WO2024105193A1 - Nouveaux composés et procédés - Google Patents

Nouveaux composés et procédés Download PDF

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WO2024105193A1
WO2024105193A1 PCT/EP2023/082116 EP2023082116W WO2024105193A1 WO 2024105193 A1 WO2024105193 A1 WO 2024105193A1 EP 2023082116 W EP2023082116 W EP 2023082116W WO 2024105193 A1 WO2024105193 A1 WO 2024105193A1
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biologically active
cell
cells
active substance
bas1
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Andrey V. KOPTYUG
Iurii G SUKHOVEI
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents

Definitions

  • the present disclosure relates, inter alia, to biologically active substances extracted from animal tissue or cell culture comprising non- pathologically proliferating cells, which substances simultaneously (i) induce and/or intensify cell proliferation; (ii) induce and/or intensify apoptosis; and (iii) have antibacterial activity, and methods of extracting said biologically active substances.
  • the present disclosure also relates to the use of such biologically active substances.
  • “Immortalization” in the present context refers to the escape from the normal limitation on proliferation of the cell population in vitro or in vivo and is not related to the fate of individual cell and possibility of its eternal life.
  • Once immortalized a cell population can be continuously cultured, or can function continuously and for a long time in vivo.
  • immortal cell lines very rarely emerge spontaneously under usual conditions.
  • activation of the nonpathogenic proliferation with simultaneous intensification of the apoptosis primarily disposing of the damaged and aging cells leads to rejuvenation of the niche.
  • Homeostasis in the space-localized proliferation niche is understood as a continuous stabilization of the average cell concentration.
  • intensification of the cell division with the stabilization of the overall cell concentration means the shift of the average cell age towards niche rejuvenation: cell division increases the number of young cells, and apoptosis aims at removing old and damaged ones.
  • increasing the lifetime of the individual cells in the proliferation niche stabilized by homeostasis leads to the suppression of the proliferation and aging of the niche.
  • the term “immortality” does not refer to the life of an individual cell, but only to the possibility of long-term maintenance of the functioning of the cell population as a whole.
  • the rejuvenation of the homeostasis-governed proliferation niches is related to the increase in the proportion of younger cells in the cell population of the niche.
  • a stem-cell niche is an area of a tissue that provides a specific microenvironment, in which stem cells are present in an undifferentiated and self-renewable state. Cells of the stemcell niche interact with the stem cells to maintain them or promote their differentiation.”
  • a proliferation niche as a space- defined tissue area or a restricted volume of culture with proliferating cells with specific or specialized microenvironment, in which dividing cells support the functioning of a cell population.
  • embryonic cells In order to increase the life span of cells in culture, and in attempts to achieve the rejuvenation of human and animal tissues a number of published techniques have included the use of embryonic cells. Such strategy is based upon different assumptions, including that embryonic cells are less differentiated than other cells, proliferate at a greater rate than differentiated cells, can maintain own proliferation for significant time, and can stimulate proliferation of other cells. Tumor cells are also known for their greater potential for proliferation, and some of the known methods use this property for promoting proliferation in cell cultures. However, the limited number of appropriate cell types and lines that can be used in such way, the limited number of phenotypes that they are able to generate, and their inherent tumorigenicity , make these types of cell lines less than ideal and unsuitable for in vivo applications.
  • One of the potential ways of cell population rejuvenation is related to the technology using cell reprogramming, claiming the possibility for stimulating cells for acquiring the stem cell properties (induction to pluripotent stem cell state) and increasing the cell population capacity for proliferation.
  • Corresponding method was initially proposed by Yamanaka and Takashi, Cell. 126(4): 663-676, 2006, and is continuously explored and developed further (see, e.g., Guo, X-L and Chen J-S, Int J Ophthalmol. 8(4): 818-825, 2015).
  • Corresponding family of the methods use so-called Yamanaka factors (four basic transcription factors in different combinations).
  • Stem cells are believed to have immense potential for therapeutic purposes. Such cells can be derived from the donor sources, including, but not limited to, embryonic, blast, tissue-derived, blood, and cord-blood cells; organ- derived progenitor cells; and stromal cells among others. It is widely believed that from a therapeutic perspective alone, such cells may be useful for the treatment of a vast array of disorders. Moreover, primary stem cells that have exhibited the most plasticity are embryonic stem cells. However, obtaining large quantities of these cells is particularly problematic and raises ethical issues. In addition, introduction of foreign cells can cause various adverse effects, and in some cases, cosmetology or therapeutic use of stem cells not derived from the same individual without proper care can lead to serious health problems.
  • Stimulation of non-pathological proliferation also presumes that corresponding means of inducing proliferation in vivo should not disturb the mechanisms maintaining the proliferation niche homeostasis. It demands that corresponding factors should simultaneously affect the apoptosis and in correlated manner with cell proliferation, preventing uncontrollable growth of the cell numbers. As a consequence, factors that are able to induce and intensify the proliferation in the niche without disturbing the homeostasis can lead to the ’’rejuvenation” of the cell niche: intensified proliferation leads to increasing share of young cells, while the apoptosis intensify ’’the removal” of older and damaged cells.
  • US 20110201105 discloses cDNA libraries obtained from Tammar wallaby (Macropus eugenii) mammary gland tissue at different times (including day 23 pregnancy, day 130 lactation, and day 260 lactation). Lactation- associated polypeptides were identified from these cDNA libraries, and bovine homologues of the wallaby proteins are also disclosed. The proteins have a range of activities including anti-apoptotic activity, pro- or anti-inflammatory activity, cathelicidin anti-microbial activity, induction of trefoil proteins (and protection of epithelial surfaces), increased cell proliferation, and induction of cell differentiation (and loss of pluripotency).
  • EP 0673653 A1 discloses a biologically active agent (BAA) based on components of cell membranes (CCM) of homogenized animal tissue which consists of organic substances and has immunomodulating properties.
  • the agent contains modified components of cell membranes with antigen structure modified during the processes of embryogenesis and/or proliferation and/or differentiation of cells, and/or during a pathology preceding or accompanying the process of regeneration and/or repatriation of the tissue.
  • a method for obtaining BAA is disclosed, as well as a pharmaceutical preparation for normalization of physiological state of human beings and animals based on the organic substances.
  • CCM of animal tissues comprises, as active agent, BAA.
  • WO 9723501 A1 relates to molecules capable of modulating apoptosis of animal cells and discloses cell homologues of viral-derived, apoptotic- inhibiting molecules which are useful in modulating apoptosis of animal cells.
  • the molecules may be used to promote or inhibit cell apoptosis depending on the exigencies of the therapeutic situation.
  • JP 2006246764 A discloses a method for producing an endogenous antibacterial substance; the method comprises culturing Paneths cells isolated from a crypt collected from a mammalian small intestine in the presence of an inducer by using a culture scaffold as a material to supplement a biological matrix function and secreting an endogenous antibacterial substance.
  • CN 112921001 A discloses an exosome containing a high-expression NAMPT protein, and a method for preparation and application thereof. The exosome containing high-expression NAMPT protein is obtained by transfecting NAMPT mRNA synthesized in vitro into iPS cells.
  • the content of the NAMPT protein in the exosome is more than 2 times of the content of the NAMPT protein in the exosome secreted by common iPS cells, and the exosome containing high-expression NAMPT protein can improve the condition of senescent cells and has a protective effect on neurons compared with the exosome secreted by common iPS cells.
  • CN 111658672 discloses a human placenta stem cell extract freeze- dried powder and a preparation method thereof, in particular, a preparation method of a human placenta stem cell extract freeze-dried powder.
