Patent Application
20080057113
The main subject of the invention is an anti-herpetic composition comprising the following components:
A virion vaccine anti-herpetic preparation—106 to 107 plaque-forming units/ml of suspension, inactivated by formalin or γ-radiation, Polyoxidonium, and also amino acids valine and lysine, as well as a combination of at least 2 amino acids selected from the group of phenylalanine, leucine, threonine, histidine, arginine, methionine. A detailed description of the new components and their importance for our composition is presented hereinbelow.
The latest-generation domestic immunomodulator—Polyoxidonium (PO)—is a very efficient immunocompetent agent. PO is a copolymer of 1,4-ethylene-piperazine N-oxide and (N-carboxyethyl)-1,4-ethylene-piperasinium bromide. It is a lyophilized porous mass having a yellowish hue, readily soluble in water, with a molecular mass of 60000 to 100000. In addition to the immunostimulating effect, PO produces pronounced detoxifying, antioxidant and membrane-stabilizing effects.
Lately we have undertaken a number of experiments with the use of PO simultaneously with using the anti-herpetic vaccine applied jointly by parenteral administration. These experiments gave positive results on model infections in laboratory animals (keratitis in rabbits, genital herpes in guinea pigs, meningoencephalitis in mice). The results of these experiments are presented in Table 1.
Our investigations have shown that the best effect for the test individuals with different forms of herpes is produced by the hi-tech synthetic drug Polyoxidonium (PO).
In vivo PO produces a more complicated and multiaspect effect on the immune system. Since the development of any immune response begins with cells of the monocytic-macrophage system, and since cytokins produced by monocytes/macrophages have pleiotropic effect, an enhancement of their functional activity under the action of PO leads to the activation of both cellular and humoral immunity. So, in particular, upon introducing PO together with low doses of an antigen a 5 to 10-fold enhancement of the synthesis of antibodies to this antigen over control takes place. It is important to note that such enhancement can be observed in animals with a genetically determined weak reaction to the given antigen. Hence, PO has an ability to bring in motion all factors of organism protection from foreign agents of antigenic nature, and this motion extends in a natural manner so as this occurs in the development of an immune response in an organism. These observations enabled us to choose just Polyoxidonium among the plethora of modern immunostimulators for its successful use in the formulation of a complex pharmaceutical anti-herpetic composition.
We have succeeded to establish empirically that essential amino acids valine and lysine, and also some other amino acids selected by us from a multitude of the known amino acids are very efficient components in the formulation of the composition.
The amino acids employed by us have the following properties apart from the generally known ones:
Histidine—is part of carnosine and anserine, plays an important role in the formation of hemoglobin, is necessary for the generation of erythrocytes. It contributes to controlling the sugar level in blood and to producing energy.
Leucine—is necessary for the growth and healing of bones, muscles. In the metabolism of leucine energy is released, it contributes to stabilization of sugar level in blood. It is found that leucine deficiency in some cases may provoke hypoglycemia, retardation of growth, reduction of bodyweight, changes in kidneys and in the thyroid gland.
Lysine—strengthens the immune system, contributes to the growth of bones and formation of collagen, improves attention, has direct anti-herpetic activity. Shortage of lysine in diet leads to disturbance of blood circulation (the number of erythrocytes decreases, the content of hemoglobin in them diminishes). Deficiency may lead to the emaciation of muscles, to disturbance of bone calcification, changes in the liver and lungs, especially in aged and senile patients.
Methionine—is used for the synthesis of choline, is known as a “lipotropic agent”, because it reduces the stock of fats in the liver and in an organism as a whole and lowers the amount of cholesterol. Builds up new bone tissue, inhibits onchypathies, protects kidneys and is a natural chelating agent for heavy metals. Methionine is of great importance for the functions of adrenal glands and for the synthesis of adrenalin, this being crucial for metabolic control in virus-affected cells.
Phenylalanine—is associated with the function of thyroidal gland and adrenal glands, participates in the formation of a core for the synthesis of thyroxin which is the main hormone of the thyroid gland, participates in the formation if adrenalin. In the organism phenylalanine can be transformed into thyrosine which is used for the synthesis of two main neurotransmitters, dopamine and epinephrine controlling cell metabolism on the neurohumoral level. Phenylalanine is effective in controlling pain and itch sensations. Besides, owing to the secretion of cholecystokinin, phenylalanine has appetite-suppressing action.
Valine—valine deficiency may lead to damage of the myeline coating of nerve fibers and to the generation of negative hydrogen balance of the organism, this being extremely dangerous for virus-infected patients.
Alanine—strengthens the immune system, participates in glucose metabolism.
Arginine—shortage of arginine may cause loss of hair, constipations, hepathopathies, and slow healing of wounds, including those caused by damage with virus toxins.
Isoleucine possesses a unique property which is used for formulating an antivirus vaccine for the first time—this amino acid received from the outside (with food) only stimulates the production in human organism (predominantly in the intestine region) of antimicrobial peptides, similar to cecropins which are capable to destroy herpes viruses.
