2--
Glu
[0266] 10. Purity (main substance content):
[0267] a) At HPLC not less than 97%:
Device: BECKMAN "Gold Nouveau Chromatography Data System" Version
6.0, Diod Array Detector Module 126. Assay--20 .mu.l of 0.1% drug
solution in the mobile phase, chromatography on the column
ULTRASPERE ODS 250.times.4.6 mm with a converted C.sub.18 phase in
isocratic conditions acetonitrile-0.1% trifluorideacetic acid
(2:98); flow rate 1 ml/min., detecting at 220 nm, scanning 190-600
nm, PDA functions--Contour Plot, 3D.
[0268] b) At the amino-acid analysis: not less than 85% (analysis
according to Item 9a with an exact weight);
[0269] c) Thin-layer chromatography is homogenous, analysis is
performed at introduction of 5 .mu.l of the 1% drug solution in the
band. There are plates Kieselgel 60.sub.f (Merck) 10.times.5 cm,
system: n.butanol--acetic acid--water (4:1:1). Development is
performed according to the standard methods--ninhydrine and
chlorine\benzidine. R.sub.f=0.15;
[0270] d) Sodium (Na) content according to the emission spectral
method is: 4.8%;
[0271] e) Platinum (Pt) content according to the mass spectrometric
analysis is 0.033%.
[0272] 11. Elements detected content, .mu.g/g:
TABLE-US-00003 Silver (Ag) <1.0 (less than 0.0001%) Aluminum
(Al) 2.0 Arsenic (As) <1.0 Barium (Ba) <0.50 Beryllium (Be)
<0.05 Calcium (Ca) 7.0 Cadmium (Cd) <0.05 Cobalt (Co) <0.5
Chromium (Cr) 1.7 Copper (Cu) <0.5 Iron (Fe) <1.0 Potassium
(K) <2.5 Selenium (Se) <2.0 Magnesium (Mg) <2.5 Manganese
(Mn) <0.2 Molybdenum (Mo) <0.2 Sodium (Na) 48 mg/g Nickel
(Ni) <0.5 Lead (Pb) <0.40 Platinum (Pt) 330 .mu.g/g Strontium
(Sr) 1.9 Titanium (Ti) <0.5 Vanadium (V) <0.5 Zinc (Zn) 0.65
Antimony (Sb) <0.5
Determination Method:
[0273] The exact assay weight (about 50 mg) is dissolved in 50 ml
of double-distilled water and the solution is used for the
analysis.
[0274] The platinum content is determined quantifiably by the
method of mass spectrometric analysis with inductively bound plasma
at a PQe device made by VG Elemental, England.
[0275] The analysis relative precision is 5%.
[0276] The content of other elements is determined quantifiably by
the method of the atomic-emission spectroscopy with inductive bound
plasma on a TRACE 61E device made by Thermo Jarell Ash, USA. The
analysis relative precision is 5%.
[0277] 12. Weight loss at drying: 10% at drying till the constant
weight at 100.degree. C. in vacuum (1 mm Hg) above CaCl.sub.2 and
P205.
(II.) Synthesis Method Description.
[0278] 1. Process chemical scheme--see FIG. 8.
[0279] 2. Method description
(III). Product (I) .gamma.-L-glutamyl-L-cysteinyl-glycine in amount
of 3.07 g (10 mmol) and N-hydroximethylbenzamide (II) in amount of
5.89 g (13 mmol) is dissolved in 30 ml of anhydrous trifluoroacetic
acid (TFA) mix at the room temperature during one hr. Then the
solvent is distilled off in vacuum at 40.degree. C., 30 ml of
anhydrous ethyl alcohol is added to the remainder; the solvent is
again distilled off in vacuum and the procedure is repeated two
times more. The product is crystallized through grinding in 50 ml
of anhydrous diethyl ether, filtered, washed on the filter with
2.times.20 ml of anhydrous ether and further it is dried in vacuum
above KOH and P205. Recrystallization is done from 90% ethanol.
Yield--5.50 g (80%). R.sub.f=0.43, Kieselgel 60.sub.f (Merck)
10.times.5 cm, system: n.butanol--acetic acid--water (4:1:1). (V).
Product (III) in amount of 4.40 g (10 mmol) is stirred in the
mixture of 15 ml of distilled water and 25 ml of dioxane; then
along with mixing, 10 ml (20 mmol) of 2 N NaOH solution is
added.
[0280] Then 3.62 g (10 mmol) of phenylalanine N-hydroxysuccinimide
ester (IV) is introduced in the reaction mixture and the stirring
is continued during 12 hrs at room temperature.
[0281] Then the mixture is evaporated in vacuum at 40.degree. C. to
dryness. The residue is dissolved in 200 ml of ethyl acetate and
washed by 2.times.20 ml of 1 N sulphuric acid, water, sodium
bicarbonate (2.times.50), water and the organic layer is above the
anhydrous chloride calcium. Then ethyl acetate sis distilled in
vacuum at 40.degree. C. to dryness and the product is crystallized
from ethyl acetate/ether.
[0282] The crystals are separated by filtration and dried in vacuum
above phosphorus pentoxide to constant weight. The product yield
(V)-4.88 g (70%). R.sub.f=0.80, Kieselgel 60.sub.f (Merck)
10.times.5 cm, system: n.butanol--acetic acid--water (4:1:1).
(VI). Product (V) in amount of 6.87 g (10 mmol) is dissolved in 20
ml of distilled trifluoroacetic acid and the solution is kept at
room temperature during two hrs. Then the product is precipitated
by absolute ether (about 200 ml), filtered and dried in vacuum
above KOH to the constant weight. The product yield--(V) 5.28 g
(90%). R.sub.f=0.48, Kieselgel 60.sub.f (Merck) 10.times.5 cm,
system: n.butanol--acetic acid--water (4:1:1). (VII). Product (IV)
in amount of 5.87 g (10 mmol) is dissolved in 100 ml of a mixture
methanol-water (1:1), then 200 ml (10 mmol) of mercury acetate
solution are added and the mixture is stirred at room temperature
during one hr. Then the hydrogen sulphur flow is sparged through
the solution during 20 min. controlling precipitation efficiency at
the assay. The mercury sulphide precipitate is filtered; the
filtrate is evaporated in vacuum up to volume of 10 ml; then 200 ml
of isopropyl alcohol is added and the product is crystallized at
cooling to 0-4.degree. C. The crystals are filtered, washed with
isopropyl alcohol, acetone and dried in vacuum above P205. The
product yield (VII)-3.72 g (82%). R.sub.f=0.30, Kieselgel 60.sub.f
(Merck) 10.times.5 cm, system: n.butanol--acetic acid--water
(4:1:1). (VIII). Product (VII) in amount of 4.54 g (10 mmol) is
suspended in 20 ml of water and along with stirring, 5 ml (10 mmol)
of 2 N NaOH solution and then 4.8 ml of 0.05% water solution of
cis-diamminedichloroplatinum (cis-[Pt(NH.sub.3).sub.2Cl.sub.2]) is
added. The solution is cooled to 18-20.degree. C. and in little
portions during about two min., 5.1 ml of 3% solution is added at
such a rate so that the temperature will not exceed 22-25.degree.
C. Thirty minutes after introduction of the whole hydrogen peroxide
amount, the solution pH is measured and its value is brought to
5.6-5.8 by adding of the necessary amount of 4N NaOH solution,
while the solution temperature is monitored (it is within
22-25.degree. C.). Then the stirring is continued without external
cooling for 30 min. and then the control analysis of the reaction
mixture is performed by HPLC. With that purpose 10 .mu.l is taken
out of the reaction solution and dissolved in 1 ml of the mobile
phase. If, according to the HPLC data, the oxidized form content is
equal or exceeds 95%, the reaction is deemed as finished.
Otherwise, the stirring at the room temperature is continued 30
min. more and the HPLC assay is repeated.
[0283] Then the reaction solution is filtered through the filter
with pore size not larger than 0.7 .mu.m and the filtrate is
lyophilized. The obtained dry product is dried out in vacuum at
40.degree. C. above anhydrous calcium chloride to the constant
weight. Yield--4.51 g (95%).
[0284] The ready substance is analyzed according to see Example
2.
Example 2
Synthesis of bis-(.gamma.-L-glutamyl)-L-cysteinyl-bis-glycine
lithium salt
(I.) General Drug Characteristics.
[0285] 1. Name: bis-(y-L-glutamyl)-L-cystinyl-bis-glycine dilithium
salt with cis-diamminedichloroplatinum. [0286] 2. Structural
formula--see FIG. 9. [0287] 3. Gross-formula:
C.sub.20H.sub.30N.sub.6O.sub.12Li.sub.2S.sub.2.[Pt(NH.sub.3).sub.2Cl.sub.-
2] [0288] 4. Molecular weight: 624.49 on
C.sub.20H.sub.30N.sub.6O.sub.12Li.sub.2S.sub.2 with Pt content
0.032% [0289] 5. Appearance: white odorless powder. [0290] 6.
Solubility: soluble in water, 0.9% isotonic solution of sodium
chloride for injections; insoluble in 95% alcohol, chloroform,
ether and other organic dissolvents. [0291] 7. Solution
transparency and color: 0.05 g of the drug solution in 10 ml of
water is transparent and colorless. [0292] 8. pH of 0.1% solution:
5.0-6.0 (potentiometry). [0293] 9. Authenticity: [0294] a)
amino-acid analysis (6 n HCl, 110.degree. C., 20 hrs.) in
correspondence: glycine--2.0 (2.0); glutamic acid--1.9 (2.0);
cysteine--1.7 (2.0). [0295] b) NMR (.sup.1H)-spectroscopy, in
correspondence: CH.sub.2 (.delta. 2.05, 2.40, 3.00, 3.80); CH
(.delta. 3.72, 4.65). [0296] c) HPLC corresponds with the standard
according to the yield time. [0297] 10. Purity (main substance
content): [0298] a) at HPLC>95%; [0299] b) at the amino-acid
analysis>85%; [0300] c) Thin-layer chromatography (TLC)
homogenous; [0301] d) Lithium (Li) content according to the
emission spectral method is 2.2.+-.0.1% [0302] e) Platinum (Pt)
content according to the mass spectrometric analysis is 0.01-0.02%
(II.) Staged scheme for the product synthesis (A.fwdarw.BC) A.
Oxidation of reduced glutathione
(.gamma.-L-glutamyl-L-cysteinyl-glycine)
[0303] A.sub.1--stage for the source reduced glutathione processing
by hydrogen peroxide at pH=8--see FIG. 10.
[0304] Source compound (I): .gamma.-L-glutamyl-L-cysteinyl-glycine
(GSH)
H-.gamma.-L-Glu-L-Cys-Gly-OH
[0305] Reagents: 1) hydrogen peroxide (35%) Fluka
[0306] 2) ammonia solution (25%)
[0307] Reaction conditions: stirring of the water solution (I) and
the reagents at 20.degree. C. (pH=8.0) during 20 min.
[0308] A.sub.2--separation of free hexapeptide
bis-(.gamma.-L-glutamyl)-L-cysteinyl-bis-glycine (see FIG. 11)
##STR00004##
[0309] Source compound: reaction mixture of the A1 stage.
[0310] Reagents: glacial acetic acid
[0311] Reaction conditions: acidification of the A1 reaction
mixture by glacial acetic acid up to pH=5.0, the solution
filtration and lyophilic drying of the product.