  • the human placental stem cell freeze-dried powder has the effects of comprehensively regulating an aged organism; it can raise the metabolism levels of an organism, raise physiological function, raise the immunity of an organism, prolong puberty period, beautify skin, prolong life and delay senility, and at the same time, the freeze-dried powder is long in storage period, not easy to degrade and convenient for use.
  • CN 101152215 discloses an ovarian granulosa cell anti-aging preparation, and a method of preparation method and application of the product.
  • the invention uses ovarian granulosa cells in preparation of anti-aging products.
  • the ovarian granulosa cell anti-aging product is in vitro cultivated and contains amplified cultivation supernatant of female mammalian animal ovarian granulosa cells and/or the cell extract, and one or more pharmaceutically acceptable medical excipient and antioxidant.
  • the invention can proliferate and secrete a plurality of bioactive substances.
  • RU 2728601 discloses a method of testing substances affecting aging processes by analyzing patient blood.
  • the method involves producing patient's mononuclear cells by standard venous blood sampling with subsequent isolation of mononuclear leukocyte (MNC) population, incubation of the patient's MNC culture with the recommended doses of the test agent and determining the ratio of cells in different phases of the cell cycle by flow cytometry in the MNC culture, followed by evaluating the obtained results using the difference in cell ratio distribution on the cell cycle.
  • MNC mononuclear leukocyte
  • the method provides the possibility of testing the activity of substances and preparations aimed at treating various pathologies, including those associated with age.
  • the present invention is based on the inventors’ discovery of biologically active substances that simultaneously combine certain characteristic activities of induction and/or intensification of cell proliferation, induction and/or intensification of apoptosis and antibacterial activity, which represents a not previously described combination of properties of bioactive factors modulating cell proliferation and promoting tissue and cell culture rejuvenation. Moreover, there is a direct correlation between the biologically active substances’ effects of inducing and/or intensifying proliferation, inducing and/or intensifying apoptosis and having antibacterial activity. In some embodiments, said biologically substances have anti-inflammatory activity.
  • the biologically active substances are distinguishable from exosomes which are extracellular vesicles that are released from cells upon fusion of an intermediate endocytic compartment, the multivesicular body (MVB), with the plasma membrane.
  • a biologically active substance extracted from animal tissue or cell culture comprising non-pathologically proliferating cells, which substance simultaneously (i) induces and/or intensifies cell proliferation; (ii) induces and/or intensifies apoptosis; and (iii) has antibacterial activity.
  • said biologically active substance has anti-inflammatory activity.
  • the biologically active substance has or comprises one or more characteristics selected from the group consisting of: a molecular weight of less than 1.5 kDa, long linear hydrocarbon chains, hydrocarbon fragments, steroid fragments, alkane fragments, high molecular weight unsaturated hydrocarbon fragments, phosphates, hydroxyl groups, amino groups and amide groups.
  • the biologically active substance has a molecular weight of less than 0.8 kDa, such as less than 0.7 kDa.
  • the biologically active substance is capable of shifting the cell cycle in a cell population such that there is an increased proportion of cells in the stages just before or just after cell division.
  • the biologically active substance of this aspect induces and/or intensifies cell proliferation and apoptosis such that there is a correlation between induction and/or intensification of cell proliferation and induction and/or intensification of apoptosis.
  • intensification of cell proliferation is correlated with intensification of apoptosis.
  • the biologically active substance is suitably not carcinogenic.
  • the biologically active substance is Biologically Active Substance 1 (hereinafter referred to as BAS1 ).
  • the biologically active substance is not BAS1 .
  • a fragment, analog or derivative of the biologically active substance according to the first aspect which fragment, analog or derivative simultaneously possess the properties defined in the first aspect.
  • composition comprising the biologically active substance according to the first aspect, or the fragment, analog or derivative according to the second aspect, and at least one pharmaceutically acceptable excipient or carrier.
  • a method of supporting, intensifying and/or activating proliferation of animal cells in vitro comprising the step of contacting said cells with one or more of the biologically active substances according to the first aspect, the fragment, analog or derivative according to the second aspect, or the composition according to the third aspect.
  • said animal cells are mammalian cells.
  • a cosmetic method of supporting, intensifying and/or activating proliferation of animal cells in vivo comprising the step of contacting said cells with one or more of the biologically active substances according to the first aspect, the fragment, analog or derivative according to the second aspect, or the composition according to the third aspect.
  • Said animal cells are typically mammalian cells, such as human cells.
  • a biologically active substance according to the first aspect, a fragment, analog or derivative according to the second aspect, or a composition according to the third aspect for use as implant coating is dental implant coating.
  • an eighth aspect of the invention there is provided a method of long-term storage of the biologically active substance according to the first aspect or the fragment, analog or derivative according to the second aspect using drying and/or lyophilization and sorption on biopolymers, such that when not exposed to direct sunlight or UV-light, said substance can be stored in dry conditions at room temperature (e.g. 18-24 °C) without degrading its activity for at least 6 months, such as for at least 12 months.
  • room temperature e.g. 18-24 °C
  • a process of extracting a biologically active substance from non-pathologically dividing cells or cell cultures comprising the steps of (i) prior to extraction, adding the biologically active substance according to the first aspect, the fragment, analog or derivative according to the second aspect, or the composition according to the third aspect to said cells or cell cultures; and (ii) extracting the biologically active substance, wherein said addition in step (i) improves the yield of the extracted biological substance.
  • the added biologically active substance may be the same as or different to the extracted biologically active substance.
  • said added biologically active substance is an autoinducer.
  • a method of identifying a substance for use as a cell proliferation inducer and/or enhancer, an apoptosis inducer and/or enhancer, an anti-inflammatory agent and/or an antibacterial agent comprising (a) providing a test substance; and (b) assessing whether the test substance shifts the cell cycle in a cell population such that there is an increased proportion of cells in the stages just before or just after cell division, wherein a test substance that shifts the cell cycle is useful as a cell proliferation inducer and/or enhancer, an apoptosis inducer and/or enhancer, an antiinflammatory agent and/or an antibacterial agent.
  • the method according to this aspect is an in vitro method.
  • the biologically active substance according to any one of the aforementioned aspects may be extracted from homogenized animal tissues or cell cultures containing actively and non-pathologically proliferating cells, followed by centrifugation, freezing-thawing, filtration and separation of the supernatant into fractions.
  • the extraction is performed by liquid chromatography with a carrier in the form of pure water or isotonic sodium chloride solution.
  • said extraction is performed by liquid phase extraction using organic solvents.
  • Figure 1 is a flow chart illustrating the production of BAS1 .
  • Figure 2 represents low (A-C) and high (D-F) resolution SEM images of typical crystallite shapes appearing after recrystallization of BASI . Embedded bars reflect the used magnification.
  • Figure 3A is a characteristic IR absorption spectrum (absorbance vs wavelength number) of dry BAS1 taken in dry chamber under argon atmosphere. The bars mark corresponding peaks belonging to organic molecules.
  • Figure 3B is an overall 1 H NMR spectrum of BAS1 in D2O.
  • Figure 3C is a 1 H NMR spectrum of BAS1 in D2O between 0.9 and 5 ppm.
  • Figure 3D is a 1 H NMR spectrum of BAS1 in D2O between 4.2 and 7.8 ppm.
  • Figure 3E is a 2D 1 H NMR spectrum of BAS1 in D2O.
  • Figure 3F is a 13 C NMR spectrum of BAS1 in D2O.