Such a composition, especially supplemented with microelements which enhance immune response to the administration of the preparation, allows using thereof also for more effective combating virus lesions by herpes of serotypes 1 and 2 and some chronic forms of this disease.
A method for using a pharmaceutical anti-herpetic composition by administering thereof to an organism affected by herpes virus is another subject matter of the invention, wherein the above-characterized composition is administered to the organism in an effective dose as a suitable dosage form in a suitable way, selected from the group: perorally, sublingually, intranasally, rectally, vaginally, parenterally, subconjuctivally or in a chewable form.
The action of microelements in the immune system in the case of herpes was studied in detail, using suppositories containing our composition.
The following mechanisms of action of microelements (MEs) exist in the immune system:
1. Effect on Specific Receptors
On receptors localized on cytoplasmic membrane: HLA-system, MHC-system (Ni, Cr, Hg).
Adhesins: selectins and integrins (Mn, Hg).
Receptors to transferrin (Al, Ga).
Receptors participating in NK-mediated lysis (Zn).
Cytokine receptors (Zn).
T-cell receptor (Zn, Hg).
Receptors to calcium and magnesium ions (Zn, Mn, Be, Cd, Hg, and others).
Immunoglobulin receptors (Zn).
Receptors localized in intracellular compartments:
Intracellular receptors to calcium on mitochondria, endoplasmic reticulum (Cd, Zn).
2. Effect on Enzyme Activity
Many essential MEs are a component of the catalytic site of a number of enzymes.
For example, Mn is an essential part of the superoxide dismutase (SOD) of immunocytes, Se forms part of the catalytic site of glutathione peroxidase (isoenzyme VI), Zn is the most important part of numerous Zn-finger proteins controlling the transcription level of other intracellular proteins. The pathways of the action of MEs on the activity of enzymes also exist, which consist in competitive inhibition or allosteric activation of metalloenzymes. For example, Zn is a competitive inhibitor of Ca+2+, Mg2+-dependent endonuclease. This action of Zn has determined its leading role in the immune system as an anti-apoptotic factor.
3. Effect on Hormone Activity
MEs as a component part of hormones.
MEs and Storage of Hormones
It is known that angiotensin-converting enzyme is Zn-dependent.
4. Effect on Carrier Proteins
Metallothioneins which are synthesized in mononuclear cells of the reticulo-endothelial system of the organism.
Stress proteins, as universal proteins synthesized in cells in response to stress effects (thermal shock, starvation, UV irradiation, effect of heavy metals, chronic infection).
5. Physical-Chemical Action of MEs on Immunocyte Membranes
It has been found that, e.g., selenium can produce an antioxidant effect, acting as a cofactor of glutathione peroxidase which provides inactivation of free oxygen species, the generation of which in the immune system ensures both destruction or elimination of a foreign agent (parasite, bacterium), and, in the case of excessive generation of singlet O2, H2O2, OH, causes damage of the membrane apparatus of the immunocytes themselves.
The generation of O2, H2O2, OH is associated with induction of Haber-Weiss and Fenton reactions under the influence of transition metals (Cu, Zn, Mn, Fe).
Thus, MEs are able through the agency of enzyme and non-enzyme mechanisms of lipid peroxidation (LPO), as well as through the activation of antioxidant mechanisms, to control the physical-chemical properties of cell membranes, including the property of semi-permeability with regard to various biological substrates (antigens, infectious agents, etc.).
6. Effect on the presentation, intracellular processing and degradation of antigens (see Item 1).
7. Effect on the formation of immunologic memory, and also probably, in the long-term existence of memory cells anti-apoptotic MEs (Zn, Se and others) are involved.
8. Effect on the production of immunoglobulins (Zn, Be).
9. Effect on the processes of chemotaxis, adhesion and phagocytosis [6, 7].
Numerous tests on laboratory animals have made it possible to select the most effective microelements for incorporation thereof into the formulation of the dosage form developed by us (suppository): 2-3 microelements selected from the group: zinc, chromium, selenium and nickel. The presence of said microelements in the formulation of the dosage form enhances the action of the immunomodulator selected by us by 25-30% over the control (without MEs).
Presented below is Table 2, which shows the level of immunologic indices in experimental animals infected with herpes virus, before and after supplementing the formulation of the composition comprising amino acids, including isoleucine with Polyoxidonium.
From the Table the effect of PO on the immunologic indices is seen, wherein the cell immunity and the level of immunocompetent proteins significantly increase. It should be noted that the incorporation into the composition of amino acids (valine and lysine, and also isoleucine, and further a combination consisting of at least 2 amino acids selected from the group: phenylalanine, leucine, alanine, threonine, histidine, arginine, methionine) in all the combinations proposed by us accelerated the re-epithelization of damaged tissues (from 15 to 36% over the prototype, see above), depending on the type of the virus. The incorporation of Polyoxidonium into the composition jointly with amino acids not only activated the immunity, which made it possible to prolong considerably the period of remission, to accelerate the process of re-epithelization and healing of skin, and in some cases to essentially succeed in curing some chronic forms of the disease in individual animals (12% of all the individuals under test).