[0312] Control for the A oxidation stage processing: by HPLC at the
Delta Pack C18 column (0.1% TFA-MeCN, 0-25%); one will check
presence of the peak (>97%) for the compound (III) (7.4.+-.0.4
min) and the peak absence for the compound (I) (3.0.+-.0.3
min).
B. Conversion of the oxidized form (III) into the lithium salt
(IV)--see FIG. 12.
[0313] Source compound: free hexapeptide (III).
[0314] Reagents: 1 N LiOH solution.
[0315] Reaction conditions: [0316] a) titration of the compound
(III) water solution by two LiOH equivalents; [0317] b) water
evaporation in vacuum at 35-40.degree. C.; [0318] c) the product
precipitation by isopropyl alcohol; [0319] d) precipitate
filtration; [0320] e) precipitate washing by acetone; [0321] f) the
product drying in vacuum (1 mm Hg) at 35-40.degree. C.
[0322] Control of the B stage processing: the reagents quantities
and technological conditions at drying is inspected.
C. Finished product quality control. 1. The main material content
according to HPLC: >97%.
[0323] The analysis is performed using 20 .mu.l of 0.1% drug
solution in the mobile phase, on the column 250.times.4.6 mm with a
converted C.sub.18 phase in isocratic conditions acetonitrile-0.1%
trifluorideacetic acid (2:98); flow rate 1 ml/min., detecting at
220 nm. The comparison is made with the standard peak obtained in
the same conditions.
2. The main material content according to the spectrophotometric
analysis data on the non-oxidized thiol groups content:
>95%.
[0324] Analysis: 0.12 ml of 0.5% drug solution is placed into a
25-ml measuring flask, 1 ml of 0.1% Tris-HCl buffer with pH=8, 0.01
M EDTA and 1 ml of 2% NaBH.sub.4 solution. The reaction mixture is
incubated at 20.degree. C. during 30 min. The reaction is
terminated through introduction of 0.6 ml of 1 M HCl during two
min. in portions of 0.05 ml along with agitation and following
introduction of 2 ml of acetone during three min. with stirring.
Then 0.25 ml of the Elman reagent is added and the volume is
brought to the mark by 0.1 M the phosphate buffer solution (pH
8.0). An optical density is measured at 412 nm, water is a
comparison solution. Simultaneously, the procedure with the drug
standard is conducted and the obtained data is compared.
3. Presence of foreign admixtures: according to TLC data the drug
is homogenous.
[0325] Analysis is performed at introduction of 5 .mu.l of the 0.1%
drug solution in the band. The plates are Kieselgel 60.sub.f
(Merck) 10.times.5 cm, system: n.butanol--pyridine--acetic
acid--water (150:100:30:120). Development is performed according to
the standard methods--ninhydrine and chlorinebenzidine.
4. Lithium (Li) content according to the emission spectral method
is 2.2.+-.0.1%.
Example 3
Pharmacokinetics and Metabolism of GSSG.cndot.Pt and GSSG in Blood
Serum and Tissues After Intravenous Introduction
[0326] The time-concentration GSSG curves and activity changes for
enzymes participating at the GSSG metabolism after the
GSSG.cndot.Pt and GSSG intravenous introduction in different doses
were studied. The variation of the oxidized glutathione
concentration was evaluated in animal blood serum, liver, kidneys,
spleen and lymphocytes during 60 min. after the single
GSSG.cndot.Pt and GSSG intravenous introduction in doses 2 mg/kg
and 20 mg/kg of body weight. In addition, the activity variation of
the enzymes participating in GSSG metabolism was evaluated
(glutathione reductase, glutathione-peroxidase,
glutathione-S-transferase, .gamma.-glutamyl-transpeptidase).
[0327] The study was performed at male CBA mice (standard body
weight--180 to 200 g). Five groups of animals (with no less than 15
mice in each) were formed. The group description is represented
below.
Control Groups:
[0328] #1--intact animals receiving a single injection of the
tested article vehicle (normal saline--(NS)) instead of the
drug;
Test Groups:
[0329] #2--animals receiving the GSSG injection (GSSG dissolved in
normal saline) in a dose of 2 mg/kg;
[0330] #3--animals receiving the GSSG injection (GSSG dissolved in
normal saline) in a dose of 20 mg/kg;
[0331] #4--animals receiving the GSSG.cndot.Pt injection
(GSSG.cndot.Pt dissolved in normal saline) in a dose of 2
mg/kg;
[0332] #5--animals receiving the GSSG.cndot.Pt injection
(GSSG.andgate.Pt dissolved in normal saline) in a dose of 20
mg/kg.
[0333] The blood samples were taken at 1, 2, 5, 10, 20, 40 and 60
min., the serum was separated and the concentration analysis was
performed according to a standard method where the main stages are
protein precipitation, removal from the sample of non-polar and
medium-polar compounds and the following chromatographic analysis
with spectrophotometric detection in conditions of isocratic and
linear gradient elution.
[0334] The GSSG content variation in the blood serum, different
organs and the lymphocytes at the drug intravenous introduction are
given in the Tables 1-4.
[0335] Activity of the enzymes participating in the GSSG metabolism
(glutathione reductase: EC.1.6.4.2; glutathione-peroxidase:
EC.1.11.1.9; glutathione-S-transferase: EC.2.5.1.18;
g-glutamyl-transpeptidase: EC.2.3.2.2) were determined by the
standard reagent kits produced by Boehringer Mannheim GmbH. The
enzyme activity variation values after the GSSG.cndot.Pt and GSSG
intravenous introduction in dose of 2 mg/kg are given at the Tables
5, 6, 7.
[0336] Comparing the drug dynamic distribution of the drugs in the
blood serum, liver, kidneys, spleen and lymphocytes a clear
advantage for the GSSG.cndot.Pt pharmacokinetics regarding GSSG is
evident. The GSSG concentration to the 10.sup.th minute is almost
equal to the initial one whereas, at the same time, the
GSSG.cndot.Pt concentration exceeds 50 times the initial parameters
and remains at the given high level till the end of the studied
period. Besides, the maximal GSSG.cndot.Pt concentration in the
blood serum and in the tissues exceeds 3 times the maximal GSSG
concentration. These features determine higher effective duration
of impact for GSSG.cndot.Pt comparing to GSSG.
[0337] As it follows out of the Tables 5, 6, 7 materials the GSSG
drug increases approximately two times the activity for the enzymes
participating in the thiol metabolism with large number of proved
indices whereas the GSSG.cndot.Pt drug does not significantly alter
the enzymes activity and the proved indices number is
insignificant. It indicates the greater GSSG.cndot.Pt drug
stability as a substrate in regard to main enzymes participating in
the glutathione metabolism and, therefore, calls out longer
presence of the glutathione oxidized form in the blood serum and
different organs.
[0338] Thus, the obtained data analysis demonstrated the higher
GSSG.cndot.Pt stability to selective impact of the main glutathione
metabolism enzymes (first of all, glutathione-reductase) that
determines new dynamics for the drug pharmacokinetics. It
facilitates manifestation of new biological-pharmacological effects
and, thereupon, new GSSG.cndot.Pt therapeutic effects.
Example 4
Table A: GSSG Pharmacokinetics
Initial Data
[0339] A graph on mean values is presented in FIG. 23, showing a
GSSG pharmacokinetic curve for intravenous introduction.
Table B: Pharmacokinetic Parameters
Table C: GSSG.cndot.Pt Pharmacokinetics
[0340] A graph on mean drug concentration values is presented in
FIG. 24, showing a GSSG.cndot.Pt pharmacokinetic curve for
intravenous introduction.
Table D: Pharmacokinetic parameters.
Example 5
Effect of GSSG.cndot.Pt and GSSG on Cytokine Production by Human
Peripheral Blood Mononuclear Leukocytes In Vitro
[0341] Oxidized glutathione (GSSG) as well as a structural analog
thereof, which is a hexapeptide with a stabilized disulfide bond,
were evaluated for their effect on cytokine production by human
peripheral blood mononuclear leukocytes in vitro.
[0342] The leukocytic cytokine production was triggered by adding a
mitogen, concanavalin A (ConA) to the cell culture immediately
after introducing the test substances. In 24 hours of the cellular
exposure to ConA and the test articles, the culture supernatants
were sampled and stored until cytokine determination at -70.degree.
C.
[0343] With the aim of evaluating the functional status of the
cells and their capacity of responding to the mitogen in the
presence of the test articles at each concentration level, the
control cell cultures, containing the test articles in identical
concentrations, were incubated for 72 hours following the initial
concomitant introduction of ConA and the test substances. Sixteen
hours prior to the incubation completion, .sup.3H-thymidine was
added, and the label rate of incorporation into DNA was interpreted
as the criterion of the cellular test system functional state.
[0344] Venous blood from male healthy volunteers was collected into
plastic heparinized tubes (endotoxin tested). PMNL fraction was
isolated by centrifugation in density gradient of Ficoll and sodium
diatrizoate (Histopaque-1077; Sigma).
[0345] Cell concentration was adjusted to 2.times.10.sup.6 per 1 mL
of culture medium (RPMI 1640, Sigma) containing: HEPES (20 mM);
L-glutamine (2 mM); Gentamicin (50 mg/mL); fetal calf serum (10%).
All the reagents used were of cell culture tested grade, Sigma.
Cell viability was estimated by the Trypan blue exclusion method
and 100 mL of cell suspension (200,000 cells) were placed into each
well of flat bottom 96-well sterile microtiter plates for tissue
cultures. Cells from each subject were placed into no less than 39
wells.
[0346] The five following final concentrations of the test articles
(GSSG, as well as GSSG.cndot.Pt) were evaluated: 5000 mg/mL; 500
mg/mL; 50 mg/mL; 5 mg/mL; and 0.5 mg/mL. Each concentration was
established in no less than six wells by adding 50 mL of medium
containing the appropriate quantity of the previously dissolved
test articles. Another six wells were used for control cultures:
only 50 mL of medium was added.
[0347] Immediately after the test articles had been introduced into
the cultures, 50 mL of medium containing ConA (Sigma, cell culture
tested) in a quantity required for a final concentration of 4.0
mg/mL, was added to all the wells excepting three additional ones
which served for evaluation of spontaneous .sup.3H-thymidine uptake
(without ConA).
[0348] After a 24-hour incubation at 37.degree. C. and 5% of
CO.sub.2, contents of three wells (from each sextuplet of identical
wells) were taken out, centrifuged, and the supernatants were
frozen and kept at -70.degree. C. until the cytokine assay was to
be performed. Cultures in the other three wells (of each sextuplet)
were incubated further under the conditions described above.
[0349] Fifty-six hours after the incubation had begun, 1.0 mCi of
.sup.3H-thymidine was added into all the remaining cultures, the
plates were incubated for another 16 hours, and then the contents
of the wells were harvested and transferred onto glass-fiber
filters which were consequently treated with 5% trichloroacetic
acid and ethanol. The filters were dried and their radioactivity
(counts per minute, cpm) was determined using liquid scintillation
counter, Betaplate 1205 (LKB).
[0350] Mean radioactivity values for triplicates of identical
cultures were used to calculate the index of mitogenic stimulation:
the ratio of averaged cpm values for ConA stimulated cultures to
averaged cpm values for unstimulated ones (three wells without
ConA). This stimulation index for wells, where the test articles
were present in various concentrations, served as a criterion of
cellular functional status, and ability of the cells to respond to
mitogenic stimulation.