  • Figure 3G is a 31 P NMR spectrum of BAS1 in D2O between -50 and 200 ppm, and its insert shows the part of the spectrum between -5 and 6 ppm.
  • Figure 4 is a typical SEM image of dried BAS1 with marked scan area and corresponding EDX (energy dispersive X-ray) spectrum with automated element abundance analysis.
  • EDX energy dispersive X-ray
  • Figure 5 is an HPLC spectrum of BAS1 .
  • Figure 6 shows two more HPLC spectra of BAS1 .
  • Figure 7 shows graphs that present the results of automated identification carried out for BAS1 by the gas chromatography system.
  • Figure 7A shows alkanes (red) and phthalates (blue), without full identification.
  • Figure 7B shows automated identification of fatty acids, cholesterol, sitosterol. Irgafos® 168 (a polymer stabilizer, most probably contamination).
  • Figure 7C shows clear identification of squalene (C30H50) and hopane (C30H52).
  • Figures 8A-B illustrate the changes induced by BAS1 in the duration of the phases of the cell cycle in the culture of human dermal fibroblasts studied by flow cytometry.
  • Figure 8A experiment without BAS1 (Control).
  • Figure 8B experiment with a single addition of BAS1 at day 0.
  • Figures 8C-D the changes induced by BAS1 in the duration of the phases of the cell cycle in the culture of human blood monocytes studied by flow cytometry, wherein Figure 8C refers to the experiment without BAS1 (Control) and Figure 8D with a single addition of BAS1 at day 0.
  • Figures 9A-D show the changes induced by BAS1 upon the cell distribution over the cell cycle phases as studied by flow cytometry for human blood mononuclear cells in cell culture (A- apoptosis; B-G0+G1 ; C-S; D-G2). Corresponding dependencies show the changes happening with addition of different concentrations of BSA1 on day 0 as compared to the control. Horizontal axis: days of incubation. Corresponding BAS1 doses are given in mkg/ml.
  • Figures 10A-D show the changes induced by BAS1 upon the cell distribution over the cell cycle phases, and Figure 10E upon cell vitality as studied by flow cytometry for human fibroblasts in cell culture. Corresponding dependencies show the changes happening with addition of different concentrations of BAS1 at day 0 as compared to the control. Horizontal axis: days of incubation. Corresponding BAS1 doses are given in mkg/ml.
  • FIGs 11 A-D show typical changes induced by BAS1 in cell cycle distribution of the human mononuclear blood cells (healthy volunteers). Single dose (20 mg) of dry BAS1 was administered orally at day 0.
  • Figures 12A-D present the measurements of the human blood mononuclear cell cycle distribution changes performed with male and female volunteers between 16 and 86 years of age according to the method described in Russian patent no. 2728601 , which suggests a quantitative criterion for the assessment of biologic age of the cell population. It is based on the distribution of the cells in the population over the cell cycle stages. It is suggested that the higher fraction of cells in the cell cycle stages immediately preceding and/or following the division, the younger is the cell population.
  • methodologically the measurements can be performed using flow cytometry. In practical application this can be used for the quantified assessment for action of any substance claimed to have rejuvenating or ageing activity by comparing the cell cycle distribution in the cell population before and after administering the substance. Such tests can be performed both in vitro and in vivo. In one of the embodiments, in vivo testing is performed using mononuclear cells extracted from venous blood.
  • Figure 13 illustrates the protocol used for mononuclear cell extraction from venous blood used for the tests on the presence of BAS1 .
  • Figure 14 illustrates the differences in the intensity of cell proliferation and apoptosis (measured by flow cytometry using reference biomarkers) in the control group (CO, no treatment), and in three test groups with the treatment using (TO) the traditional method; (TR) the BAS1 and hyaluronate gel composition; and (BAS1 +GI) hyaluronate gel only (Gl).
  • Figure 15 illustrates the differences in intensity of cell proliferation and apoptosis in the control group (with no treatment, CO, control) and the group treated with gel only (Gl), used in further experiments as a carrier for BAS1 .
  • Figure 16 illustrates the differences in intensity of cell proliferation and apoptosis (measured by flow cytometry using reference biomarkers) in the groups treated in the traditional way (TR) and with hyaluronate gel (carrier) and BAS1 as active ingredient (BAS1 + Gl).
  • Figure 17 present the results of the WST-1 cell vitality tests in the case of undiluted and 1 :10 diluted tested substances. Graphs represent the effect of undiluted (left) and tenfold diluted (right) hyaluronic acid preparations on PDLSC cell viability. The absorbance value is measured using the WST-1 cell proliferation reagent, proportional to the number of viable cells. Star indicates the results that were showing p ⁇ 0.05 as compared to control.
  • Figure 18 presents the results of the effect of tested substances on stimulating spontaneous osteogenic differentiation based on Koss densitometry.
  • the samples were analyzed by densitometry.
  • the darkening in the corresponding image is directly proportional to the degree of mineralization.
  • Mineralization in the HYL and ALC cases is significantly higher compared to the control (Cont) and NTS * p ⁇ 0.05 vs control, # p ⁇ 0.05 vs NTS.
  • Figure 19 presents analysis of the alkaline phosphatase activity.
  • the absorbance is proportional to ferment activity as measured using an ALP reagent.
  • Figure 20 presents the results of inflammation assessment after 31 days of treatment by used preparations.
  • Control group was injected with saline solution, test groups with the solution of hyaluronate (HYL), hyaluronate (carrier) and BAS1 as an active agent (BAS1) and NST solution (NST).
  • Induction of the periodontitis was performed at day 0 on the left side of the mouth.
  • the difference between treatment groups is significant (p value ⁇ 0.05 versus linked side control). Within the groups, the difference between the left and right sides was compared and evaluated. A significant difference was found between the left (induced) and right (not induced) sides only in the control group.
  • Figures 21 A-C present the comparison of the treatment results using BAS 1 -hyaluronate composition and commercial composition FlexBarrier, and comparison between both results. Test results indicate that both BAS1 + hyaluronate and FlexBarrier compositions are equally suitable for the treatment of interdental papillary defects, with BAS1 + hyaluronate showing better clinical results.
  • Figures 22A-H illustrate correlated action of BAS1 upon the cell cycle stages and apoptosis of the cultivated human monocytes.
  • Monocytes were extracted from the venous blood of healthy human volunteers aged between 15 and 61 years.
  • Figures 22A-D illustrate the difference in action of the PGA and BAS1 at different concentrations (graphs represent the ratios of the corresponding values characterizing the cell stage population and apoptosis intensity);
  • Figures 22E-F illustrate the difference in action of the PGA and BAS1 on the rest and proliferation phases. In the graphs of Figures 22A-F, the data averaged for all subjects without separation into age sub-groups).
  • Figures 22G-H illustrate the correlation of the action of PGA and BAS1 upon the cell stages (synthesis, S and proliferation, P) for the young (age 15 to 20) and mature (aged 21 to 61 ) volunteer sub- groups.
  • Graphs represent the ratios of the corresponding values characterizing the cell stage population and apoptosis intensity, which are averaged for the corresponding age subgroup.
  • Figure 23 shows Tables 1 and 2, wherein Table 1 provides a summary of the EDX (energy dispersive X-ray) analysis of the elemental content from different sites on the individual crystallites of the dried BAS1 . Typical spectrum at the site SO is shown in Figure 4. Table 2 provides a resulting elemental content of the BAS1 crystallites averaged from different sites analyzed by EDX.
  • Table 1 provides a summary of the EDX (energy dispersive X-ray) analysis of the elemental content from different sites on the individual crystallites of the dried BAS1 .
  • Typical spectrum at the site SO is shown in Figure 4.