The above-characterized composition can be embodied as dosage forms in which solid, soft or liquid substances can be used as a carrier.
The final form of the composition with the use of solid carriers comprises a tablet, dragee, granule, sachet or powder placed into a capsule.
The final product produced from the composition with the use of liquid carriers comprises a solution, gel, emulsion, suspension, mixture, syrup or liniment.
The composition with the use of soft carriers comprises an ointment, crème, paste, suppository, implant or chewing tablet, or pastille.
The above-said pharmaceutical forms were produced with the help of conventional procedures and auxiliary substances commonly adopted in such cases.
A method of preparing a suppository based on the above-said pharmaceutical composition with the help of a conventional suppository-making technology is a further subject matter of the invention, wherein the active components of the above-described pharmaceutical composition and 2-3 microelements selected from the group: zinc, chromium, selenium and nickel are further introduced into a suppository mass based on cocoa oil by following a conventional technology. A set of microelements as soluble chelate forms is introduced into the formulation of the suppository in an amount of 0.01 to 0.08% based on the total weight.
The results of testing the suppositories thus produced in a group of 76 experimental animals showed that using the new preparation not only activated the immunity, but made it possible to prolong appreciably the period of remission, to accelerate the process of re-epithelization and healing of skin and mucous tissues, and in some cases to essentially succeed in curing of the disease in particular individuals (12% of all the test subjects with the chronic form of the disease).
In our opinion, the incorporation of vitally important ingredients controlling the metabolic processes on all levels, namely, of PO, valine and lysine, isoleucine stimulating the production of virus-blocking peptides (of cecropin-type) by the intestinal cells, of vitamins of all groups, and also of MEs: zinc, chromium, selenium and nickel into the suppository formulation played an important role in this result.
The invention is supported by particular tests on animals.
Major performance criteria (over the close analog). The time of attaining topical convalescence (complete re-epithelization) reduced by 15-36%, the duration of remission increased to 6-7 months on an average, the absence of virus in the smear (PCR-diagnostics) in 98.7% of cases, activation of the antiviral immunity, essentially complete curing in 12% of the follow-ups of the animals.
Among the test animals (herpetic keratitis in rabbits, genital herpes in guinea pigs) 52 individuals were affected with herpes simplex virus of serotypes 1 and 2 in acute form and 24 individuals were affected with chronic form of herpes simplex. The animals with the acute form were divided into three groups: in the 1st group treatment was carried out with the herpetic vaccine described in the prototype; in the 2nd group—with the new pharmaceutical composition, in the control group the animals received chemotherapy in the form of ointments and solutions containing the known anti-herpetic preparations. The group of the animals with the chronic form received new suppositories with a complete set of amino acids, vitamins and microelements.
Results. In the 1st group complete re-epithelization took place on the 6th day of treatment on an average, while in the 2nd group complete re-epithelization was noted on the 4th-5th day of treatment on an average. In the control group complete topical convalescence was stated on the 7th-10th day from the start of treatment on an average. The virus in control smears was not detected in 95.2% of the 1st group, in 97.8% of the 2nd group, and in 92.5% of the control group. The duration of remission of up to 4 months was observed in 92% of cases in the first group and of up to 6-7 months in 85% of cases in the second group. At the same time, in the control group the duration of remission of up to 4 was observed in 28% only. A highly intensive immune response was registered in the 1st group in 92.1% of cases, while in the 2nd group this was the case in 98.6% with a clear-cut significance (p<0.01). Thus after the treatment a tendency to an increase in the percentage and absolute counts of natural killer cells, T-lymphocytes of CD8+ phenotypes was noted in the blood serum of the test animals of both groups (in the second group by 11-18% higher) as well as an increase in the virus-induced interferon-α and mitogen-induced interferon-γ production was revealed.
The obtained pharmaceutical suspension taken in an amount of 20 ml is mixed with 0.01-0.03 ml of each of the solutions of salts of 2-3 MEs and cocoa oil to give the total weight of 100 g, from the resulting mass suppositories are prepared by a conventional method, each suppository weighing 0.15-0.20 g. Such suppository contains ≦400 μg/g of protein, has a residual humidity ≦2.2% and physiological pH value 7.3±0.2, is nontoxic and capable of inducing the synthesis of the virus-neutralizing antibodies in rats, with the neutralization index equal to 3.0 lg TCD50/ml in terms of HSV-1 and to 2.0 lg TCD50/ml in terms of HSV-2, where TCD50 denotes the dose producing cytopathic effect in 50% of test tubes with a cell monolayer, infected with the virus.
Owing to the development of the new highly immunogenic composition, the antigenicity and stability of the specific activity of the preparation are preserved, the anti-herpetic properties of individual organs at the cell level are enhanced, the organism resistance is increased and prolonged not only in acute but also in chronic herpes infection.
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| Number | Date | Country | Kind |
|---|---|---|---|
| 2003131814 | Oct 2003 | RU | national |
| 2004128635 | Sep 2004 | RU | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/RU04/00414 | 10/20/2004 | WO | 00 | 9/27/2007 |