[0351] Supernatants of 24-hour culture triplicates were
subsequently assayed for cytokine content only if their 72-hour
matched control culture triplicates developed mitogenic response to
ConA with value of the stimulation index in the range from 15 to
50.
[0352] Concentrations of interleukin-1b (IL-1b), interleukin-2
(IL-2); interleukin-3 (IL-3); interleukin-4 (IL-44); interleukin-6
(IL-6), interleukin-8 (IL-8); interleukin-10 (IL-10);
interleukin-12 (IL-12); tumor necrosis factor-.alpha., g
(TNF-.alpha., g), and interferon-.alpha., g (IFN-.alpha., g) were
determined by ELISA using commercial reagent kits (Medgenix,
Belgium) and were expressed in pg/mL of culture supernatants.
[0353] The salient findings given in Tables 8, 9. As one can see
from Tables 8 and 9, the adding of GSSG and GSSG.cndot.Pt into the
culture media resulted in statistically significant and
dose-dependent stimulation of the cytokine production by human
mononuclear leukocytes. However, GSSG.cndot.Pt stimulating
influence was more significant (1.5-2 times as much) on the studied
cytokine production with stimulation and regulation for production
of the wider cytokine range in comparison with the GSSG effect. One
can clearly see correlation of the interrelated cytokine changes
(increasing of IL-1b, IL-2, TNF-.alpha., g along with decreasing of
IL-4, IL-10) in the Tables 8 and 9.
[0354] Thus, the GSSG.cndot.Pt impact on the human peripheral
mononuclear leukocytes in vitro was manifested with considerable
stimulation of the wider cytokine range release into culture media
considering their reciprocal regulative effect, and, thereby, it
confirmed the GSSG.cndot.Pt stimulatory and regulatory effect on
the natural cytokine-producing capacity of the human blood
cells.
Example 6
Effect of GSSG and GSSG.cndot.Pt on Cytokine and Hemopoietic Factor
Production as Well as on Hemopoiesis and Immunity Parameters in
Cyclophosphamide-Induced Hemo- and Immunodepression
[0355] 1. The oxidized (GSSG) glutathione as well as the structural
analog thereof, which is the hexapeptide with the stabilized
disulfide bond, were evaluated in a murine model of hemo- and
immunodepression induced by a single administration of cytostatic
agent Cyclophosphamide (CP).
[0356] The study was designed to evaluate the effect of a five-day
long administration of the test articles on the capability of the
CP-treated murine splenocytes to produce interleukin-1
(IL-1.alpha.,b); interleukin-2 (IL-2); interleukin-3 (IL-3);
interleukin-4 (IL-4); interleukin-6 (IL-6), interleukin-8 (IL-8);
interleukin-10 (IL-10); interleukin-12 (IL-12); tumor necrosis
factor-.alpha., g (TNF-.alpha., g), interferon-.alpha., g
(IFN-.alpha., g) and G-CSF, M-CSF, GM-CSF in vitro. In addition,
the blood leukocyte and lymphocyte count and the bone marrow
cellularity (karyocyte count) were determined at eight days after
CP administration. Some animals receiving CP were then challenged
with sheep red blood cells (SRBC), and the humoral immune response
to the antigen was evaluated.
[0357] Male CBA mice (180 to 200 g body weight) were given a single
introperitoneal injection of CP in a dose of 50 mg/kg. Four groups
of animals (with no less than 15 mice in each) were formed. The
group description is represented below.
Control Groups:
[0358] #1--intact animals receiving a single injection of normal
saline (NS) instead of CP injection, which further were treated
with test article vehicle (normal saline);
[0359] #2--animals receiving a single CP injection, which further
were treated with test article vehicle (normal saline);
Test Groups:
[0360] #3--animals receiving a single CP injection, which further
were treated with the test article (GSSG dissolved in normal
saline) in a dose of 5 mg/kg;
[0361] #4--animals receiving a single CP injection, which further
were treated with a GSSG.cndot.Pt (dissolved in normal saline) in a
dose of 5 mg/kg.
[0362] Twenty-four hours after the CP injection, five animals in
each group were immunized with SRBC (10.sup.7 cells in 0.5 mL of
NS, intra-peritoneally).
[0363] On day 3 after the CP injection (24 hours after the
immunization) the intraperitoneal injections of the test or
reference articles were started (as it has been described above).
Injections were performed during five days: once a day, daily.
[0364] Twenty-four hours after the completion of five-day treatment
course (on the 8.sup.th day after the CP injection), mice were
euthanized and splenocyte cultures were aseptically prepared for
assessment of spontaneous cytokine and hemopoietic factor
production by the spleen lymphocytes in vitro.
[0365] Simultaneously, blood and marrow samples were collected for
blood leukocyte, lymphocyte, and marrow nucleated cell counts.
[0366] Serum samples from immunized animals were tested on level of
SRBC agglutinins (day 8 after the CP injection, and day 7 after the
immunization).
[0367] Table 10 shows the parameters of cytokine and hemopoietic
factor production by splenocytes, bone marrow and blood count
indices, and the immune response to sheep red blood cells in mice
receiving the test articles against the background of
cyclophosphamide induced hemo- and immunodepression.
[0368] According to the Table 10 data, the GSSG.cndot.Pt
administration set to norm the cytokine and hemopoietic factor
production while GSSG performed only exiguous stimulating effect.
Besides, GSSG.cndot.Pt stimulated production for the wider cytokine
and hemopoietic factor range as well as had significant regulatory
influence on shift with respect of the cytokine status that was
confirmed with the positive correlation of the interrelated
cytokine changes at the corresponding pathologic process.
[0369] Thus, the GSSG.cndot.Pt use in CP-induced hemo- and
immunocompromised animals results in a prominent stimulation and
regulation on the cytokine and hemopoietic factor endogenous
production along with restoration of the bone marrow and blood
cellular indices as well as immune response development to sheep
red blood cells.
2. The purpose of the present study was to explore the GSSG_Pt
efficacy on the cyclophosphamide-induced cytopenia (myelopenia)
model.
[0370] The study was conducted on the white male rats weighing
160.0 gr. Cyclophosphamide was introduced once subcutaneously at
the back in doses 50 mg/kg (a vehicle was water for
injections).
[0371] Four groups of animals (with no less than 15 mice in each)
were formed. Group description is represented below.
Control Groups:
[0372] #1--intact animals receiving a single injection of normal
saline (NS) instead of CP injection, which further were treated
with test article vehicle (normal saline);
[0373] #2--animals receiving a single CP injection, which further
were treated with test article vehicle (normal saline).
Test Group:
[0374] #3--animals receiving a single CP injection, which further
were treated with GSSG.cndot.Pt (dissolved in normal saline) in a
dose of 5 mg/kg.
[0375] On day 2 after the CP injection the intraperitoneal
injections of the test or reference articles were started (once a
day, during 10-15 days).
[0376] At the end of each series (10 and 15 days) the experimental
groups were euthanized through ether overdose and peripheral blood
(tail veins) and bone marrow (femora) were taken for the analysis.
The hematological studies were conducted with the unitized standard
methods. The peripheral blood analysis results are represented in
the Table 11; myelogram analysis results are given in the Table
12.
[0377] Analyzing Table 11 results one can note that
cyclophosphamide in the dose 50 mg/kg was found to perform the
marked cytopenic effect at all of the formed blood elements with
absolute and relative lymphopenia depending on the observation
terms (maximally exhibited at the day 15).
[0378] It should be noted that four animals died: on the 9.sup.th,
10.sup.th, 12.sup.th, and 14.sup.th days. In fact, all these
animals demonstrated flabbiness, hypodynamia, weight loss.
[0379] The GSSG.cndot.Pt introduction provided the significant
stimulating effect. The general state improvement, positive weight
changes were observed, and the immature cell form appearing in
blood indicated the bone-marrow hemopoiesis activation. The death
of the animals was not noted.
[0380] Analysis of the myelogram revealed that cyclophosphamide in
the dose 50 mg/kg was found to perform the significant myelotoxic
effect. Erythro-, trombocyto- and lymphopoiesis are especially
suppressed. Myelosuppression is the most marked at the 15.sup.th
day.
[0381] The GSSG.cndot.Pt administration had the considerable
myelostimulating effect.
[0382] The drug GSSG.cndot.Pt in the dose 15 mg/kg being introduced
as the daily course during 10-15 days exerts the marked
myelostimulating effect at the cyclophosphamide-induced cyto- and
myelopenia. The drug administration improves the general state, it
positively influences at the body weight changes and lowers
mortality by about 30%.
Example 7
Effect of GSSG and (Li.cndot.GSSG.cndot.Pt) on Cytokine and
Hemopoietic Factor Production as Well as on Hemopoiesis and
Immunity Parameters in Radiation-Induced Hemo- and
Immunodepression
[0383] Both oxidized (GSSG) and the structural analog thereof,
which is the lithium salt of the hexapeptide with the stabilized
disulfide bond (Li.cndot.GSSG.cndot.Pt), were evaluated in a murine
model of hemo- and immunodepression induced by a single irradiation
in a total dose of 1 Gy.
[0384] The study was designed to evaluate efficacy of seven-day
daily administration of the test articles (with the dosing started
two hours post-exposure) on the capability of the splenocytes from
mice exposed to radiation to produce interleukin-1 (IL-1.alpha.,b);
interleukin-2 (IL-2); interleukin-3 (IL-3); interleukin-4 (IL-4);
interleukin-6 (IL-6), interleukin-8 (IL-8); interleukin-10 (IL-10);
interleukin-12 (IL-12); tumor necrosis factor-.alpha., g
(TNF-.alpha., g), interferon-.alpha., g (IFN-.alpha.,) and G-CSF,
M-CSF, GM-CSF in vitro. In addition, the blood leukocyte and
lymphocyte counts and the spleen and bone marrow cellularity
(karyocyte count), as we well as splenic and medullary
colony-stimulating capacity, were determined at the 8.sup.th day
post-exposure.
[0385] Male CBA mice (18 to 20 g body weight) were irradiated with
single dose of 180 kV X-rays filtered with 0.5 mm Cu (at 15 mA,
distance--70 cm, duration two min. and 28 sec.). The total absorbed
dose comprised approximately 1 Gy.
[0386] Four groups of animals (with no less than 12 mice in each)
were formed. Group description is represented below.
Control Groups:
[0387] #1--intact animals receiving a sham irradiation procedure to
reproduce a stress impact, which further were treated with the test
article vehicle (normal saline);
[0388] #2--control animals irradiated in a dose of 1 Gy, which
further were treated with test article vehicle (normal saline).
Test Groups:
[0389] #3--animals irradiated in a dose of 1 Gy, which further were
treated with the test article (GSSG dissolved in normal saline) in
a dose of 5 mg/kg;
[0390] #4--animals irradiated in a dose of 1 Gy, which further were
treated with the test article (Li.cndot.GSSG.cndot.Pt dissolved in
normal saline) in a dose of 5 mg/kg.
[0391] Two hours after the irradiation the intraperitoneal
injections of the test or reference articles were started (as it
has been described above). Injections were performed during seven
days: once a day, daily.