  • Table 2 provides a resulting elemental content of the BAS1 crystallites averaged from different sites analyzed by EDX.
  • Figure 24 shows Table 3, which presents results of the high- performance liquid chromatography (HPLC) studies summarized as a list of suggested identifiable compounds semi-automatically generated using the embedded instrument features and linked substance databases.
  • Figure 25 shows Table 4, which presents results of two more HPLC analyses of BAS1 , summarized as the semi-automatically generated identifiable compound listings.
  • Figure 26 shows Tables 5A-D, which provide the list of the identifiable compounds resulting from liquid and gas chromatography studies of BAS1 , separated into few categories (hormone-like substances, amides-amines, phosphates, aliphatic substances).
  • Figure 27 shows Tables 6-8, wherein Table 6 provides the experimental data (average values of Bactericidal Activity Index) on the antibacterial activity of BAS1 for different strains of bacteria taken from a large bacteria database. Tables 7 and 8 provide the experimental data on sensitivity of the bacteria to the action of BAS1 with the concentration 100 pg/ml for different clinical strains of bacteria.
  • biologically active substance can be used interchangeably with “cell extract”.
  • “BAST’ or “biologically active substance 1" is a non-limiting example of a biologically active substance of the invention.
  • Cell population is the number of cells in a given area, both in cell culture and in live tissues and organs.
  • exosomes can be used interchangeably with “extracellular vesicles”.
  • proliferation may include proliferation to the point of production of a continuous cell line (e.g., immortalization).
  • the biologically active substances of the present invention have an effect on cells in contact with the substance as described and claimed herein. Upon contact with the biologically active substance, cells in the population are induced to achieve a proliferation rate that is higher than the normal in vitro cell proliferation rate, thereby increasing the cells’ potential for proliferation.
  • non-pathologicaf in relation to proliferation means that after division new cells do not show any abnormalities and do not have functional or structural deviations from the norm for that particular cell type.
  • non-pathogenic in relation to proliferation means that no pathogens are generated during the division process or by the cells resulting from this process.
  • autoinduce is meant a compound capable of inducing or stimulating its own synthesis.
  • animal cell proliferation induce is meant a compound capable of inducing and stimulating the division of animal cells, such as mammalian cells. It will be appreciated that the animal cell proliferation inducer is able to induce and/or intensify animal cell division in vitro (in cell culture) and in vivo.
  • the biologically active substance may be an autoinducer and/or an animal cell proliferation inducer. It will be appreciated that while all biologically active substances of the present invention are animal cell proliferation inducers, only some of them are also autoinducers.
  • apoptosis is primarily understood to be mitochondrial apoptosis.
  • “Immortalization” in the present context refers, as hereinbefore described, to the escape from the normal limitation on proliferation of the cell population in vitro or in vivo and is not related to the fate of individual cell and possibility of its eternal life.
  • Proliferation niche refers, as hereinbefore described, to a space- defined tissue area or a restricted volume of culture with proliferating cells with specific or specialized microenvironment, in which dividing cells support the proliferation functioning of a cell population.
  • Homeostasis is understood according the original definition given in Waddington, C. H. 1956. Principles of Embryology. See also Chuang, J. S. et al. PNAS, 2019, 116(30): 14852-14861 , meaning the stabilization in time of a chosen parameter value within a defined system.
  • the “homeorhesis” is understood as the capability of a system to maintain the value of chosen parameter following given dependency on time.
  • homeostasis refers to the static and homeorhesis- dynamic regulation over the chosen parameter in the system.
  • “Lysis” refers to the disintegration of a cell by rupture of the cell wall or membrane.
  • the term “extraction” encompasses conditioning of tissue or cells, such as cleaning, separation from damaged sections, homogenization and centrifugation, as well as more advanced separation stages (e.g. sedimentation, filtration and chromatography) and concentration (e.g. drying).
  • the extraction does not forcedly change the original structure of the extracted biologically active substance.
  • the extraction does not involve any chemicals other than water, isotonic and biological buffer solutions.
  • “Induction of proliferation or apoptosis” in the context of present invention means the stimulation of these processes, including the action leading to separately or jointly starting (literally induction) or stimulation (enhancement) of the proliferation and apoptosis.
  • the present invention relates generally to biologically active substances extracted from animal tissue or cell culture comprising non- pathologically proliferating cells, which substances simultaneously (i) induce and/or intensify cell proliferation; (ii) induce and/or intensify apoptosis; and (iii) have antibacterial activity.
  • said substances have anti-inflammatory activity.
  • the present disclosure is based on the inventors’ discovery and development of biologically active substances that advantageously can be used as cell proliferation inducers and/or enhancers, apoptosis inducers and/or enhancers, anti-inflammatory agents and/or antibacterial agents.
  • an antibacterial agent is to be understood to be a substance that, similar to antibiotics, reduces the growth of, or kills, undesirable bacteria (e.g. disease-causing microorganisms), which may include both gram-positive and gram-negative bacteria. See e.g., Example 5, which describes the antibacterial activity of BAS1 .
  • an “anti-inflammatory agent’ is to be understood as a substance that reduces inflammation, such as redness, swelling and pain, in the body, e.g. by blocking certain substances in the body that cause inflammation. See, for example, Example 4, which describes preclinical tests performed to assess the potential of BAS1 and other preparations containing BAS1 for tissue regeneration and inflammation prevention.
  • the present inventors have advantageously managed to discover and derive, e.g. by extraction and purification, biologically active substances that possess the above-mentioned desirable characteristics and are typically suitable for both in vitro and in vivo applications.
  • the biologically active substance of the invention has or comprises one or more characteristics selected from the group consisting of: a molecular weight of less than 1.5 kDa, long linear hydrocarbon chains, hydrocarbon fragments, steroid fragments, alkane fragments, high molecular weight unsaturated hydrocarbon fragments, phosphates, hydroxyl groups, amino groups and amide groups.
  • said biologically active substances have a molecular weight of less than 0.8 kDa, such as less than 0.7 kDa.
  • the biologically active substance described herein is preferably capable of shifting the cell cycle in a cell population such that there is an increased proportion of cells in the stages just before or just after cell division. In this regard, it will be appreciated that the higher the fraction of cells in the cell cycle stages immediately preceding and/or following the division, the younger is the cell population.
  • the biologically active substance of this aspect induces cell proliferation and apoptosis in the proliferation niche such that there is a correlation between induction and/or intensification of cell proliferation and induction and/or intensification of apoptosis.
  • the increase in the numbers of divided cells in a separated volume of the proliferation niche is accompanied with the correlated intensification of the apoptosis leading to the increased numbers of cells subjected to “programmed death”.
  • the biologically active substance is preferably not carcinogenic. Typically, the biologically active substance does not cause allergy reactions, does not show any, or only very mild, side effects and is typically compatible with most tested medicinal drugs.
  • the biologically active substance is BAS1 which specific composition and properties are described in Example 1 , including Figures 1 -7, and Tables 1 -5.
  • the biologically active substance is not BASI .
  • BAS1 has been exemplified herein, other biologically active substances are also encompassed by the invention as defined by the claims.
  • the cells that the biologically active substances are extracted from may be of any type of non-pathologically proliferating cells.
  • the proliferating cells may range in plasticity and may include, for example, blast cells, fertilized ova, non-fertilized gametes, embryonic stem cells, adult stem cells, precursor or progenitor cells, and highly specialized cells.
  • the biologically active substances are extracted from cells that possess one or more proliferation activation receptors. See, e.g., latropoulos M. J. and Williams, G. M. “Proliferation markers”. Review. Exp Toxicol Pathol. 48(2- 3): 175-81 , 1996.
