WO2015020567A1

WO2015020567A1 - Laminaria angustata-based product for enhancing regeneration and proliferation of cells, method of producing and use the same

Info

Publication number: WO2015020567A1
Application number: PCT/RU2014/000575
Authority: WO
Inventors: Vladimir Georgievich Nesterenko; Vadim Igorevich POSPELOV
Original assignee: Limited Liability Company "Nearmedic Plus"
Priority date: 2013-08-06
Filing date: 2014-07-31
Publication date: 2015-02-12
Also published as: RU2540487C1; WO2015020567A8

Abstract

The present invention relates to the field of pharmacy and medicine and is directed to the provision of a biologically active product for enhancing regeneration and proliferation of cells, wherein the product is a 5-10 molecular weight kDa fraction of a Laminaria angustata water-isobutanol extract subjected to ultrafiltration. The invention also relates to a method for producing said product, and a medicament for regeneration and/or proliferation of cells, comprising said product and a pharmaceutically acceptable additive. The medicament can be used in complex therapy in the treatment of immunodeficiency conditions of different origins, including a health condition after cancer chemotherapy.

Description

LAMINARIA ANGUSTA 7 -BASED PRODUCT FOR ENHANCING REGENERATION AND PROLIFERATION OF CELLS, METHOD OF

PRODUCING AND USE THE SAME

The invention relates to the field of pharmacy and medicine and is directed to a Laminaria angustata-based product for enhancing regeneration and proliferation of cells, a method for producing such a product and the use thereof in a medicament for restoration and improvement of health in complex therapy of immune disorders of various origins, including a health condition after chemotherapy in oncological diseases.

Background

Algae-based preparations are commonly used in nutritional therapy as a source of micronutrients, iodine and biologically active compounds, which have a health improvement effect, increase performance capacity, regulate the immunity and have other favorable effects. It has been noticed that the longevity is higher in regions where the diet contains seafood products, including algae.

A positive effect from the administration of algae and algae-based preparations on the human body is observed in immune-deficiency disorders, oncologic diseases and in aging. There are numerous studies on production of biologically active preparations from algae (V.G. Bespalov, 2005; V.G. Bespalov, V.B. Nekrasova, 2000).

The process of producing some preparations from algae is known to comprise thermal treatment in the presence of aggressive acids (patent RU 2030885, C I, 20.03.1995). Such treatment destroys many biologically active compounds and leads to accumulation of inert polysaccharides and agarose related compounds.

There is a known method of treating algae Laminaria angustata to produce nutrition product "Lamifaren" with the maximum content of unbound alginate in the form of sodium and calcium salts (36%) and macro- and micronutrients in the bio- organic maximally assimilable form (RU 2230464, 20.06.2004). The nutrition product "Lamifaren" is used as a source of macro- and micronutrients and as a sorbent. The method of producing "Lamifaren" comprises preparing algae, treating thereof in acid medium with pH 6 at an algae/solution weight ratio of 1 : 1.5, and ageing for from 5 to 6 hours, and washing the algae mass by a fresh- water digestion four times at temperature of 20°C for from 10 to 30 minutes at an algae/water weight ratio of 1 :3. Then water is allowed to drain out, the algae mass is treated with indirect steam at temperature of water of 40-60°C at an algae mass/water weight ratio of 1 : 1 for 16 to 24 hours under continuous stirring, followed by homogenization of the obtained algae mass to prepare a fine jellylike mass. The use of acidic medium to produce the nutrition product "Lamifaren" also leads to destruction of biologically active compounds and multicomponent composition of the product.

There are data that many of proteins in algae are tightly bound to the cell-wall polysaccharides, and are not transferred into the solution during the aqueous digestion, but remain in the cell walls.

The effectiveness of the treatment of various immunodeficiency conditions, anticancer chemotherapy and prognosis of a disease are known to depend mainly on health condition and conducted maintenance therapy. In recent years, the use of biologically active additives (BAA) and preparations based on natural biologically active compounds as an accompanying therapy for the prevention and treatment of immunodeficiency conditions and oncological diseases became widespread in practice. However, the lack of science-based criteria for the use of such preparations and inability to determine accurately an appropriate dose of a used drug due to its multicomponent composition lead to an empirical prescription of such drug, which sometimes leads to an opposite result in its administration due to side effects caused by the multicomponent composition of the administered drug.

The use of biologically active preparations having a narrow-component composition established by biochemical and/or chemical methods would enhance the effectiveness of the treatment of various diseases, including accompanying therapy in oncological diseases.