[0392] Twenty-four hours after the completion of seven-day
treatment course (on the 8.sup.th day after the irradiation), mice
were euthanized and splenocyte cultures were aseptically prepared
for assessment of spontaneous cytokine and hemopoietic factor
production (interleukin-1 (IL-1.alpha.,b); interleukin-2 (IL-2);
interleukin-3 (IL-3); interleukin-4 (IL-4); interleukin-6 (IL-6),
interleukin-8 (IL-8); interleukin-10 (IL-10); interleukin-12
(IL-12); tumor necrosis factor-.alpha., g (TNF-.alpha., g),
interferon-.alpha., g (IFN-.alpha., g) and G-CSF, M-CSF, GM-CSF) by
the spleen lymphocytes in vitro.
[0393] Simultaneously, blood, spleen and marrow samples were
collected for blood leukocyte and lymphocyte, and spleen and marrow
nucleated cell counting.
[0394] Additionally, hemopoietic colony-formation ability of spleen
and bone marrow cells was assessed by the method of colony-forming
unit (CFU) direct count in the spleens of irradiated singenic CBA
mice receiving intravenously spleen or bone marrow cells obtained
from animals of control or test groups.
[0395] Splenocytic levels for the cytokine and hemopoietic factor
production, blood, bone marrow, and spleen cellular indices as well
as the colony-forming parameters (colony-forming units, CFU) at the
bone marrow and spleen of the irradiated animals at the 8.sup.th
day post-exposure, are summarized in Table 13
[0396] As is evident from the table data, the
Li.cndot.GSSG.cndot.Pt administration results in statistically
significant recovery of the cytokine and hemopoietic factor
production by splenocytes, whereas GSSG produces less significant
effect. However, Li.cndot.GSSG.cndot.Pt influences endogenous
production for the wider cytokine and hemopoietic factor range as
well as regulates the cytokine status alterations with respect of
the corresponding pathologic process.
[0397] Thus, the Li.cndot.GSSG.cndot.Pt usage as an applied method
in animals with developed radiation-induced hemo- and
immunodepression results in pronounced stimulation-regulation of
the endogenous cytokine and hemopoietic factor production, and also
leads to an effective recovery for the cellular compositions of the
blood, lymphoid and hemopoietic organs as well as the bone marrow
and spleen colony-forming activity.
Example 8
Effect of the GSSG.cndot.Pt Composite and Salts Thereof on
Processes of Phosphate Modification as Well as on Content of
Lymphocytes Bearing IL-2 Receptors
[0398] The molecular mechanisms of the reproduction of the
immuno-biochemical effects of the cytokines with the GSSG.cndot.Pt
composite and the salts thereof were studied.
[0399] At the study action of the GSSG.cndot.Pt composite and the
salts thereof--sodium (Na), lithium (Li) and magnesium (Mg)--was
evaluated at the murine model of hemo- and immunodepression induced
by single administration of cytostatic cyclophosphamide (CF).
[0400] At this study the effect of a five-day long administration
of the test articles on the capability of the phosphorylating level
of the lymphocyte cytosol proteins on tyrosine and content of the
"active" lymphocytes-carriers of IL-2-receptors were evaluated.
[0401] Male CBA mice (18 to 20 g body weight) were given a single
intraperitoneal injection of CP in a dose of 50 mg/kg. After the CF
injection the animals were introduced with the tested articles in
dose of 5 mg/kg 24 hours later. The tested articles were introduced
during five days (daily, once a day). Twenty-four hours after
completion of the tested article introduction the mice were
euthanized and blood samples were collected to conduct the
study.
[0402] A fraction of mononuclear leukocyte was obtained by
centrifugation in gradient of ficoll-metrizoat (Histopaque, Sigma).
Cell concentration was adjusted to 2.times.10.sup.6 cells per 1 ml
of cell culture medium (RPMI 1640), containing 20 mM HEPES, 2 mM
glutamine, 50 mg/mL gentamicin and 10% fetal calf. Cell viability
was estimated by the Trypan blue exclusion method, then the cell
suspension was placed into wells of 96-well microliter
plates--200,000 cells per well.
[0403] Content of the lymphocytes-carriers of the IL-2-receptors
was determined according to Horgan A. F. (1994) on smears of
mononuclear slip. Mononuclear antibodies to chains p55 and p75 of
the IL-2-receptor were used as the first antibodies. To reveal the
first antibodies the polyclonal rabbit antibodies against murine
immunoglobulins marked with were used. Count of the
lymphocytes-carriers of the IL-2-receptors was made in percentage
to the number of total lymphocytes.
[0404] For metabolical marking lymphocytes were cultivated in the
Igla medium with addition of 10% cattle serum. The metabolical
marking with [32P]ortho-phosphoric acid was performed by cell
incubation during 10-12 hrs. in the phosphorusless medium DME
containing 100 .mu.Ci/ml of [32P]ortho-phosphate. On each sample
0.2 ml of the medium with isotope were added. After incubation
cells were destroyed by pipetting and centrifuged at 6000 g for 30
min. The obtained supernatant was used for immunoprecipitation with
polyclonal antibodies to phosphotyrosine at Fu method (1992).
Protein A-sefarose was used for the precipitation of immune
complex. The precipitate was washed three times and the precipitate
activity was counted on "Gamma" counter.
[0405] According to the results of the conducted study (Table 14),
it was found that action of the GSSG.cndot.Pt composite on the
isolated lymphocytes causes (see Table 14) at 10 minute a
significant increase of the phosphorylating level on tyrosine of
the lymphocyte cytosol proteins, which is the integral indication
of the activity of the signal-transducing systems. These changes
due to the GSSG.cndot.Pt composite action, largely due to the
GSSG.cndot.Pt composite derivative action determine the modulation
of the redox-sensitive gene expression, first of all,
immunologically important genes, responsible for the synthesis of
the cytokines and hemopoietic factors.
[0406] In Table 15 the data is given on phosphorylating level on
tyrosine of the lymphocyte cytosol proteins and percentage of the
lymphocytes-carriers of IL-2-receptors in CBA-line mice received
the tested articles having cyclophosphamide induced hemo- and
immunodepression.
[0407] Application of the GSSG.cndot.Pt composite salts results in
the increase of the percentage of the lymphocytes-carriers of
IL-2-receptors and almost normalizing their quantity (normal one is
18.3.+-.1.6%). The similar regularity was found when
phosphorylating level on tyrosine of the lymphocyte cytosol
proteins was studied.
[0408] Application of the GSSG.cndot.Pt composite salts results in
restoration of the percentage of the lymphocytes-carriers of
IL-2-receptors in immunodeficiency conditions modeled by
cyclophosphamide. There is the increase of the phosphorylating
level on tyrosine of the lymphocyte cytosol proteins of the
signal-transducing systems that can be one of the factors of the
described immunostimulating actions of the articles tested.
[0409] Thus, the example provides evidence of GSSG.cndot.Pt to
reproduce (imitate) the regulatory effects of the range of
cytokines, first of all, IL-2. We are intending to mean the
induction by the GSSG.cndot.Pt composite of the intracellular
mechanisms performing regulatory cytokine signals on the system of
immunocompetent and hemopoietic cells as the IL-2 effect
reproduction. The conducted studies have shown that changing of the
phosphorylating level on tyrosine of the lymphocyte cytosol
proteins of the signal-transducing systems of the cells of the
organs immunogenesis and hemopoiesis in conditions of
cyclophosphamide modeled immunodeficiency causes the effect of the
dynamic normalization of the active lymphocyte content.
[0410] Therefore, application of the GSSG.cndot.Pt composite and
derivatives thereof in the form of therapeutically purposed
medicinal drugs not only stimulates the cytokine and hemopoietic
factor endogenous production but also provides the reproduction of
the immune-biochemical cytokine effects, especially in case of
receptors desensitization observed mainly in oncological and
retro-virus pathology.
Example 9
Stimulation of Endogenous Cytokine Production and the Therapeutic
Effect of the GSSG_Pt Application in a Patient with a Stomach
Cancer, Peritoneal Metastases, Ascites, Splenomegaly and
Cholestatic Hepatitis
[0411] A 33-year old patient was diagnosed as having stomach
neoplasm for more than two years (adenocarcinoma of moderate
differentiation degree). In 1993 the patient was operated for
malignant stomach ulcer and numerous dense lymph nodes were found
in the porte hepatis which were considered to be metastases.
[0412] In January 1994 the course of chemotherapy (5-FU) was
complicated by the severe cholestasis and percutaneous drainage of
the left and right liver ducts was undertaken, that six months
later was followed by the choledochoejunostomy with changeable
transliver drains with Brown's anastomosis.
[0413] In November 1995 the patient's state worsened. According to
the examinations the patient experienced an active secondary
hepatitis. The liver was enlarged and painful and protruded from
the costal arch up to 5-6 cm. Blood chemistry indices proved to be
persistently abnormal and hardly corrected by the performed
treatment: bilirubin--40.0 due to indirect (up to 31.0); activity
of amino-transferases--approximately six times higher than upper
normal limit, hypoalbunemia was up to 26%; and there was also
hypergammaglobulinemia; hypercholesterolemia was up to 10.2
mmol/l.
[0414] During fibrogastrocopy (November, 1995) a stomach cancer
located in the middle area of the stomach body and extended about 8
cm was confirmed. The tumor was solid-like type. Stomach walls were
rigid. Histology examination defined the tumor as adenocarcinoma of
moderate degree differentiation. In December, 1995, the patient had
an explorative laparotomy. Ascites was found with plural metastases
all over the peritoneum, splenomegaly. The patient's case was
identified as inoperable.
[0415] A decision was taken to apply GSSG drug form. The drug was
injected parenterally (intramuscularly and intravenously), and
additionally, the drug form was used via local injections around
the tumor tissue through an endoscope. An average doses which were
used for intramuscular and intravenous injections--0.1-0.5 mg/kg,
and for local injections--up to 50 mg in situ. Parenteral
injections of the drug were applied every other day, b.i.d.
(intravenous injections at the morning, and intramuscular ones at
the evening), during three weeks, and after that, two times a week
during four weeks. The drug introduction through the endoscope was
performed once in seven days. Two months after the beginning of the
treatment with the drug form used the fibrogastroduodenoscopy
showed: esophagus was passable, mucous membrane was pink, cardia
rosette was partly closed. On empty stomach moderate amount of
foamy secretion was in the stomach, which was intensively colored
with bile. The tumor extent was 4.8 cm. At the same time,
substantial improvement of hematology and blood chemistry indices
was found and the liver size decreased up to 3 cm (below the costal
arch).
[0416] Six months after the treatment completion (July, 1996) the
patient's state worsened significantly. According to the
examination the secondary hepatitis relapsed. The liver was
increased in size and tender, protruded 4 cm beyond the costal
arch. Blood chemistry indices were abnormal and hardly corrected by
the performed treatment: bilirubin--360 due to indirect (up to
28.0); activity of amino-transferases--approximately four times
higher than upper normal limit, hypoalbunemia was up to 21%; and
there was also hypergammaglobulinemia; hypercholesterolemia was up
to 9.42 mmol/l.
[0417] At the fibrogastrocopy (July, 1996) a stomach cancer located
in the middle area of the stomach body and extended about 6 cm was
confirmed. The tumor was solid-like type. Stomach walls were rigid.