  • a person of skill in the art will be familiar with current and more recently discovered proliferation activation receptors and cells possessing one or more of these receptors.
  • the substances are isolated from the tissue intercellular (interstitial) fluid without cell destruction. See, for example, Wu, W., Yenkie, K.M. & Maravelias, C.T. BMC Chem Eng 1 (21 ), 2019, or from the cells themselves through their destruction, followed by extraction, typically, but not exclusively, with a liquid agent. See, e.g., “Natural Bioactive Compounds. Technological Advancements”. Academic Press, 2020. Pages 409-433, Chapter 21 - “Advances in extraction technologies: isolation and purification of bioactive compounds from biological materials”.
  • the destruction of cells is achieved by osmotic destruction of membranes, also referred to as “lysis”, under the action of enzymes, compression of tissues or homogenization of varying degrees of intensity. Freeze-thaw cycles and/or ultrasound may also be used to destruct the cells.
  • the substances may be extracted with a liquid agent, such as water or an aqueous solution (with varying acidity), or with organic compounds or complex mixtures at different temperatures.
  • a liquid agent such as water or an aqueous solution (with varying acidity), or with organic compounds or complex mixtures at different temperatures.
  • the extract is subsequently filtered or separated by other methods from undissolved sediment.
  • a centrifugation step is performed after extraction to efficiently separate the phases. Extraction from animal tissue
  • the tissue Prior to extracting the biologically active substances from animal tissue, the tissue is typically preliminarily crushed or separated to remove connective tissues, fat and defective fragments. Homogenization is typically carried out with a homogenizer, e.g. a paddle homogenizer, with the addition of a cold or warm extracting agent. Suitably, homogenization is repeated if fragments of non-homogenized tissue remain. In some cases, additional agents may be added to the homogenate and mixed with it. Centrifugation of mixtures is typically undertaken at 10,000 g for 30-60 minutes. The extract is subsequently filtered through specialized filters, gauze, filter paper or glass wool.
  • a homogenizer e.g. a paddle homogenizer
  • liquid extraction is accompanied by sparging with gases, sonication, or microwave irradiation.
  • crystallization and various sorption methods using molecular or ionic sorbents are used to isolate substances from the extract.
  • the most selective sorption separation method used with liquid extracts is liquid chromatography.
  • Testing for the induction of proliferation is performed through testing the differences in the expression levels of genes that control cell proliferation without (control) and with (test) corresponding inducer compound present or added (Whitfield, M. et al., Nat Rev Cancer 6, 99-106, 2006).
  • an autoinductive factor or autoinducer is capable of inducing or stimulating its own synthesis.
  • autoinduction of the respective biologically active factors means that the presence of these factors induces and intensifies cell division.
  • these factors are synthesized, thereby supporting the process of division and “production” of these factors.
  • the more intense cell division is, the more intensely the divisioninducing factor is synthesized.
  • testing for the autoinductive properties of the factor can be performed by monitoring its concentration dynamics in the closed volume containing corresponding growth media and proliferating cells. If in such closed volume cell division proceeds and the concentration of the proliferationinducing factor is stable or grows, it indicates autoinduction. If the concentration of the inducing factor in the presence of continually proliferating cells is at least stable, but in the presence of corresponding growth media and absence of proliferating celis its concentration falls with time (due to some factor degradation or destruction mechanisms, including but not limited to the oxidation or chemical reactions with the components of growth media), it also points to the presence of autoinduction.
  • apoptosis is a form of programmed cell death that occurs in multicellular organisms and cell cultures. Biochemical events lead to characteristic cell changes and death. These changes include blabbing, cell shrinkage, nuclear fragmentation, chromatin condensation, DNA fragmentation, and mRNA decay (see, e.g. Programmed Cell Death (Apoptosis). Alberts B, Johnson A, Lewis J, et al. Molecular Biology of the Cell. 4th edition. New York: Garland Science, 2002).
  • Apoptosis is a highly regulated process that can be initiated through one of two major pathways. In the intrinsic pathway, the cell kills itself because it senses cell stress, while in the extrinsic pathway the cell is eliminated because of external signals. Weak external signals may also activate the intrinsic pathway of apoptosis (Alberts B et al., 2008).
  • apoptosis is primarily meant mitochondrial apoptosis.
  • Induction or enhancement of the cell proliferation or apoptosis is performed through testing and quantification of the samples taken from the cell population by any accepted methods, preferably by flow cytometry.
  • samples are taken from the cell population before and after it is subjected to the action of any substance or activation method claiming the capacity for cell proliferation or apoptosis enhancement or induction.
  • any substance or activation method claiming the capacity for cell proliferation or apoptosis enhancement or induction.
  • multiple samples from the cell population are taken at regular time intervals after the action of the tested substance or the activation method.
  • Another method for assessing the apoptosis induction/enhancement in vitro uses the monitoring of cell debris in the sample with cells suspended in the liquid media. Corresponding optical density of the sample according to the methods can be used as a quantitative measure or as a qualitative indicator (e.g. Image-Based Quantification of Cell Debris as a Measure of Apoptosis, M. Olander ef a/. Anal. Chem. 91 : 5548-5552, 2019).
  • tissue samples extracted before and after the method claiming the actions changing the cell properties in the tissues are using the cells extracted from the blood.
  • Corresponding method uses the samples, collected before and after the action, claiming rejuvenating activity, and analyzing the relative proportions of the cells in different cycle stages.
  • Corresponding samples can be tested immediately after extraction, or kept for a certain time in the cell culture state.
  • Conclusion on the presence/absence of claimed rejuvenation or anti-aging activity and intensity of such action can be derived from the changes in relative proportion (percentage) of the cells in the division cycle stages just preceding or just following the division.
  • Methods of testing substances for the antibacterial properties are well established and may include Diffusion methods (Agar disk, Agar well and Agar plug-diffusion methods, Antimicrobial gradient method or Etest, Cross streak method, Poisoned food method); Thin-layer chromatography (TLC)- bioautography methods (Agar diffusion, Direct bioautography, Agar overlay bioassay); Dilution methods (Broth dilution method, Agar dilution method); Time-kill test or time-kill curve method; ATP bioluminescence assay and Flow cytofluorometric method. All these methods are well described in a number of publications and manuals, and will be familiar to the skilled person.
  • fragment is a shorter or truncated portion of the biologically active substance that essentially retains the activity and function of the substance.
  • an analog or a derivative is a substance with slight variations in its composition that essentially retains the same activity and function as the biologically substance described herein.
  • composition comprising the biologically active substance, or the fragment, analog or derivative, and at least one pharmaceutically acceptable excipient or carrier.
  • composition is to be used in its widest sense, encompassing all compositions comprising at least one biologically active substance, or fragment, analog or derivative thereof, and optional excipients, carriers, adjuvants, constituents etc.
  • a method of supporting, intensifying and/or activating proliferation of animal cells in vitro comprising the step of contacting said cells with one or more of the herein defined biologically active substance, fragment, analog, derivative or composition.
  • the method suitably comprises the step of contacting a target cell or cells with the biologically active substance defined herein, including but not limited to BAS1 , such that the proliferation factor induces or promotes the proliferation of the cells.
  • the substance is added directly to the cell culture.
  • the contact with the specified substance is indirect, for example, when the media containing the substance or the media containing the substance and the cell culture are separated by a semi-permeable membrane allowing only the passage of relatively small molecules, including the biologically active substance.