Thus, the objective of the present invention is to provide a Laminaria angustata- based biologically active product that enhance regeneration and proliferation of cells and does not cause side effects induced due to its multicomponent composition.

Summary of the invention

The present invention relates to a biologically active product for enhancing regeneration and proliferation of cells, wherein the product is a 10.5 kDa molecular weight fraction of a Laminaria angustata water-isobutanol extract subjected to ultrafiltration. The present invention also relates to a method of producing said product, comprising preparing a Laminaria angustata water- isobutanol extract subjected to ultrafiltration, followed by separation of a 10.5 kDa molecular weight fraction.

In addition, the present invention relates to a medicament for enhancing regeneration and/or proliferation of cells, comprising said product and pharmaceutically acceptable additives.

The medicament can be used to restore and improve a health condition in complex therapy of immunodeficiency conditions of various origins, including a health condition after chemotherapy in oncologic diseases.

Detailed description of the invention

A biologically active product according to the present invention is produced under conditions that maintain the biological activity of extracted compounds, by the following method. A dried and granulated preparation of algae Laminaria angustata is mixed with isobutanol and incubated, then water is added thereto, and the resulting mixture is heated preferably to 55-65°C and incubated for a day. Then, the supernatant is collected, centrifuged, the supernatant is again collected and subjected to vacuum drying, the resulting powder is diluted with water, and the obtained solution is centrifuged, the supernatant is collected and subjected to ultrafiltration, followed by separation a 10.5 kDa molecular weight fraction from the obtained ultrafiltrate.

A medicament for enhancing regeneration and/or proliferation of cells is produced by combining the product according to the present invention, taken in an amount effective for regeneration and/or proliferation of cells, with pharmaceutically acceptable additives. Depending on the selection of pharmaceutically acceptable additives, the medicament may be a dosage form such as a suppository for rectal administration; a tablet or capsule for oral administration, or a solution for intravenous or intramuscular injections. The amount of the product according to the present invention in a unit dosage form is preferably from 0.1 to 10 mg. Acceptable additives for preparing suppositories usually is a solid fat; lactose monohydrate, potato starch, and calcium stearate are generally used as additives in tablets. When the product is administered in capsules, it generally comprises as additives compounds such as talc, magnesium stearate and silica as well as gelatin and titanium dioxide; and preservatives such as sodium lauryl sulfate and methyl parahydroxybenzoate. Capsules also may comprise various food dyes. For intravenous or intramuscular injections, the product is diluted with physiological saline. A treatment scheme (regimen and an administered dose) is selected by a physician depending on the disease severity, patient's condition and age, and is adjusted during administration.

The produced medicament can be used to restore and enhance regeneration and/or proliferation of cells, in particular to restore and improve a health condition in complex therapy of immunodeficiency conditions of various origins, including a health condition after chemotherapy in oncologic diseases.

Example of preparation

A powder of Laminaria angustata (1 part, 100 g) is added to isobutanol (2.5 parts, 250 mg) and stirred to obtain a homogenous mixture (uniform dark-green mixture), the mixture is incubated for 10 minutes at temperature of 18 to 25°C, then 250 ml of distilled water is added to the mixture and thoroughly mixed to obtain a homogenous solution. The obtained mixture is heated on a water bath or in a thermostat for 1 hour at temperature of 55 to 65°C and then incubated for 22-28 hours at 18 to 25°C, the supernatant is collected and centrifuged for 30 minutes at 3000 rpm at a temperature of from 2 to 8°C, after that the supernatant is collected, placed to a rotary evaporator and flushed with argon. Vacuum drying in the rotary evaporator lasts for 1 hour at a temperature of from 18 to 25°C, gradually reducing pressure, then the temperature in the rotary evaporator is elevated to 55-65°C, and the product is kept at said temperature until complete drying. The obtained powder is diluted with distilled water to prepare a 3% solution, followed by centrifugation at 5000 rpm and then at 3000 rpm at 4 to 8°C, and the supernatant is collected. The obtained solution is subjected to ultrafiltration and a 5-10 kDa molecular weight fraction is collected.

The obtained product is a light yellow solution. The solution comprises low molecular weight proteins, polypeptides and separate peptide fragments, as established by chromatography analysis and filtration procedures.

To study the biological activity of the product, sodium chloride is added to the obtained solution to a final concentration of 0.09%, or the obtained product is mixed with physiological saline at a ratio of 1 :1.