Histology examination defined the tumor as a moderate degree
differentiation adenocarcinoma. In August, 1996, the patient had an
explorative laparotomy. Ascites was found with plural metastases
all over the peritoneum, splenomegaly. Considering the previously
conducted therapy the decision was taken to apply the new drug
GSSG.cndot.Pt that is the structural analog of the former
administered GSSG drug. The drug was introduced according to the
identical regime: parenterally (intramuscularly and intravenously),
and additionally, the drug form was introduced via local injections
around the tumor tissue through an endoscope. An average doses
which were used for intramuscular and intravenous
injections--0.1-0.5 mg/kg, and for local injections--up to 50 mg in
situ. Parenteral injections of the drug were applied every other
day, b.i.d. (intravenous injections at the morning, and
intramuscular ones at the evening), during three weeks, and after
that, two times in a week during four weeks. The drug introduction
through the endoscope was performed once in seven days.
[0418] Two months after the treatment initiation with the applied
drug: the liver protruded 1 cm beyond the costal arch, tenderless
at palpation. According to the ultrasound examination data: there
is fibrous tissue at the place of previously determined cancer
sites. At the fibrogastrocopy: the esophagus was passable, mucous
membrane was pink, cardia rosette was partly closed. The gastric
walls are elastic. There was moderate amount of foamy secretion in
the stomach with saliva in empty stomach. The tumor extent was 1.5
cm. The duodenum was freely passable. At the same time, substantial
improvement of hematology and blood chemistry indices was
indicated.
[0419] Comparing the therapeutical efficacy of the drugs
GSSG.cndot.Pt and GSSG using of the former one was found to be
advantageous that was manifested by the positive changes at the
clinical, biochemical, hematological and immunological indices,
fibrogastrocopy data (the tumor size decrease at 75% while applying
GSSG.cndot.Pt comparing to the 40% decrease after the GSSG
administration) (Tables 16, 17). Moreover, at the Table 17 one can
see that GSSG.cndot.Pt stimulates production of the wider cytokine
and hemopoietic factor range having a regulatory influence on their
content change.
[0420] Thus, the treatment according to the present invention
resulted in considerable regress of tumor process with simultaneous
obvious beneficial changes in hematology, blood chemistry and
immunology parameters, and significant improvement of the life
quality.
Example 10
Therapeutical Efficacy the GSSG.cndot.Pt Application for Treatment
of Lung Cancer
[0421] No. 1
[0422] Year of birth: 1938.
[0423] Diagnosis: Cancer of the right lung upper lobe.
[0424] Histological diagnosis: No. 45760 (State Research Center on
Pulmonology)--small cell cancer.
[0425] Case-history: No. 4024.
[0426] Complaints on admission: coughing with hard discharged
mucous sputum, dyspnea on little exertion.
[0427] Objective examination: The patient's state is satisfactory.
Peripheral lymph nodes are not enlarged. There are coarse breath
sounds weakened at the upper and medium regions of the right lung.
There are rare dry rales, dyspnea on the slightest exertion.
[0428] Roentgenography (initial data): The upper mediastinum shadow
is broadened due to enlarged right paratracheal lymph nodes. The
indistinct shadow of the upper right root part is broadened. There
is an additional shadow in the peripheral S.sub.3 region of the
right lung against the background of marked interstitial changes in
both lungs. The right interlobar borders are thickened. Conclusion:
there are signs of metastases into lymph nodes of the root and the
mediastinum with lymphangoitis sings.
[0429] Treatment course: there were applied three
immunochemotherapy courses using GSSG.cndot.Pt drug.
[0430] After the treatment: the patient's state has improved
significantly: the mild weakness is still present, there is no
dyspnea.
[0431] Objective examination: The state is satisfactory. The
peripheral lymph nodes are not enlarged. There is a weakened breath
sounds in the medium departments of the right lung. The breath
sounds in other departments are vesicular. There are no rales.
[0432] Roentgenography (after the treatment performed): The lung
fields are particularly clear. There are mild infiltrative signs at
the S.sub.3 department of the right lung. The roots are not
enlarged. There are no additional formations at the right root
projections. The upper mediastinum shadow is not enlarged. The
solitary paratracheal lymph nodes can be determined.
[0433] No. 2
[0434] Year of birth: 1945.
[0435] Diagnosis: Right lung cancer, hepatic metastases.
[0436] Histologic diagnosis: No. 45998, small cell cancer
[0437] Case-history: No. 4076
[0438] Complaints on admission: coughing with hard discharged
mucous sputum, dyspnea on mild exertion, weakness, constant pains
in the lumbar region extending to stomach. During last six months
the patient lost 6 kg.
[0439] Objective examination: The state is medium severe. Scleras
are icteric. The breathing sounds are coarse, weakened in the right
lung upper and medium departments. In the right supraclavicular
zone one can palpate enlarged lymph nodes (solid consistency,
hardly movable, size--3.0 and 1.0 cm, painless). At auscultation
there are coarse breathing sounds, weakened in the medium
departments in the right. There are solitary dry rales, dyspnea on
the slightest exertion. The liver protruded 3.5 cm over the costal
arch.
[0440] At examination there were found: middle-lobe bronchus
cancer, middle lobe hypoventilation, pneumonitis. At ultrasound
examination there are sings of metastatic liver impairment.
[0441] Roentgenography (initial data): The right lung middle lobe
is decreased in size (state of hypoventilation). Against the
background of the increased lung pattern there is intensive, almost
homogenous infiltration with distinct margin along the horizontal
interlobar pleura. The right root cannot be determined. The upper
and lower lobes of the right lung and the left lung are without any
particular features. The mediastinal organs are not noticeably
displaced.
[0442] Treatment course: there were applied three
immunochemotherapy courses using GSSG.cndot.Pt drug.
[0443] After the treatment: The patient's state has improved
significantly: there are no weakness and dyspnea, appetite has
appeared, he gained 5 kg. The blood indices have restored.
[0444] Objective examination: The state is satisfactory. The
peripheral lymph nodes are not enlarged. There is a weakened breath
sounds in the medium departments of the right lung. The breath
sounds in other departments are vesicular. There are no rales.
[0445] According to the liver ultrasonography and CAT scan data:
there is full mass process regression.
[0446] Roentgenography (after the treatment performed): There is
the insignificant atelectasis of the middle lobe at the thoracic
X-rays picture. The roots are structural and not enlarged, the
right one is slightly displaced downward. The heart is not enlarged
in size.
[0447] Comparing to the initial data there is considerable positive
development: at the right the pulmonary tissue has become more
transparent (reduction of the hypoventilation signs), there are no
infiltrative shadows.
[0448] Conclusion:
Evaluating Therapeutical Effectiveness of the GSSG.cndot.Pt Drug
the Following was Found:
[0449] 1. Clinical restoration of indices (general state
improvement, pathologic absence of symptoms, body weight
gaining);
[0450] 2. Roentgenologic picture changes (pulmonary tissue
transparency increase, infiltrative absence of shadows,
disappearing of atelectasis and hypoventilation);
[0451] 3. Hematologic restoration of indices (increase of the
erythrocyte count and hemoglobin content, restoration of white
blood count);
[0452] 4. Changes of the ultrasound and CAT scan data (full tumor
process regression);
[0453] 5. Immune indices restoration and increase of the
CD16.sup.+, CD25+ counts indicating restitution of the antitumor
surveillance system.
[0454] 6. Induction of the wide cytokine range synthesis as well as
modulation of their content mutual regulation (correlation of the
content changes of IL-1b, IL-4, TNF-.alpha.).
[0455] Thus, in the given clinical Examples it has been
demonstrated that the GSSG.cndot.Pt drug application has provided
faster restoration of clinical, roentgenologic, hematologic and
immune indices ensuring more effective restitution of the immunity
and hemopoiesis systems. The aforementioned indicates the tumor
process regression that, eventually, calls out significant increase
of the patient's quality of life.
Example 11
Therapeutical Efficacy the GSSG.cndot.Pt Application for Treatment
of Chronic Viral Hepatitis B (CHBV)
[0456] No. 3
[0457] Date of birth: 1945
[0458] Diagnosis: CHBV, replicative phase (PCR HBV+) with moderate
activity grade.
[0459] Case-history: No. 1068.
[0460] Complaints at admission: weakness, discomfort under the
right costal arch, nausea, no appetite.
[0461] Anamnesis morbi: during last six years the patient noted
periodically appeared dull pains under the right costal arch,
weakness, urine color changes. At examination there was found:
hyperbilirubinemia up to 34 mmol/L, ALT increase to 5.4 mmol/hr.L.
CHBV serologic markers and PCR HBV(+) were determined at the
hospital examination.
[0462] Objective examination: The patient's state is satisfactory.
The liver protruded 2 cm beyond the costal arch. The liver margin
is firm and tender.
[0463] Previous treatment was not performed.
[0464] Treatment course: There was performed the treatment course
with administration of the GSSG drugs according to the regime.
[0465] State after the performed treatment course: There were noted
the following positive changes--significant general state
improvement, no weakness and nausea, diminution of the discomfort
sensation. The liver protruded 0.5 cm beyond the costal arch. The
liver margin is soft and tenderless.
[0466] No. 4
[0467] Year of birth: 1964
[0468] Diagnosis: CHBV, replicative phase (PCR HBV+), moderate
activity degree.
[0469] Case-history: No. 1043.
[0470] Complaints on admission: considerable weakness, no appetite,
sweating, urine darkening.
[0471] Anamnesis morbi: The patient feels sick beginning from
January 1996 when for the first time there appeared dull pains
under the right costal arch, temperature increase up to
38.7.degree. C., vomiting, urine darkening. Acute viral hepatitis B
was diagnosed and confirmed serologically. The patient was
administered with detoxicating and antibacterial therapy. However,
afterwards there were observed increased Hbs Ag and ALT values,
persistent viral replicative activity. Being examined at the last
time there were found: hyperbilirubinemia to 78 mmol/L, ALT
increase to 6.2 mmol/hr.L. CHBV serological markers and PCR HBV(+)
were determined at the hospital examination.
[0472] Objective examination: The general state is satisfactory.
Skin and scleras are icteric. The liver protruded 2.5 cm beyond the
costal arch. The liver margin is firm and tender.
[0473] Previous treatment: from 17.09.97 the patient was
administered with acyclovir during 21 days. After the course
completion there were increased ALT--up to 2.1 mmol/hr.L, bilirubin
to 32 mmol/L. The Hbs Ag degree did not change. The patient's state
was defined by the significant asthenic-vegetative syndrome.
[0474] Treatment course: There was performed the treatment course
with administration of the GSSG.cndot.Pt drugs according to the
regime.
[0475] State after the performed treatment course: There were noted
the vivid following positive changes--significant general state
improvement, no weakness, sweating and nausea. The skin and scleras
were not icteric. The urine color became normal, diminution of the
discomfort sensation. The liver protruded 0.5 cm beyond the costal
arch. The liver margin became softer and tenderless.
Comparison (Tables 20, 21):
[0476] At the comparative analysis on the therapeutical efficacy of
the GSSG.cndot.Pt and GSSG drugs the former was shown to be
advantageous that was manifested by the following:
[0477] 1. Biochemical indices normalization (ALT and bilirubin
decrease);
[0478] 2. Significant HBs Ag decrease and replication
termination;
[0479] 3. Immune indices normalization and increase of the CD95+
content indicating the apoptosis process activation in the
virus-transformed cells;
[0480] 4. Considerable regulation for the wider cytokine range.
Example 12
Therapeutical Efficacy the GSSG.cndot.Pt Application for Treatment
of Acute Viral Hepatitis B (AHBV)
[0481] No. 5
[0482] Sex: female.
[0483] Age: 20.