  • a cosmetic method of supporting, intensifying and/or activating proliferation of human cells in vivo comprising the step of contacting said cells with one or more of the biologically active substance, fragment, analog, derivative, or composition described herein.
  • a biologically active substance a fragment, analog or derivative, or a composition for use as a cell proliferation inducer and/or enhancer, an apoptosis inducer and/or enhancer, an anti-inflammatory agent or an antibacterial agent.
  • a biologically active substance, fragment, analog or derivative, or composition for use as implant coating may be used for coating any type of implant that has been implanted or surgically placed in the human body.
  • Said implant may be made of any type of material, such as metals and alloys, ceramics, polymers and composites.
  • said implant is a dental implant.
  • Example 4 describe preclinical in vitro ( Figures 17-19) and in vivo ( Figures 20-21 ) tests performed to assess the potential of the preparations containing BAS1 for tissue regeneration and inflammation prevention. The aim of these studies was to assess the potential of preparations comprising BAS1 for coating of the implants and preventing and treating inflammations associated with implantation.
  • a method of long-term storage of the biologically active substance, or the fragment, analog or derivative using drying and/or lyophilization and sorption on biopolymers such that when not exposed to the direct sunlight or UV-light, said lyophilized substance can be stored in dry conditions at room temperature without degrading its activity for at least 6 months, such as for at least 12 months.
  • a process of extracting a biologically active substance from non-pathologically dividing cells or cell cultures comprising the steps of (i) prior to extraction, adding the biologically active substance, the fragment, analog or derivative, or the composition to said cells or cell cultures; and (ii) extracting the biologically active substance, wherein said addition in step (i) improves the yield of the extracted biological substance.
  • the added biologically active substance may be the same as or different to the extracted biologically active substance.
  • said addition in step (i) improves the yield of the extracted biological substance compared to if it had been extracted by the process without step (i).
  • a method of identifying a substance suitable for use as a cell proliferation inducer and/or enhancer, and apoptosis inducer and/or enhancer, an anti-inflammatory agent and/or an antibacterial agent comprising (a) providing a test substance; and (b) assessing whether the test substance shifts the cell cycle in a cell population such that there is an increased proportion of cells in the stages just before or just after cell division, wherein a test substance that shifts the cell cycle is suitable for use as a cell proliferation inducer and/or enhancer, an apoptosis inducer and/or enhancer, an anti-inflammatory agent and/or an antibacterial agent.
  • the biologically active substance of the invention may be extracted from homogenized animal tissues or cell cultures containing actively and non-pathologically proliferating cells, followed by centrifugation, freezing-thawing, filtration and separation of the supernatant into fractions.
  • said extraction is performed by liquid chromatography with a carrier in the form of pure water or isotonic sodium chloride solution. Said extraction may be performed in a liquid phase using organic solvents.
  • the biologically active substances of the invention may be used in various applications, and in particular for the intensification and/or continuous support of cell division in tissues, and long-term maintenance of functionally competent cell cultures; in biofactories (increasing productivity of biosynthesis); in laboratory research; for the preservation and production of cells and tissues in cell therapy and reconstructive surgery; in bioprinting (three-dimensional printing of tissues and organs); for extracorporeal division activation of cells taken from a patient with their subsequent return to the body; in medicine and in the production of drugs and other agents (in particular, anti-aging drugs, enhancers and restorers of immunity, wound healing agents, new families of antibiotics); in substances, tinctures, gels and ointments for promoting and enhancing tissue regeneration and wound healing; in activating and functionalization of coatings for implants (orthopedics, dentistry); in cosmetics and cosmetology (preparations for skin care and skin revitalization); and in veterinary applications. Continuously cultured cell lines will also be invaluable as a
  • BAS1 A substance with the properties of proliferation inducer (including autoinducer) and simultaneous enhancer of programmed cell death (apoptosis) was extracted, and studied. Along with the two properties of supporting and most probably defining the homeostasis in the proliferation niche, BAS1 also possessed strong antibacterial properties towards both gram-positive and gram-negative bacteria, and in some applications anti-inflammatory activity. Dried BAS1 is in a form of irregular shape crystallites with light brown or light amber color. BAS1 has excellent solubility in water at 20°C (better than 200 mg/ml) and rather limited solubility in alcohols and oils. Toxicology tests of BAS1 in vitro and in vivo have shown its biocompatibility and the absence of cytotoxicity.
  • BAS1 is not carcinogenic and that it has no tendency to promote existing lesions (e.g., it does not induce cancer and does not aggravate existing cancer).
  • Preclinical tests have shown that BAS1 and preparations containing it do not cause allergy reactions, does not show any side effects and is compatible with most, if not all, tested medicinal drugs.
  • BAS1 was used in dried form, in combination with gels or solutions. BAS1 can be used both in neutral solutions (pure water, standard biological buffers, isotonic sodium chloride solution, etc.), and in different media with a pH from about 1 .5 (human stomach) to about 9. Verification of the capacity for long-term storage of the substances with certain biological activity was carried out using, for example, the methods listed below.
  • a liquid or gel-based sample with known biological activity was stored in one of the recognized ways (at different temperatures, in the light or in the dark, flashed with an inert gas, nitrogen or CO2, or without it etc.) and the corresponding activity after certain periods tested.
  • a liquid, gel-based or solid sample with known biological activity was frozen in test vials and stored in one of the recognized ways at various negative temperatures. After certain periods, the sample was thawed, and its activity checked.
  • BAS1 induces and/or intensifies non- pathological and non-pathogenic cell proliferation.
  • Figure 3A shows IR absorption spectrum of the crystalline BAS1 taken in dry chamber under argon atmosphere. Corresponding conditions were chosen to exclude the potential background from NaCI. Analysis summary
  • Figure 3B-G shows NMR spectra of BAS1 in solution. Both 1 H and 13 C NMR spectra were acquired with long accumulation time to provide better signal-to-noise ratio. In 1 H NMR spectra narrow single lines around 1.5 ppm commonly belong to CH3 groups; the ones around 2.8-3.0 ppm commonly belong to NCH3 groups. 31 P NMR spectrum indicates the presence of phosphate groups, possible in a number of different surroundings.
  • Table 1 provides a summary of the EDX data taken from different sites of individual crystallites (Figure 4 shows the analysis at the site SO) and control (pure NaCI crystals). Sites SO and S1 do not show abundant organic content, and for the analysis of organic substances these data are inconclusive. Sites with organic (carbon) presence do not show balanced content of chlorine and potassium that is characteristic to free NaCI. Site SO shows Na and Cl balance close to pure NaCI with some presence of oxygen, but no organic content. Table 2 provides content analysis data averaged over the sites S1 to S9.
  • Organic part of the substance according to EBSD and EDX is dominated by carbon, oxygen and sulphur. Such atoms like hydrogen and nitrogen are not easily showing in the common EBSD and EDX spectra, so no conclusion on their presence and abundance in the samples can be drawn.
  • Inorganic part is represented by sodium with smaller amounts of potassium and magnesium, and even smallest amounts of calcium and silicon. Presence of Ca, Si and Mg may be a reconstruction artefact or may be due to impurities coming from the sample holding glass plate.
  • XRD and other X-ray diffractometry results are dominated by the features of pure NaCI, unlike the results of EDX analysis. It can be explained by the fact that EDX is capable of only shallow (mainly surface) analysis as the electrons have much smaller penetration depth as compared to X-rays. Liquid chromatography
  • FIG. 5 presents HPLC spectra of BAS1.
  • Table 3 presents suggested automatic interpretation of the compounds (fragments) using most intense peaks as semi-automatically generated by the embedded instrument features using linked substance databases.