Examples of studying the biological activity of the obtained product

Study of an effect of the obtained product on cell proliferation 10 ml of cells H-9 used in this test were placed in each well of a 24-well culture plate at 250x10³ cells/ml. The obtained solution of the test product was added to wells at 10 μΐ/ml. Control wells were filed with the corresponding amount of a culture medium comprising RPMI-1640, supplemented with 10% fetal bovine serum, glutamine (14.6 mg per 500 ml) and gentamicin (20 mg per 500 ml).

The cells with and without the added solution of the product were incubated for 24 hours in a C0₂ incubator at 37°C. After the incubation, the number and percentage of died cells were counted. The obtained results are shown in Table 1.

Table 1. Study of an effect of the obtained product on cell proliferation

As can be seen from Table 1 , the obtained product at a concentration of 10 μΐ/ml causes cell proliferation.

It has been found that after one-year storage of the product at -80°C, its activity to induce cell proliferation is completely retained.

Contrary to the product according to the invention, similar study of the known preparation "Lamifaren" has shown that said preparation does not possess the ability to induce cell proliferation.

Study of the action of the obtained product on bone marrow-derived mesenchymal stem cells

The action of the obtained product on bone marrow-derived mesenchymal stem cells (MSC) has been studied. The material derived from donors was collected into sterile bags containing an anticoagulant. A mononuclear cell fraction was separated using Ficoll. The separated cells were divided into 3 portions: 1. control (cultivation of bone marrow cells without the test product), 2. cultivation of bone marrow cells with the test product used at 10 μΐ/ml, and 3. cultivation of bone marrow cells with a test substance (preparation) used at 50 μΐ/ml.

The bone marrow cells were cultured in sterile flaks at 37°C under absolute humidity and 5% C0₂. 80-100xl0⁶ bone marrow-derived mononuclear cells were placed into 30 ml of a growth medium contained a culture flask (bottom diameter of 175 mm). After a certain time, the flask was washed to remove non-adherent cells. The stromal progenitors were passaged one time a week.

In the study, the obtained product was used at concentrations of 10 and 50 μΐ/ml. The product was added at the initiation point and in each passage during the whole period of culturing the cells. The culture was passaged on Day 14 from the beginning of cultivation. The results were assessed according to the number of MSCs collected from a passage.

CE (cloning efficiency) of the primary culture was determined on Day 14 day by the number of colonies of stromal progenitors per 10⁵ explanted cells. The cell population dynamic was estimated by a cellular growth rate as a ratio of the number of cells obtained at a given passage to the number of cells seeded on the previous passage.

The results of the study of an effect of the obtained product on proliferation and percentage of early progenitors of the umbilical cord blood at a long-term cultivation are shown in Table 2.

Table 2. Effect of the obtained product on proliferation and percentage of early progenitors of the umbilical cord blood at a long-term cultivation

- cloning efficiency

TN - total number of cells in a group

The results provided in the Table demonstrate that the addition of the obtained product to the culture stimulates proliferation of stromal progenitors. The test product leads to an increase in the number of MSC progenitors in the primary culture.

Study of the colony-stimulating activity of the obtained product

An effect of the obtained product on proliferation and percentage of early progenitors of the umbilical cord blood were studied on the following cells used as a target cell: stem cells of the umbilical cord blood of a mature newborn infant whose colony-forming activity is well known, the cells being selected using Ficoll.

In in vitro studies, the obtained product was used in a concentration of 10 μΐ/ml.

The study of action of the obtained product on long-term cultures of haematopoietic progenitors was performed in CellGenix Kit Systems for expansion of haematopoietic stem cells (HSC). The cultivation in said system is always conducted by using a standard set of colony-stimulating factors, and the action of the test agents is analyzed against said factors.

The test product was added to the culture at the initiation point (Day 0).

The culture was analyzed on Day 0, further on Days 14 and 28 (from bag 1), on Days 42 and 56 (from bag 2), and on Days 70 and 84 (from bag 3). Colonies were counted under an inverted microscope (x40), and the number of CD34+ cells was determined on a FACSCalibur™ flow cytometer (Becton Dickinson, USA).

The action of the obtained product to haematopoietic stem cells was studied in a methylcellulose culture system. Cultivation in methylcellulose provides an opportunity to study the nature of action of the obtained product on proliferation of the full range of haematopoietic progenitors and on the direction of their differentiation: early, mixed colony-forming (CFU-Mix), intermediate granulocyte/macrophage colonies (CFU-GM), and late macrophage and erythroid colonies (CFU-G, CFU-M and CFU-Er). Cultivation in this system is always carried out using a standard set of colony-stimulating factors, and the action of the test compounds is analyzed against said factors.