[0484] In-patient card: 678
[0485] Diagnosis: Acute viral hepatitis B (HBs Ag "+"), replicative
phase (PCR HBV "+"), prolonged form; chronic viral hepatitis D,
replicative phase (PCR HDV "+").
[0486] Complaints during examination: weakness, appetite
decrease.
[0487] Anamnesis morbi: The patient felt sick in August 1997, when
she noticed sharp weakness, malaise, back bone aching, temperature
raising up to 38.8.degree. C. Dark urine and sclera icterus
appeared 10 days later. The patient was admitted to the viral
infectious clinic, where she received course of the detoxicating,
spasmolytic, antibacterial therapy. However, replicative viral
activity and increased GPT level were still present. The prolonged
cytolytic syndrome gave foundation for administration of the drug
GSSG.cndot.Pt.
[0488] Previous treatment was not performed.
[0489] Treatment with GSSG.cndot.Pt drugs: from 30.10.97 to
23.11.
[0490] Patient's state after the treatment course completion:
[0491] The patient's state is satisfactory. She noted the appetite
increase, weakness reduction. Conclusion (Tables 22-24):
[0492] The immunomodulating course with the GSSG.cndot.Pt drugs has
provided the following positive changes: biochemical indices
normalization; termination of HBV and HDV replication; termination
of HBs Ag persistency; virus-infected cell apoptosis induction;
general state improvement; stable therapeutic effect.
Example 13
Therapeutical Efficacy the GSSG.cndot.Pt Application for Treatment
of Chronic Viral Hepatitis C (CHCV)
[0493] No. 6
[0494] Sex: male.
[0495] Age: 18.
[0496] Patient's case: No. 1043
[0497] Diagnosis: Chronic viral hepatitis C, replicative phase (PCR
HCV "+"), moderately manifested activity; chronic viral hepatitis
B, integrative phase (PCR HBV "-"); narcotic intoxication,
narcomania.
[0498] Complaints during examination: weakness, pains at the right
under the ribs, at knee-joints, the backbone and wrist joints.
[0499] Anamnesis morbi: The patient noticed pains in the
knee-joints and the backbone at the beginning of August, 1997. On
blood test an increase of the bilirubin level up to 34 mmol/L and
GPT level up to 2.1 mmol/hr.L. were found. During examination in
the hospital from Aug. 15, 1997, anti-HCV IgG and the replicative
activity of the hepatitis C virus were found.
[0500] Anamnesis vitae: The patient started using drugs at 14. To
the examination time he uses up to 2 g of heroin per day. He is at
the state of the narcotic abstinence.
[0501] Previous treatment was not conducted.
[0502] Immunomodulating therapy course with GSSG.cndot.Pt drugs:
from Aug. 15, 1997, to Sep. 7, 1997.
[0503] Patient's state after the treatment course completion: The
patient's state is satisfactory. He noticed significant reduction
of weakness, no pains in the right under the ribs and in the
joints. As patient said the narcotic abstinence state diminished
almost without pain and in less time. Biochemical indices
normalization and absence of the viral replicative activity was
marked.
[0504] Conclusion: The immunomodulating therapy course with the
GSSG.cndot.Pt drug provided positive changes, which were indicated
by: biochemical and serologic indices normalization; termination of
HCV replication. Immune indices and cytokine status parameters
correlate to the infectious process controlling and viral
replication absence. Examination of the patient's peripheral blood
lymphocytes by liquid chromatography with monoclonal antibodies to
FasAg (CD95.sup.+) after the treatment revealed the CD95.sup.+ cell
increase indicating activation of the programmed cell death process
in virus-infected cells. At supervision at one and three months
after the treatment stabilization of this state was noted.
[0505] Concomitant drug intoxication and an abstinence state at
application of the GSSG.cndot.Pt drugs were corrected faster and
were less excruciating for the patient.
[0506] The immunomodulating course with the GSSG.cndot.Pt drugs for
chronic hepatitis C with replicative activity and concomitant drug
intoxication has provided the following results: [0507] biochemical
indices normalization in blood; [0508] hematological indices
normalization; [0509] termination of HCV replication; [0510]
normalization of the immune blood indices and cytokine status;
[0511] apoptosis process induction in the peripheral blood
lymphocytes; [0512] rapid correction for the drug abstinence state;
[0513] stable therapeutic effect
Example 14
Comparative Analysis of the GSSG and GSSG.cndot.Pt Effects on
Growth Development and Apoptotic DNA Degradation at Normal and
Transformed Cells
[0514] The GSSG and GSSG.cndot.Pt effects on growth development and
apoptotic DNA degradation at normal and transformed (HL-60) cells
were comparatively analyzed. To that end, GSSG and GSSG.cndot.Pt
were incubated for 24 hours with HL-60 myeloid line cells and
normal human lymphocytes obtained from healthy volunteers'
blood.
[0515] Venous blood of healthy volunteers was collected into
heparinized test-tubes, which had been tested for endotoxin. A
mononuclear fraction of blood leukocytes was obtained by
centrifugation in ficoll-metrizoat gradient (Histopaque, Sigma).
Cell concentration was adjusted to 2.times.10.sup.6 cells per 1 ml
of cell culture medium (RPMI 1640), containing 20 mM HEPES, 2 mM
glutamine, 50 mg/mL gentamicin and 10% fetal calf serum. Cell
viability was estimated by the Trypan blue exclusion method, then
the cell suspension was placed into wells of 24- and 96-well
microliter plates--250,000 cells per well.
[0516] The HL-60 myeloid line cells were grown in RPMI-1640 medium
with addition of 10% fetal calf serum. Cultivation was carried out
in closed flasks, the medium volume was 12 mL, it was replaced
every four days. Directly before testing the cell suspension in the
fresh medium was brought into 24-well microliter plates (cell
concentration for each well was 250.000 cells per well) and the
tested articles--GSSG and GSSG.cndot.Pt--were added into the
corresponding wells up to final concentration 100 mg/mL.
[0517] The effect testing for the testing articles was performed
24, 48 hours after addition into the culture.
[0518] The analysis procedure involved the following: after 24-48
hour incubation there were calculated the total cell count and the
dead cell count by the Trypan blue exclusion method; afterwards the
cell suspension was centrifuged (at 12.000 g, in Eppendorf
test-tubes during 10 min). The cell pellet was frozen and kept at
-70.degree. C. before the DNA separation. The DNA separation was
conducted by Kirbi-Georgiyev phenol method. To the cell pellet
there were added 0.5 ml of 10% SDS and 0.5 ml of TE-buffer
containing 0.1M EDTA and 0.01 M Tris-HCl with pH 8.0 ("A" buffer),
and the pellet was resuspended by the tube shaking during 15 min.
Then the equal phenol amount was added adjusted by 0.01 M Tris-HCl
with pH 8.0, the product was mixed during two min. and centrifuged
in the Eppendorf test-tubes at 12.000 g during 15 min. After the
centrifugation completion the upper water phase was phenol-treated
one more time. After the phenol treatment the upper water phase was
twice treated with phenol-chloroform mixture (1:1) that was mixed
with the water phase in equal amount. The water phase was separated
by centrifugation at 12.000 g during 10 min., pumped out and once
mixed with the equal chloroform amount. Afterwards it was
centrifuged at 12.000 g, the upper phase was separated, mixed with
double amount of distilled ethyl alcohol frozen to -20.degree. C.
and kept for one night at -20.degree. C. The DNA pellet was
gathered by centrifugation at 12.000 g during 10 min., the
supernatant was removed and the pellet was washed by 200 mL of 70%
ethyl alcohol frozen to -20.degree. C. during five min.,
centrifuged one more time at the same conditions and afterwards the
pellet was air-dried during one hour. Then it was diluted in 10 mL
of the A buffer; the DNA amount was determined by Dishe method and
electrophoresis was performed in 2% agarose gel (agarose with NA
grade made by "Pharmacia LKB, Biotechnology Inc" (Austria) was
used). The DNA electrophoretic separation was made in a block of 2%
agarose gel in a device made by "Pharmacia LKB, Biotechnology Inc"
(Austria). As a buffer solution 0.04 M tris-HCl buffer, pH 7,
containing 0.02 M of sodium acetate and 0.02 M EDTA was applied.
Agarose (2%) was prepared at an electrode buffer. The DNA samples
(2-5 mg) were placed into the gel slots. The electrophoresis was
conducted with an electrical field intensity of 6 W/cm during three
hours. The sample propulsion was observed due to bromine-phenol
blue motion. On the electrophoresis completion the gel block was
taken out of the device and introduced into a tray with etidium
bromide solution (3 mg/mL H.sub.2O) for 30 minutes in dark place.
After the incubation completion the gel was rinsed with water and
examined in the transmitted ultraviolet radiation with the
wave-length 254 nm at a transilluminator made by "Pharmacia LKB,
Biotechnology Inc" (Austria). The gel was photographed by Zenit E
camera with a red colour filter.
[0519] The study results are given in the Tables 28 and 29 and
FIGS. 13 and 14. As one can see in the Tables 28 and 29 data, the
GSSG and GSSG.cndot.Pt influence on normal and transformed cells is
of an alternative character. GSSG and GSSG.cndot.Pt stimulated the
normal cell proliferation (Table 28). The electrophoresis of DNA
obtained from the normal cells (Table 28) revealed presence of only
traceable quantities for apoptotic fragments at the background of
homogenous high-molecular fraction hcaracteristic for the viable
cells.
[0520] In the contrary, in the myeloid origin (HL-60) cancer cell
culture, the apoptosis activation and the cell division inhibition
were observed due to the influence of both drugs (Table 29, FIG.
14). The diversities of the effects were of the quantitative
nature.
[0521] Thus, the dead HL-60 cell quantity after the GSSG impact was
reliably lower than the one after the GSSG.cndot.Pt effect (Table
29). An evident indication for stronger impact efficacy
(GSSG.cndot.Pt comparing to GSSG) on the HL-60 cells is the
apoptotic fragment's character obtained after the electrophoretic
analysis. The DNA electrophoresis of the HL-60 cells non-incubated
with the drugs demonstrated presence of highly-molecular,
practically homogenous DNA characteristic for the viable cells
(FIG. 14, band #2). The DNA electrophoresis of the cells incubated
during 24 and 48 hours with the GSSG.cndot.Pt and GSSG drugs
revealed the DNA oligonucleosomic degradation (FIG. 14, bands 1 and
3, respectively), i.e., an apoptotic ladder, that is indisputable
sign for the programmed cell death. However, the apoptotic ladder
for the HL-60 cells treated with GSSG contained considerable amount
of the high molecular DNA characteristic for the viable HL-60 cells
(FIG. 14, band #1), whereas the high molecular DNA for the cells
treated with GSSG.cndot.Pt was virtually absent (FIG. 14, band
#3).
[0522] The HL-60 cells are defective in the p53 gene (p53 gene
deletion) and apoptosis induction through the GSSG and
GSSG.cndot.Pt treatment can occur only without involving the p53
product. Therefore, one can state that the GSSG and GSSG.cndot.Pt
drugs activate an inner contour of the programmed cell death
irrespectively of the p53 gene product. The p53 defect is present
approximately at half of cancer pathology cases. The experimental
results appeared to denote efficacy of GSSG and, especially,
GSSG.cndot.Pt for these tumor chemotherapy.
