CH717767A2 - Method of stimulating human immune cells. - Google Patents

Abstract

The invention disclosed herein relates to the field of medicine, particularly immunology, and can be used to stimulate cells of the human immune system in the treatment of various diseases related to disorders of the immune system. A substance for use in a therapeutic method for stimulating cells in the human immune system and a method for producing this substance are disclosed.

Description

The invention relates to the field of medicine, particularly immunology, and can be used to stimulate cells of the human immune system in the treatment of various diseases related to disorders of the immune system.

A known immunopotentiation method uses Panagene (No. JICP-004429/08) administered in the form of tablets or intramuscular solution, which stimulates the endogenous production of cytokines and blood-forming factors (patent: RU 2498821 Cl, published 04.10.2012) . The drug is double-stranded fragmented human genomic DNA with fragments ranging from 200 to 6000 base pairs. Highly efficient immunopotentiation cannot be achieved with this method due to the low bioavailability of the drug and losses during passage through segments of the digestive system when taken orally. On the other hand, like any foreign DNA, it can be actively destroyed; and since it is a specific nucleotide sequence, it can also induce negative responses by interfering with the DNA structure in human cells.

Panagene is in tablet form for one to fourteen days from initiation or until therapeutic benefit is achieved, at three tablets per day containing 5 mg of the active ingredient. When taken in tablet form, the drug based on foreign DNA may lose much of its active ingredient and effectiveness.

Our proposed method aims to create a drug for stimulating human immune cells that is free from the drug's known shortcomings, with higher bioavailability and cell penetrating ability both due to the small size of its active ingredients (5-10 kD) and the lower losses during passage through the digestive system due to the chemical structure of its active ingredients, which mainly include polysaccharides.

To circumvent this problem, a human immune cell stimulant is obtained by leaching the homogenate of algae Laminaria japonica and Laminaria angustata with aqueous and isobutanolic solution having a mixing ratio of 1 g : 2.5 ml : 2.5 ml, µm obtaining a homogeneous mixture, which is then post-treated at 60°C for 1 hour and incubated at room temperature for 10-15 hours; then the supernatant is drawn off and centrifuged for 30 minutes at 3000 rpm and +4°C. The centrifuged supernatant is placed in a vacuum evaporator, purged with argon, then vacuum dried for one hour at room temperature and then at 60°C until all the liquid fraction is evaporated. The resulting sediment is diluted with water to a 5% solution and then ultracentrifuged at 100,000 rpm for one hour; the resulting supernatant is withdrawn and centrifuged in 10 kD filter tubes at 5000 rpm for one hour, after which the lower percolated fraction is withdrawn and centrifuged in 5 kD filter tubes at 5000 rpm for one hour. Then the upper fraction retained by the filter is withdrawn and diluted in 500 ml of NaCl-saline to a final concentration of 10 mg/ml.

In another embodiment, after centrifugation at 3000 rpm and +4 °C for 30 minutes and before the centrifuged supernatant is placed in a vacuum evaporator and purged with argon, the centrifuged supernatant is ultracentrifuged at 100,000 rpm for one hour .

A second aspect of the invention includes the substance produced by the method described above.

Another aspect of the invention includes the substance produced by the method described above for use in a therapeutic method for stimulating cells in the human immune system. In a preferred embodiment, this substance is administered orally for 14 days, preferably in dosages of 1 ml once a day, for example before meals.

Enterocytes and goblet cells found in the extrathoracic trachea, as well as in the pancreas and parotid ducts, and in portions of the digestive system are three times more permeable to molecules of 5-10 kD than other cells of the digestive system, including stem and paneth cells. Consequently, the cell pools responsible for the manifestations of tissue immunity and the protective functions of the digestive system, as well as for the penetration of substances from the digestive system into the bloodstream, prove to be the cell pools most permeable to the seaweed extract compared to others. This allows the active algae extract to enter the bloodstream both paracellularly and transcellularly (via the enterocytes). The greater permeability of certain cells of the digestive system and the small particle size of the seaweed extract (5-10 kD) and the absence of a complicated 3D structure ensure high bioavailability. Studies on the effectiveness of the drug showed that 1 ml oral administration once a day before a meal is the safest and most effective method of treatment.

The above method for obtaining individual fractions of Laminaria Japonica and/or Laminaria Angustata consisting of 5 to 10 kD molecules appears optimal and allows a more than 10,000-fold enrichment of the fraction from its starting content.

The algorithm of the proposed method is as follows: In order to obtain the fractions mentioned from dried and powdered homogenate of algae (Laminaria Japonica and Laminaria Angustata) by leaching with butanol-water mixtures, powdered Laminaria is dissolved in water and isobutanol (1 g : 2.5 ml : 2.5 ml). To this, 500 ml of isobutanol is placed in a 12 L bottle, then 200 g of powdered laminaria is added, followed by stirring until the solution is homogeneous and dark green. Ten minutes later, 500 mL of distilled water is added to the flask, followed by stirring again until homogeneous. The resulting mixture is heated to 60°C in a water bath or thermostat and then incubated at room temperature for 10-15 hours; then the supernatant is drawn off and centrifuged in an Eppendorf 5804 R centrifuge at 3000 rpm and +4° C. for 30 minutes. The supernatant thus obtained should be drawn off, placed in a vacuum evaporator and purged with argon. The resulting supernatant is then vacuum dried as follows: at room temperature for one hour to avoid boiling on suction and then at 60°C until complete evaporation. Then, a 5% aqueous solution is prepared and ultracentrifuged at 100,000 rpm for one hour. The resulting supernatant should be withdrawn and centrifuged again in 10 kD filter tubes at 5000 rpm on the Eppendorf Centrifuge 5804 R for one hour. The lower percolated fraction is then drawn off and centrifuged again for one hour in 5 kD filter tubes at 5000 rpm on the Eppendorf Centrifuge 5804 R7. Then the upper fraction retained by the filter is drawn off and 500 ml of NaCl-saline solution are added. The drug obtained is used as follows: 1 ml of the drug is diluted in 20 ml of water and taken once a day before a meal for two weeks. The drug consists of molecules of 5 to 10 kD.