The culture was analyzed on Day 14. Colonies were counted under an inverted microscope (x40).

The results of culturing were analyzed by counting the number of each type colony-forming progenitors per 10⁵ explanted cells. The following parameters were analyzed:

1. CFU-Mix - mixed colonies (early progenitors)

2. CFU-Er - erythroid progenitors

3. CFU-GM - granulocyte/macrophage colonies

4. CFU-G - granulocyte progenitors

5. CFU-M - macrophage progenitors

CE (cloning efficiency) was determined by the sum of colony-forming progenitors of all types per 10⁵ explanted cells. The results of the analysis of an effect of the obtained product on proliferation and percentage of early progenitors of the umbilical cord blood in long-term cultivation are shown in Table 3.

Table 3. Effect of the obtained product (P) on proliferation and percentage of early progenitors of the umbilical cord blood in ling-term cultivation

Doses TC CFU per 10^¾ mononuclear cells (M) CD34 CD1

(xl O⁶) Mix G M G/M E CE (%) 33

(%)

Bag 1 (Day 0)

Control 18.0 3.2 15.1 18.3 8.1 6.9 51.6 0.20 0.09

Long-term exposure (Day 14)

Control 180.3 4.6 18.9 20.5 12.0 8.7 64.7 1.31 0.22

P, 10 μΐ 350.2* 26.3 32.1 33.5 23.1 22.5 137.5* 1.73* 0.41 *

Long-term exposure (Day 28)

Control 201.1 4.4 17.2 18.9 10.0 8.4 58.9 1.29 0.20

P, 10 μΐ 494.6* 36.9 44.2 42.0 27.8 35.6 186.5* 3.45* 0.79*

Bag 2 (Day 28)

Control 20.0 4.4 17.2 18.9 10.0 8.4 58.9 1.29 0.20

_/ong-term exposure (Day 42)

Control 22.8 4.7 18.6 19.1 TD 8.2 61.7 1.30 0.21

P, 10 μΐ 100.2* 22.4 30.3 29.1 22.6 19.3* 123.7 1.70 0.38

Long-term exposure (Day 56)

Control 19.3 3.9 16.0 17.5 8.4 7.2 53 1.30 0.20

P, 10 μΐ 121.5* 34.3 43.8 41.0 26.9 32.7* 178.7 2.44* 0.51 *

Bag 3 (Day 56)

Control 19.3 39 16.0 17.5 8.4 7.2 5.3 1.30 0.20

Long-term exposure (Day 70)

Control 15.3 1.9 14.0 16.0 8.0 6.3 46.2 0.4 0.02

P, 10 μ] 19.4 2.8 20.0 31.0 1 1.2 18.0 83.0 0.6 0.1 Long-term exposure (Day 84)

Control 10.3 0.8 5.2 8.3 6.3 2.9 23.5 0.1 0.01

P, 10 μΐ 15.1 2.0 15.6 17.3 10.2 8.4 53.5 0.4 0.05

TC - total cellularity (the number of nuclear cells)

Mix - mixed colonies

G - granulocyte colonies

M - macrophage colonies

G/M - granulocyte/macrophage colonies

E - erythroid colonies

CE - cloning efficiency

TD - threshold dose

* - Significant differences from control (by the Student t test): p<0.05.

As can be seen from the data presented in Table 3, in a long-term contact (for 14, 28, 42 and 56 days of continuous exposure), the obtained product used at 10 μΐ/ml have a statistically significant, pronounced stimulatory effect on proliferation of early hematopoietic progenitors (mostly on CD34+ and CD133+ cells), CFU-Mix and CFU- GM. The expansion rate of hematopoietic cells (total cellularity of culture) was significantly higher than in the control when the obtained product was added to the culture medium.

The obtained product provides a delayed natural loss of surviving cells compared with the control (56 days). The number of stem cells and their proliferative capacity are reduced in a long-term cultivation; however, the action of the obtained product leads to an observed delayed degradation of the cultures (70 and 84 days), whereas in the control this effect is observed much earlier (already after 56 days).

Study of the haemostimulating activity of the obtained product

The haemostimulating activity of the obtained product was studied on stem cells of the umbilical cord blood of a mature newborn infant whose colony-forming activity is well known, wherein the cells, which were used as a target cell, were selected using Ficoll. In this study, the obtained product was used at a concentration of 10 μΐ/ml. The study of action of the obtained product on long-term cultures of haematopoietic progenitors was performed in CellGenix Kit Systems for expansion of haematopoietic stem cells (HSC). The cultivation in this system is always conducted by using a standard set of colony-stimulating factors, and the action of the test agents is analyzed against said factors.