[0523] Conclusion: The performed studies allowed to obtain data
indicating capacity of the GSSG and GSSG.cndot.Pt drugs to increase
normal cell (lymphocytes) viability and, contrariwise, to induce
apoptosis in ransformed cells, i.e., exercise an antitumor
activity. Besides, the GSSG.cndot.Pt activity regarding to the
transformed cell apoptosis induction excelled significantly the one
for the GSSG drug according to the objective viability criterion,
i.e., presence of the high molecular DNA that appeared considerably
after the GSSG treatment and was almost absent after the
GSSG.cndot.Pt treatment. Thus, the data obtained on cells of the
myeloid line HL-60 defective in the p53 gene that is the crucial
agent in apoptosis induction allows us to state that: [0524] The
GSSG and GSSG.cndot.Pt drugs induce the inner contour of the
apoptosis development regardless of the p53 gene product; [0525]
The GSSG.cndot.Pt drug based on composite possesses higher
chemotherapeutic activity (due to the apoptotic induction in the
transformed cells) versus the first generation drug GSSG.
Example 15
Analysis of the GSSG.cndot.Pt Effects on Growth Development and
Apoptotic DNA Degradation at Normal and Transformed Cells Defective
in the p53 Antioncogene with Increased Ras-Gene Expression
[0526] The GSSG.cndot.Pt effects on growth development and
apoptotic DNA degradation of different lineage of transformed cells
(HL-60, C-8, A-4) were comparatively analyzed depending on the p53
defect that is the key factor for the apoptosis development and the
ras-gene that is a multipotent factor for cell reaction. The HL-60
cell culture is human cells of myeloluekosis origin defective in
the p53 gene. Cell cultures C-8 and A-4 are transformed murine
fibroblasts having a plasmid with the ras gene and a gene of the
EL1a expression enhancement factor that is an adenovirus antigen
fragment. At that, the A-4 cells are defective at the p53 gene and
the C-8 ones contained the intact p53 gene. A donor blood
lymphocyte slip was used as a control of the human cells with the
intact p53 gene.
[0527] The HL-60 (p53--) myeloid line cells were grown in RPMI-1640
medium with addition of 10% fetal calf serum. The cell suspension
cultivation was carried out in closed flasks, the medium volume was
12 mL, it was changed every four days. Directly before testing the
cell suspension in the fresh medium was brought into 24-well
microliter plates (cell concentration for each well was 250.000
cells per well) and the tested article--GSSG.cndot.Pt--was added
into the corresponding wells up to final concentrations 10-100
mg/mL.
[0528] Venous blood of healthy volunteers was collected into
heparinized test-tubes, which had been tested for endotoxin. A
blood leukocyte mononuclear fraction was obtained by centrifugation
in ficoll-metrizoat gradient (Histopaque, Sigma). Cell
concentration was adjusted to 2.times.10.sup.6 cells per 1 ml of
cell culture medium (RPMI 1640), containing 20 mM HEPES, 2 mM
glutamine, 50 mg/mL gentamicin and 10% fetal calf serum. Cell
viability was estimated by the Trypan blue exclusion method, then
the cell suspension was placed into wells of 24- and 96-well
microliter plates--250,000 cells per well.
[0529] The murine transformed fibroblasts were grown in DMEM medium
with addition of 10% fetal calf serum. The cell suspension
cultivation was carried out in closed flasks, the medium volume was
12 mL, it was changed every four days. Directly before testing the
cell suspension in the fresh medium was brought into 24-well
microliter plates (cell concentration for each well was 50.000
cells per well) and the tested article--GSSG.cndot.Pt--was added
into the corresponding wells up to final concentrations 10-100
mg/mL.
[0530] The effects testing for the testing articles was performed
24, 48 hours after introduction into the culture.
[0531] The analysis procedure involved the following: after 24-48
hour incubation, the total cell count and the dead cell count were
calculated by the Trypan blue exclusion method; afterwards the cell
suspension was centrifuged (at 3.000 g, in Eppendorf test-tubes
during 10 min). The cell pellet was frozen and kept at -70.degree.
C. before the DNA separation. The DNA separation was conducted by
Kirbi-Georgiyev phenol method. To the cell pellet there were added
0.5 ml of 10% SDS and 0.5 ml of TE-buffer containing 0.1M EDTA and
0.01 M Tris-HCl with pH 8.0 ("A" buffer), and the pellet was
resuspended by the tube shaking during 15 min. Then the equal
phenol amount was added adjusted by 0.01 M Tris-HCl with pH 8.0,
the product was mixed during two min. and centrifuged in the
Eppendorf's test-tubes at 12.000 g during 15 min. After the
centrifugation completion the upper water phase was phenol-treated
one more time. After the phenol treatment the upper water phase was
twice treated with phenol-chloroform mixture (1:1) that was mixed
with the water phase in equal amount. The water phase was separated
by centrifugation at 12.000 g during 10 min., pumped out and once
mixed with the equal chloroform amount. Afterwards it was
centrifuged at 12.000 g, the upper phase was separated, mixed with
double amount of distilled ethyl alcohol frozen to -20.degree. C.
and kept for one night at -20.degree. C. The DNA pellet was
gathered by centrifugation at 12.000 g during 10 min., the
supernatant was removed and the pellet was washed by 200 mL of 70%
ethyl alcohol frozen to -20.degree. C. during five min.,
centrifuged one more time at the same conditions and afterwards the
sediment was air-dried during one hour. Then it was diluted in 10
mL of the A buffer; the DNA amount was determined by Dishe method
and electrophoresis was performed in 2% agarose gel (agarose with
NA grade made by "Pharmacia LKB, Biotechnology Inc" (Austria) was
used). The DNA electrophoretic separation was made in a block of 2%
agarose gel in a device made by "Pharmacia LKB, Biotechnology Inc"
(Austria). As a buffer solution 0.04 M tris-HCl buffer, pH 7,
containing 0.02 M of sodium acetate and 0.02 M EDTA was applied.
Agarose (2%) was prepared at an electrode buffer. The DNA samples
(2-5 mg) were placed into the gel slots. The electrophoresis was
conducted with an electrical field intensity of 6 W/cm during three
hours. The sample propulsion was observed due to bromine-phenol
blue indicator motion. On the electrophoresis completion the gel
block was taken out of the device and introduced into a tray with
etidium bromide solution (3 mg/mL H.sub.2O) for 30 minutes in dark
place. After the incubation completion the gel was rinsed with
water and examined in the transmitted ultraviolet radiation with
the wave-length 254 nm at a transilluminator made by "Pharmacia
LKB, Biotechnology Inc" (Austria). The gel was photographed by
Zenit E camera with a red color filter.
[0532] The study results are given in the Tables 30, 31, 32, 33. As
is evident from the Table 31, GSSG.cndot.Pt induces apoptosis in
the HL-60 cell culture defective at the p53 gene. The effect was
rather developed at the concentration of 10, however, it was more
evident at 100 mg/mL. Also there were observed conglomeration of
DNA apoptotic fragments multiple of DNA nucleosome size that is the
indisputable sign of programmed cell death.
[0533] In contrary, GSSG.cndot.Pt made stimulating effect on normal
cells, i.e., there was certain proliferation observed (Table 30).
Electrophoresis of DNA obtained from normal cells exhibited that it
was represented by a homogenous high-molecular fraction
characteristic for viable cells.
[0534] Thus, differences of the drug action on normal and
transformed cells were basically divergent. Mechanism for the
GSSG.cndot.Pt divergent action might be conditioned by activation
of the p53-independent apoptotic pathway through the
ras-signal-transducing system. The ras-system is capable to
stimulate cell proliferation and differentiation through the
mitogenic factor cascade and induce apoptosis (programmed cell
death) through another cell signal cascade.
[0535] To check it there were compared the GSSG.cndot.Pt effects on
murine fibroblasts with enhanced ras-gene expression but different
in the intact p53 gene (wild type) presence--cells C-8(p53++), or
the p53 gene absence (a genetic defect)--(cells A-4(p53--)). As one
can see from the Tables 32 and 33, the GSSG.cndot.Pt effect
appeared in both cell lineages. The apoptosis induction was
significantly exhibited. It indicates activation of the
p53-independent apoptotic pathway. Presence of the activated ras
gene in both of the cell lineages can be an explanation for the
apoptotic exhibition that was even more marked in the transformed
fibroblasts than in the HL-60 cells. It confirmed the apoptosis
induction through the ras-signal-transducing system. The
GSSG.cndot.Pt effect on the A-4 and C-8 cells was not different at
concentration of 10 mg/mL. The significantly superior GSSG.cndot.Pt
effect on the A-4 cells comparing to the C-8 cells was noted at the
concentration 100 mg/mL. The p53-protein absence appeared even to
enhance the ras-signal-transducing pathway for apoptosis induction
in tumor cells.
[0536] The p53 gene defect occurs in approximately half of the
cancer disease cases. The results of these experiments can imply
the GSSG.cndot.Pt effectiveness for chemotherapy of these
tumors.
[0537] Conclusion: The data from these studies indicate the
capacity of the GSSG.cndot.Pt drug to increase normal cell
(lymphocytes) viability and, contrariwise, to induce apoptosis in
transformed cells, i.e., exercise an antitumor activity. Besides,
the GSSG.cndot.Pt activity regarding to the transformed cell
(defective at the p53 gene) apoptosis induction even excelled the
one for the cells with the intact p53 gene at the high drug
concentration. According to the objective viability criterion,
i.e., presence of the high molecular DNA that appeared in
considerable amounts after the GSSG.cndot.Pt treatment of the
normal donor cells (take from the lymphocyte slip), the cell death
was practically absent whereas in case of the GSSG.cndot.Pt impact
on transformed cells there was observed the DNA apoptotic
degradation, i.e., the sign of the irreversible apoptotic death
induction even in the cells defective at the p53 gene (with the
stimulation especially). Previously, in the Example 9 there was
shown activation of the cytokine range after the GSSG.cndot.Pt
treatment. Considering that the cytokine action may be determined
with the ras-signal-transducing pathway activation the cytokine
stimulation can also cause the antitumor effect through an
interaction with the ras-protein.
Example 16
Analysis of the GSSG.cndot.Pt Effects on Growth Development and
Apoptotic DNA Degradation at Murine Transformed Cells Cultures
Defective at Antioncogenes
[0538] The GSSG.cndot.Pt effects on growth development and
apoptotic DNA degradation of transformed fibroblasts of a cell
lineage with activated ras-gene but an intact p21 gene (p21++-C-8
cells) and murine cell lineage with knockout p21 gene (p21--).
[0539] The p21++ lineage cells are murine transformed fibroblasts
having a plasmid with the ras gene and a gene of the E1a expression
enhancement factor that is an adenovirus antigens fragment but have
intact p53 and p21 genes. The p21 (--) lineage has the intact p53
and ras genes but it is defective at the p21 gene. It allows
evaluating the GSSG.cndot.Pt impact on the apoptosis induction in
conditions of impaired regulation for the cell division G1 phase.
The cell cultures were grown in DMEM medium with addition of 10%
fetal calf serum. The cell suspension cultivation was carried out
in closed flasks, the medium volume was 12 mL, it was changed every
four days. Directly before testing, the cell suspension in the
fresh medium was brought into 24-well microliter plates (cell
concentration for each well was 50.000 cells per well) and the
tested article--GSSG.cndot.Pt--was added into the corresponding
wells up to final concentrations 100 mg/mL.