  • analysis suggests presence of certain compounds that is not possible to identify with reasonable probability.
  • Figure 6 shows two more examples of BAS1 HPLC spectra (graphs) while Table 4 below shows suggested automatic interpretation using most intense peaks done by the embedded instrument features using linked substance databases.
  • Liquid chromatography results are rather inconclusive. It is partially because HPLC methods of detection are sensitive to both the present molecules and their fragments. Therefore, analysis reveals the presence of long hydrocarbon chain aliphatic compounds with different active groups (acids, ethers). There are indications of the presence of basic amino acids, peptides, complex amines, phosphates and pyrophosphates. There is a presence of human hormone precursors (squalene, pregnane) and their derivatives. The most useful is the conclusion about the largest possible molecules (highest molecular weight) of the substances present in the test samples.
  • Aromatic compounds were not present at all or identified in trace amounts. Analysis using linked databases revealed a significant number of unidentified compounds.
  • Figure 7A-C show the elements of automatically generated report that presents the results of the automated identification carried by the system.
  • Cytoskeletal domain Junction plakoglobin; Keratin, type I cytoskeletal;
  • Extracellular domain Annexin; Cytoplasmatic Actin.
  • Glycolysis domain glyceraldehyde-3-phosphate dehydrogenase; pyruvate kinase; 2-phospho-D-glycerate hydro-lyase.
  • Ion transport Voltage-dependent anion-selective channel protein 1 .
  • BAS1 is most likely present or contained as an active part a mineral-organic complex having a complicated composition with the presumable presence of the following organic molecular fragments:
  • Figure 8A-D shows experimental data illustrating the changes induced by BAS1 upon the duration of the phases of the cell cycle in the culture of human dermal fibroblasts studied by flow cytometry.
  • Figure 9A-D shows experimental data illustrating the changes induced by BAS1 upon the cell distribution over the cell cycle phases and cell vitality as studied by flow cytometry for human blood mononuclear cells in cell culture. Corresponding dependencies show the changes happening with addition of different concentrations of BAS1 as compared to control.
  • Figure 11A-D show typical changes induced by BAS1 in cell cycle distribution of the human mononuclear blood cells (volunteers). Mononuclear cells were isolated from venous blood and then cultured. See below for the corresponding procedure of mononuclear cells isolation.
  • Figure 12A-D present the measurements of the human blood mononuclear cell cycle distribution changes performed with 18 male and 18 female volunteers between 16 and 86 years of age according to the method described in Russian patent no. 2728601 summarized above.
  • Mononuclear cell extraction from the venous blood can be performed according to any protocol accepted in the microbiology.
  • the protocol used in the present case is outlined in Figure 13.
  • Venous blood is taken from the ulnar vein in the morning before any meals. The tests were conducted with volunteers between the ages of 16 and 86.
  • the separation of mononuclear cells from the whole blood is performed by gradient centrifugation using DIACOLL-1077 (density gradient cell separation medium).
  • the heparinized blood is diluted 3-fold with the Versene solution (an EDTA solution, a non- enzymatic cell dissociation reagent), then layered on top of the DIACOLL-1077 density gradient and centrifuged at 300 G for 45 minutes.
  • the interphase containing mononuclear cells is collected and washed with RPMI-1640 medium three times.
  • Human dermal fibroblast cultures were prepared using skin biopsies from volunteers aged 18 to 86 years according to the following protocol.
  • a uniform consistency substance obtained from the skin was placed in a DMEM medium with type II collagenase (Gibco No. 17101 -015, USA) at a concentration of 1 mg/ml was placed in a CO2 incubator at 37°C for 1 .5 hours. Periodically, every 15-20 minutes, the tube was shaken.
  • the DMEM medium with 10% FBS fetal calf serum
  • FBS fetal calf serum
  • the culture was added to the Petri dishes (10 cm in diameter) at a density of 1x10 5 -2x10 5 cells/cm 2 and placed under standard incubation conditions. Standard cultivation was carried out at 37°C in an atmosphere of 5% CO2 using a CO2 incubator (Sanyo, Japan).
  • fibroblasts were incubated for 7 days until the formation of a complete monolayer. At this stage, microscopic examination was carried out to separate skin fragments from the attached fibroblast cells. Harvested tissue microexplants serve as additional sources of fibroblasts.
  • Optical inspection of fibroblasts and visualization of their preparations fixed and stained according to Romanovsky-Giemsa were performed using an inverted microscope CKX-41 (Olympus, Japan). Cell viability after each experiment was assessed by microscopy by staining them with trypan blue. Control over the processes of apoptosis and necrosis was carried out by assessing the number of apoptotic and necrotic cells by flow cytometry on a CytoFLEX flow cytometer (Beckman Coulter, USA) using the ANNEXINV- FITCKit (Immunotech, France) according to the manufacturer's instructions. Corresponding method allows the identification of living, necrotic and apoptotic cells.
  • Immunophenotyping of cultured fibroblasts was performed with a CytoFLEX flow cytometer (Beckman Coulter, USA) using monoclonal antibodies to CD29, CD44, CD90, CD105 (markers specific for MSCs), and CD34, CD45 (markers of hematopoietic cells).
  • the resulting suspension of cells was diluted with a complete culture medium at the rate of 1 million cells in 1 ml of medium, and a 96-well plate was filled for studying the phases of the cell cycle.
  • control 24 wells (200 pl of RPMI-1640 with 10% fetal calf serum is added to 200 pl containing 200,000 cells.
  • the content of the vials is transferred to cytometry.
  • the animals were divided into 4 groups of 20 rabbits each.
  • the edges of the wounds were treated with a Lugol’s iodine solution, the wound was treated with a 3% hydrogen peroxide solution, dried, and then dressing was performed after applying a substance specific for each group or no substance for control group: Group 1 , control 1 , gel base only;
  • control 2 traditional treatment (in the inflammation phase - traditional water-based healing preparations; in the regeneration phase - traditional fatbased healing preparations);
  • FIG. 14 illustrates the differences in the intensity of cell proliferation and apoptosis in the control group (no treatment), and in three test groups with the treatment using: traditional method and using the BAS 1 -hyaluronate gel composition and hyaluronate gel only.
  • Figure 15 illustrates the differences in the intensity of cell proliferation and apoptosis in the control group (with no treatment, CO, control) and the group treated with gel only (Gl).
  • Figure 16 illustrates the differences in the intensity of cell proliferation and apoptosis in the groups treated in traditional way (TR) and with gel, and BAS1 + hyaluronate gel composition (BAS1 +GI). Corresponding data indicate a serious potential of preparations containing BAS1 for the intensification of tissue regeneration.
  • NTS gel is a commercial hyaluronate-based composition designed and used for supporting tissue regeneration and guided bone regeneration in oral implantology.
  • Figure 17 presents the results of the WST-1 vitality tests in the case of undiluted and 1 :10 diluted tested substances.
  • Figure 18 presents the results of the effect of tested substances on stimulating spontaneous osteogenic differentiation (measurements based on Koss densitometry).
  • Figure 19 shows analysis of alkaline phosphatase activity for tested substances as compared to control.
  • test materials 0.02 ml I injection
  • results of the treatment were also assessed using histological studies and micro-CT.
  • induction of the periodontitis was only performed at one side of the animal jaw teeth.
  • Tissue samples for the histologic and staining analysis were removed under local anesthetic after 31 days of treatment.
  • Figure 20 presents the results of inflammation assessment after 31 days of treatment.