The obtained product was added to the culture at the initiation point (Day 0) and repeatedly on Day 84. The culture was analyzed on Day 14 after the beginning of cultivation. Colonies were counted under an inverted microscope (x40), and the number of CD34+ cells was determined on a FACSCalibur™ flow cytometer (Becton Dickinson, USA).

The action of the obtained product to haematopoietic stem cells was studied in a methylcellulose culture system, which provides an opportunity to study the nature of action of the obtained product on proliferation of the full range of haematopoietic progenitors and on the direction of their differentiation: early, mixed colony-forming (CFU-Mix), intermediate granulocyte/macrophage colonies (CFU-GM), and late macrophage and erythroid colonies (CFU-G, CFU-M and CFU-Er). Cultivation in this system is always carried out using a standard set of colony-stimulating factors, and the action of the test compounds is analyzed against said factors.

The results of cultivation were analyzed by counting the number of each type colony-forming progenitors per 10⁵ explanted cells. The following parameters were analyzed:

CFU-Mix - mixed colonies (early progenitors)

CFU-Er - erythroid progenitors

CFU-GM - granulocyte/macrophage colonies

CFU-G - granulocyte progenitors

CFU-M - macrophage progenitors

CE (cloning efficiency) was determined by the sum of colony-forming progenitors of all types per 10⁵ explanted cells.

In addition, an effect of the obtained product on the viability of haematopoietic progenitors was studied on thawed cultures after a long-term cultivation in bags.

The results of the study of an effect of the obtained product on proliferation and percentage of early progenitors of the umbilical cord blood in long-term cultivation are shown in Table 4.

Table 4. Effect of the obtained product on proliferation and percentage of early progenitors of the umbilical cord blood in ling-term cultivation

TC - total cellularity (the number of nuclear cells) of a colony

Mix - mixed colonies

G - granulocyte colonies

M - macrophage colonies

G/M - granulocyte/macrophage colonies

E - erythroid colonies

CE - cloning efficiency

As can be seen from the data presented in Table 4, the repeated addition of the obtained product to the culture did not have an additional stimulatory effect on proliferation of haematopoietic progenitors.

Addition of the obtained product to the thawed cultures leads to an improved viability of the cells in culture compared with the control.

Thus, data obtained in these studies demonstrate a pronounced ability of the obtained product to enhance regeneration and proliferation of cells, especially of early hematopoietic progenitors.

It has been found that the obtained product at 10 μΐ/ml induces cell proliferation in the culture.

After one-year storage of the product at -80°C, its activity to induce cells proliferation is completely retained.

The addition of the obtained product to the culture stimulates proliferation of stromal progenitors. Its action leads to an increase in the number of progenitors of bone marrow-derived mesenchymal cells in the primary culture.

In a long-term contact with the cell culture (for 14, 28, 42 and 56 days of continuous exposure), the obtained product even at a concentration of 10 μΐ/ml demonstrates a pronounced stimulatory effect on proliferation of early hematopoietic progenitors. The action of the obtained product also increases the total cellularity of cultures.

The obtained product provides a delayed natural loss of surviving cells. The action of the obtained product leads to an observed significantly delayed degradation of the cell cultures. The obtained data are of fundamental importance for the manufacture of cell preparations for anti-aging and possibly for rejuvenation; they demonstrate that this product can be used for restoration and improvement of a health condition, which is essential in the treatment of immunodeficiency conditions of various origins, including a health condition of cancer patients after chemotherapy.

Repeated addition of the obtained product to the culture does not provide an additional stimulatory effect on proliferation of hematopoietic progenitors; however, the addition of the obtained product to a thawed culture leads to an improved viability of cells in the culture. This effect is very important when the obtained product is used for the manufactory of cell preparations for therapeutic applications in future.

Claims

1. A product for enhancing regeneration and/or proliferation of cells, the product being a 5-10 kDa molecular weight fraction of a Laminaria angustata water-isobutanol extract subjected to ultrafiltration.

2. A method for producing a product according to claim 1 by processing algae, characterized by: preparing a water-isobutanol extract of algae Laminaria angustata, subjecting the prepared extract to ultrafiltration, and separating a 5-10 kDa molecular weight fraction.

3. A medicament for enhancing regeneration and/or proliferation of cells, comprising a product according to claim 1 and pharmaceutically acceptable additives.

4. The medicament of claim 3 intended for restoration and improvement of a health condition in complex therapy of immunodeficiency conditions of various origins, including a health condition after chemotherapy in oncologic diseases.

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