implementation examples

example 1

[0012] A 40-year-old patient was referred to an immunologist by her GP because she had frequent episodes of ARD (4-5 per year) for the past three years and complained of fatigue, drowsiness, and persistent weakness. Her medical history was excellent, with two pregnancies, both successfully completed (aged 23 and 27); physical examination revealed no abnormalities. The blood count and chemistry tests as well as the clinical urine test also found nothing. The immunologist ordered an examination of their immune status, which found a reduced pool of natural killer cells and B lymphocytes, while their absolute levels remained normal. Treatment of the patient with our medication was initiated, 1 ml administered orally in 20 ml of water before meals, once daily for two weeks. One month after starting treatment, the patient noticed a subjective improvement in her condition with less weakness and fatigue; she found it easier to get up in the morning (while her daily routine stayed the same). Objectively, a re-examination of her immune status showed an increase in her natural killer cell and B-lymphocyte pools, which doubled and tripled, respectively. T lymphocytes (CD3+) 68 66 66-76 1.4-2*10^9 B lymphocytes (CD19+) 5 10 12-22 0.3-0.5*10^9 T helper cells (CD3+, CD4+) 39 35 33-41 0.7-1.1*10^9 Cytotoxic T cells (CD3+, CD8+) 29 33 27-35 0.6-0.9*10^9 Natural killer cells (CD3+, CD16+, CD56+) 2 6 4-27 0.1-0.5*10^9 CD4/CD8 1.3 1.3 1-1.5 Ig A, mg/mL 2.0 2.5 1.03-4.61

example 2

A male patient aged 60 years with no abnormal history consulted the immunologist alone and reported occasional joint pain, malaise and sleep problems (difficulty falling asleep and waking up). The examination in the internal medicine of a private clinic did not reveal any abnormalities. The examination of the immune status showed a (relatively) reduced number of natural killer cells and humoral immunity factors (IgA and IgM). Treatment of the patient with our medication was initiated, 1 ml administered orally in 20 ml of water before meals, once daily for two weeks. One month after starting treatment, the patient reported better sleep (falling asleep quickly without nighttime awakenings) and improved mood. Objectively, one month after the start of treatment, an increase in humoral immunity factors and normalized lymphocyte population ratios were noted. T lymphocytes (CD3+) 75 76 66-76 1.4-2*10^9 B lymphocytes (CD 19+) 15 17 12-22 0.3-0.5*10^9 T helper cells (CD3+, CD4+) 32 32 33-41 0.7-1.1*10^9 Cytotoxic T cells (CD3+, CD8+) 31 29 27-35 0.6-0.9*10^9 Natural killer cells (CD3+, CD16+, CD56+) 3.5 10 4-27 0.1-0.5*10^9 CD4/CD8 1.3 1.3 1-1.5 Ig A, mg/ml 0.5 2.7 1.03- 4.61 Ig G, mg/mL 7.5 8 6.2-14.7 Ig M, mg/mL 0.3 0.51 0.61-1.64

The above examples show that the medicament, the active ingredient of which is 5-10 kD molecules of Laminaria japonica and Laminaria angustata, leads to a normalization of the immune status of the patients and a subjective feeling of improved health.

Claims (6)

1. A process for preparing a substance, the process comprising leaching the homogenate of the algae Laminaria japonica and Laminaria angustata with aqueous and isobutanolic solution having a mixing ratio of 1 g : 2.5 ml : 2.5 ml to form a homogeneous mixture obtained, which is then post-treated at 60°C for one hour and incubated at room temperature for 10-15 hours; the supernatant is withdrawn and centrifuged for 30 minutes at 3000 rpm and +4°C, after which the centrifuged supernatant is placed in a vacuum evaporator and flushed with argon, then vacuum dried for one hour at room temperature and then at 60°C, until all the liquid fraction is evaporated, after which the resulting sediment is diluted with water to a 5% solution and then ultracentrifuged at 100,000 rpm for one hour; the resulting supernatant is withdrawn and centrifuged in 10 kD filter tubes at 5000 rpm for 1 hour, after which the lower percolated fraction is withdrawn and centrifuged in 5 kD filter tubes at 5000 rpm for 1 hour, after which the upper fraction retained by the filter is withdrawn and diluted in 500 mL of NaCl-saline to a final concentration of 10 mg/mL. The method of claim 1, further comprising, after centrifugation at 3000 rpm and +4°C for 30 minutes and before the centrifuged supernatant is placed in a vacuum evaporator and purged with argon, ultracentrifuging the centrifuged supernatant at 100,000 rpm one hour. 3. Substance produced by the method according to any one of claims 1 or 2. 4. A substance according to claim 3 for use in a therapeutic method for stimulating cells in the human immune system. 5. A substance for use in a therapeutic method for stimulating cells in the human immune system according to claim 4, wherein the substance is administered orally for 14 days. 6. Substance for use in a therapeutic method for stimulating cells of the human immune system according to claim 5, wherein the substance is administered orally for 14 days, preferably in dosages of 1 ml once a day, for example before meals.