  • Control group was injected with saline solution, test groups- with the solution of hyaluronate (HYL carrier), solution of hyaluronate with BAS1 and NST solution (NST). Induction of the periodontitis was performed at the left side. Micro-CT was used to assess the long-term results of the treatment. Persisting inflammation leads to some bone loss, and the distance between the cemented enamel junction (CEJ) and the edges of the alveolar bone increases. Corresponding distances were measured after four weeks of treatment at seven positions of the lower jawbone. Effect of the treatment by BAS1 with hyaluronate carrier was at least as good as that of the NST treatment. Tissue histology was performed after 31 days from the treatment initiation.
  • BAS1 -based composition is superior to the other two in its effect on PDLSC cells. BAS1 -based composition also proved to be most effective in spontaneous osteogenic differentiation. Judging by cell adhesion, cytokine production and apoptosis markers, the use of BAS1 -based composition has no side effects. Among other drugs used, BAS1 -based composition showed the most significant changes in the expression level of the hyaluronic acid receptor CD44.
  • BAS 1 -hyaluronate composition demonstrates significant potential for effective treatment and healing of severe lesions.
  • BAS1 -based compositions When used together with high molecular weight hyaluronates drying to the glass consistency BAS1 -based compositions have a significant potential as a bioactive coatings for implants facilitating better implant integration and preventing certain side effects such as inflammation.
  • Figures 21A-B present the comparison of the treatment results using BAS1 - hyaluronate composition and commercial composition FlexBarrier, and comparison between both results.
  • Antibacterial activity of BAS1 at different concentrations was tested using eight database strains of gram-negative and gram-positive bacteria (Escherichia coli K12, E. coli 25922, Pseudomonas aeruginosa 27853, Staphylococcus aureus 25923, S. aureus 6538P, S. xylosus, S. epidermidis 711 , Micrococcus luteus var. lysodeikticus 15307) and 120 clinical strains of gram-negative microorganisms highly resistant to antibiotics (22 strains of Escherichia coli, 55 strains of Acinetobacter baumannii and 43 strains of Pseudomonas aeruginosa).
  • Suspension of the bacterial cells taken from the one-day cultivated in agaric media in the isotonic solution was used. 25 pl of suspension is introduced into the wells of a plastic 96-well sterile plate, and 25 pl of BAS1 water solution is added to the wells (concentrations of 50, 100, 150 and 200 pg/ml). It corresponds to the final BAS1 concentration of the wells of 25, 50, 75 and 100 pg/ml. 25 pl of isotonic NaCI solution were added to the control wells. After that, the plates were incubated for 20 minutes at 37 °C, and then 200 pl of cultivation broth is added to all wells.
  • Antibacterial activity of BAS1 are presented in Tables 6-8 using the following parameters:
  • BAI Bactericidal Activity Index
  • BAI (ODc - ODe) / ODc * 100%, where ODc and ODe are the optical densities of the control and experimental cultures, respectively. Strains with BAI> 10% were considered susceptible for the antibacterial action of BAS1 .
  • the primary source (raw material) for obtaining biologically active substances from animal raw materials according to present invention is any tissue or cell culture with non-pathologically proliferating cells. 2. Defective agglomerates or tissue areas are identified by visual inspection and removed.
  • the washed tissues are placed in fresh isotonic solution and homogenized at room temperature with a Medi machine automatic mechanical tissue homogenization system (Becton Dickinson). When using cell culture, the homogenization step is omitted.
  • a Medi machine automatic mechanical tissue homogenization system Becton Dickinson
  • the suspension is centrifuged for 25 minutes at 1500 rpm and filtered. The filtered supernatant is frozen.
  • the completely frozen supernatant is thawed at room temperature.
  • the supernatant is centrifuged for 25 minutes at 1500 rpm and separated from the residue by filtration for later use.
  • the collected purified supernatant is separated into fractions by liquid chromatography. Chromatographic separation is carried out using most gentle liquid carriers, such as isotonic solution or distilled water. 10. Fractions corresponding to the characteristic peaks of the chromatographic separation are collected and each fraction is tested for specific biological activity.
  • Corresponding fraction containing BAS1 is selected using the test for the characteristic set of activities, namely the influence upon cell proliferation and apoptosis and antibacterial properties.
  • Examples of possible tests for biological activity of biologically active compounds acquired by the described method may include (but are not limited to): tests for the influence on cell proliferation or apoptosis; antimicrobial activity test (verification is carried out using a standard method using appropriate bacterial cultures); antimycotic or antifungal activity test (verification is carried out in a standard manner using appropriate fungal cultures); antibacterial activity tests.
  • BAS1 possesses specific biological activity, namely influence upon cell proliferation and apoptosis.
  • Corresponding example outlined below describes one of the possible methods of testing used.
  • Such test can be performed for any substance, having the activity similar to that of BAS1 .
  • Particular example refers to BAS1 for better clarity.
  • the test is performed by using the isolated fraction to the cell culture (for example, human fibroblasts, and human blood mononuclear cells).
  • the original cell culture is divided into two identical lines maintained under identical conditions; one is used to check the activity of the corresponding fraction, the other for control.
  • Corresponding amount of tested fraction is added to the cell culture, and amount of the sediment (dying cells and debris) is assessed.
  • Comparison may include an assessment of the number of active cells and associated debris, an assessment of cell viability, an assessment of the life span of cells in culture.
  • One of the definitive options of comparison is carried out by determining the ratio of the number of cells in different stages of the cell cycle, for example, using flow cytometry or method similar to that described in Russian patent no. 2728601 (Method for testing substances affecting aging processes by blood analysis).
  • Flow cytometry methods allow for both qualitative determination of the presence of substance with chosen biologic activity (activities), and for the quantification of such activity (activities).
  • Mononuclear cells and human fibroblasts were extracted according to the protocols described in EXAMPLE 2 (Mononuclear cell extraction; Preparation of the human dermal fibroblast cultures).
  • the graphs shown in Figures 22A-H represent the data acquired for the mononuclear cells received from a group of 80 healthy volunteers aged between 15 and 61 years.
  • the substance BAS1 has five features that are simultaneously exhibited: i. substance induces/enhances proliferation ii. substance induces/enhances apoptosis iii. induction/enhancement of proliferation and that of apoptosis are coupled supporting the homeostasis in the cell population (e.g. supporting cell number maintenance) iv. substance has the property of autoinduction (self-amplification) v. substance has antibacterial activity against gram-positive or gramnegative microorganisms or against both.
  • BAS1 and exosomes are both derived from the supernatants extracted from the cultures of actively dividing cells and some of the properties reported for the exosomes coincide with those of the BAS1 .
  • BAS1 is not a membrane-based vesicle. BAS1 does not lose its activity after drying and soldering. Extraction yields of BAS1 dramatically exceed those of exosomes. Although some of the properties of BAS1 are aligned with those of the extracellular vesicles, the simultaneous presence of the key features of BAS1 , namely synchronized intensification of the proliferation and apoptosis, antibacterial activity, and especially the autoinduction, has not been reported for exosomes.

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Abstract

La présente divulgation concerne, entre autres, des substances biologiquement actives extraites d'un tissu animal ou d'une culture cellulaire comprenant des cellules proliférant de manière non pathologique, lesquelles substances, de manière simultanée, (i) induisent et/ou intensifient la prolifération cellulaire; (ii) induisent et/ou intensifient l'apoptose; et (iii) ont une activité antibactérienne, et des procédés d'extraction desdites substances. La présente divulgation concerne également l'utilisation de telles substances biologiquement actives.
PCT/EP2023/082116 2022-11-17 2023-11-16 Nouveaux composés et procédés Ceased WO2024105193A1 (fr)

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