[0540] The effect testing for the testing articles was performed
24, 48 hours after introduction into the culture.
[0541] The analysis procedure involved the following: after 24-48
hour incubation, the total cell count and the dead cell count were
calculated by the Trypan blue exclusion method; afterwards the cell
suspension was centrifuged (at 3.000 g, in Eppendorf test-tubes
during 10 min). The cell pellet was frozen and kept at -70.degree.
C. before the DNA separation. The DNA separation was conducted by
Kirbi-Georgiyev phenol method. The cell lysis was performed by
addition of 0.5 ml of 10% SDS. The equal phenol amount was added
there adjusted by 0.01 M Tris-HCl with pH 8.0, the product was
mixed during two min. and centrifuged in the Eppendorf test-tubes
at 13.000 g during 15 min. The phenol deproteinization was repeated
twice. Afterwards the water phase was twice treated with
phenol-chloroform mixture (1:1) and once with chloroform. Nucleic
acids were precipitated by the addition of two volumes of 96% ethyl
alcohol at 20.degree. C. overnight. The DNA was gathered by
centrifugation at 13.000 g during 30 min., decanted, washed with
70% ethyl alcohol and air-dried. After the drying the precipitate
was dissolved in TE buffer. The DNA concentration was determined in
the obtained solution (by Dishe method). Fractional content of the
nucleic acids was determined by electrophoresis in 2% agarose gel
(agarose with NA grade made by "Pharmacia LKB, Biotechnology Inc"
(Austria) was used). The DNA electrophoretic separation was made in
a block of 2% agarose gel in a device made by "Pharmacia LKB,
Biotechnology Inc" (Austria). The electrophoresis was performed in
TAE buffer pH 7.4 (0.04 M tris, 0.02 M of sodium acetate, 0.02 M of
EDTA) at an electrical field intensity of 6 W/cm during three
hours. The sample propulsion was observed due to bromine-phenol
blue indicator motion. The electrophoresis results were examined in
the transmitted ultraviolet radiation (y=254 nm) at a
transilluminator made by "Pharmacia LKB, Biotechnology Inc"
(Austria) after the gel dying with etidium bromide solution (5
mg/ml).
[0542] The study results are given in the Tables 34, 35. As it is
evident from the Table 34, GSSG.cndot.Pt induces apoptosis in the
p21++ cell culture with activated ras-gene manifested by
conglomeration of DNA apoptotic fragments multiple of DNA
nucleosome size.
[0543] In the p21 (--) cells having non-active
ras-signal-transducing system but defective at the control of the
cell cycle delay in the G1 phase GSSG.cndot.Pt induced apoptosis to
less extent that in p21++ cells (Tables 34, 35). It might be
implicated by the fact that the ras-system is capable of stimulatin
cell proliferation and differentiation through the mitogenic
factors' cascade and inducing apoptosis (programmed cell death)
through another cell signals' cascade. Absence of the p21 gene
expression can cause changes in interrelations of specific cascades
of the ras-signal-transducing pathway and, thereupon, lessen the
apoptotic stimulation by the drug in cases of p21 gene defects.
[0544] The p21 gene defect does not often occur in oncological
diseases that considering the performed experiments' issues can
imply the GSSG.cndot.Pt effectiveness for chemotherapy of these
tumors except cases with lower effectiveness at tumors with
mutations in p21 gene.
[0545] Conclusion: The performed studies allowed obtaining data
indicating capacity of the GSSG.cndot.Pt drug to induce apoptosis
actively in transformed cells, i.e., exercise an antitumor
activity. The enhanced ras-gene expression facilitates the
apoptosis induction in the transformed cells C-8 indicating
implementation of the GSSG.cndot.Pt antitumor activity through the
ras-signal-transducing system. The lesser drug effect in case of
p21 gene expression absence can be determined by the redistribution
of factors in mitogenic and apoptotic cascades of the
ras-signal-transducing pathway.
[0546] Nevertheless, presence of the DNA apoptotic degradation,
i.e., the sign of the irreversible apoptotic death induction, is
found after the GSSG.cndot.Pt action even in p21-defective
cells.
[0547] The aforesaid allows us to state that the GSSG.cndot.Pt
composite-based drug:
[0548] Realizes chemotherapeutic activity regarding the
tumor-transformed cells through induction of the ras-dependent
apoptotic pathway;
[0549] Induces the apoptosis' development in the tumor-transformed
cells including p21-defective cells.
Example 17
Therapeutical Effect of the GSSG.cndot.Pt Vanadium Salt in Patient
with Diabetes Mellitus
[0550] No. 7
[0551] Gender: female
[0552] Age: 37
[0553] Out-patient card: No. 63
[0554] Diagnosis: Diabetes mellitus. Insulin-independent type--type
II. Diabetic angiopathy, grade IV.
[0555] Anamnesis morbi: Firstly the high blood sugar was revealed
in the age of 31. In October 1994, the patient was admitted into
the Endocrinologic Department of the Hospital #16. There were
diabetes cases in the patient's hereditary history on the mother's
side. The disease developed gradually, the blood glucose level
fluctuated from 12.1 to 15.7 mmol/L, glucosuria to 7%.
[0556] Previous treatment: At the onset of the disease, the patient
was frequently admitted to hospitals; in 1994 during six months she
was administered with Insulin-lente S.N.C.-32 IU and peroral
hypoglycemic agents of the sulfonylureas group; then Insulin was
cancelled and the patient used antidiabetic agents--Bucarban,
Diabeton. The blood glucose changed from 10.7 to 15.4 mmol/L,
glucosuria 3-7%.
[0557] In January 1998 the patient was taken to the hospital and
the following treatment regime was administered: Diabeton 0.08 g--2
tab twice a day, Glucobay 0.1.times.3 times per day.
[0558] The blood glucose content:
TABLE-US-00004 9 12.6 mmol/L 12 12.0 mmol/L 14 15.3 mmol/L 17 19.1
mmol/L 6 11.5 mmol/L
[0559] Glucosuria--to 3%.
[0560] Because of the previous treatment inefficacy, it was decided
to use the drug of the GSSG.cndot.Pt vanadium salt.
[0561] Hospital treatment regime for the GSSG.cndot.Pt vanadium
salt: from 17.01.98.
[0562] During 10 days once a day--intravenous injections of the
GSSG.cndot.Pt vanadium salt (daily dose--0.01-0.5 mg/kg).
[0563] During the following 10 days--intravenous injections of the
GSSG.cndot.Pt vanadium salt every other day (daily dose--0.01-0.5
mg/kg).
[0564] During the following 10 days (the third
decade)--intramuscular injections of the GSSG.cndot.Pt vanadium
salt once a day (daily dose--0.01-0.5 mg/kg).
[0565] The GSSG.cndot.Pt vanadium salt treatment was performed
along with changed treatment regime by Diabeton and Glucobay.
Diabeton was administered 0.08 twice a day, Glucobay 0.05.times.3
times a day.
[0566] The blood glucose restored to normal values and did not
exceed 8.2-10.6 mmol/L after meals. After the discharge the patient
received the GSSG.cndot.Pt vanadium salt treatment as an
out-patient during one month.
[0567] Ambulant treatment regime for the GSSG.cndot.Pt vanadium
salt:
[0568] For one month there were administered intramuscular
injections of the GSSG.cndot.Pt vanadium salt once a day (daily
dose--0.01-0.5 mg/kg).
[0569] During two sequential months of treatment with the
GSSG.cndot.Pt vanadium salt drug there were noted two episodes of
the glucose content decrease to 1.5-2.5 mmol/L, thereat the
antidiabetic drugs dosages were lowered. After two months of the
treatment, Glucobay was cancelled.
[0570] Patient's state after the treatment course completion: After
four months of the treatment with administration of the
GSSG.cndot.Pt vanadium salt drug as a support for the basic
therapy, the patient's general state improved significantly and was
evaluated as satisfactory. Development of hematologic, biochemical,
immunologic blood indices is provided in the Tables 36, 37.
[0571] Comments to the indices development of glucose, cAMP/cGMP,
thiol-disulfide ratio and other analyzed parameters.
[0572] The glucose content development is an integrative criterion
for the impact effectiveness of the antidiabetic drugs. The
combined therapy by the peroral antidiabetic drugs (Diabeton and
Glucobay) along with the GSSG.cndot.Pt vanadium salt drug called
forth the blood glucose normalization, decrease of the Diabeton
therapeutic dosage and the Glucobay cancellation. Character of the
hematologic, biochemical and immune parameter changes indicated
restoration of the metabolism and the systemic reactions in general
that does not allow to determine the antidiabetic constituent for
the GSSG.cndot.Pt vanadium salt drug. The following indices were
used--cAMP/cGMP and thiol-disulfide ratio (TDR)-- that allowed to
characterize basically a molecular mechanism of the GSSG.cndot.Pt
vanadium salt drug action on cellular reaction complex stabilizing
the blood glucose level. In particular, the key intracellular
messengers--cAMP and cGMP--determine intensity of the glucose flow
into intracellular metabolic processes. At that, cGMP-dependent
enzymatic cellular systems determine the cellular glucose intake
intensity. In its turn, the cGMP level is determined by the
cellular oxidative potential. In particular, oxidants increase the
cGMP level, in the contrary, antioxidants perform depressive action
on its content. Thus, the intracellular thiol-disulfide ratio (TDR)
reflecting the balance of the anti- and pro-oxidants determines
value of the intracellular cAMP/cGMP index. Taking into account an
organism as a whole we can analyze these indices in blood as
integrative internal medium interrelated with the glucose level
fluctuations, because the glucose metabolism is an indefeasible
constituent for functioning of all cell types at our organism along
with thiol-disulfide metabolism and cyclic nucleotide systems.
[0573] The results obtained indicated the glucose-lowering
influence of the GSSG.cndot.Pt vanadium salt drug. Besides, this
effect follows the cGMP content increase development (lowering of
the cAMP/cGMP index) and the TDR value decrease (increase of the
thiol oxidized forms). Considering regulatory possibilities of the
thiol oxidized forms and the cGMP in blood glucose regulation, one
can state the basic mechanism of the regulatory hypoglycemic effect
of the GSSG.cndot.Pt vanadium salt drug. Inter alia, the regulatory
impact of the GSSG.cndot.Pt vanadium salt drug on the cellular
redox contour, provides an increase of the oxidant constituent
level that, in its turn, redistributes balance in the cyclic
nucleotide system in favor of cGMP (guanylat-cyclase induction and
inhibition of phosphodiesterase, cGMP synthesis and destruction
enzymes, respectively). The cGMP regulatory impact stimulates the
glucose transport processes into insulin-dependent tissues calling
forth the blood glucose decrease.
[0574] Conclusion: Application of the GSSG.cndot.Pt vanadium salt
drug in the scheme for the combined diabetes treatment allowed to
obtain the following therapeutic effects: [0575] the quality of
life improvement and the blood glucose level stabilization; [0576]
dosage decrease for the administered glucose-lowering drugs; [0577]
restoration to normal values of the hematologic, biochemical and
immunologic indices.
[0578] Those skilled in the art would readily appreciate that all
parameters listed herein are meant to be examples and that actual
parameters will depend upon the specific application for which the
methods and apparatus of the present invention are used. It is,
therefore, to be understood that the foregoing embodiments are
presented by way of example only and that, within the scope of the
appended claims and equivalents thereto, the invention may be
practiced otherwise than as specifically described.
* * * * *