METHOD OF OBTAINING STABLE SUSPENSIONS OF HETEROCRYSTALS OF TITANIUM DIOXIDE OR PARTICLES OF SILICON DIOXIDE AND STABLE SUSPENSIONS OBTAINED BY THIS METHOD FOR INITIATION OF ACTIVE FORM OF OXYGEN IN BODY AT USE IN MEDICAL FORMS
20240358746 ยท 2024-10-31
Inventors
Cpc classification
B01F23/04
PERFORMING OPERATIONS; TRANSPORTING
B01F23/56
PERFORMING OPERATIONS; TRANSPORTING
B01F23/511
PERFORMING OPERATIONS; TRANSPORTING
International classification
B01F23/00
PERFORMING OPERATIONS; TRANSPORTING
Abstract
The method of obtaining stable suspensions of heterocrystals of titanium dioxide and particles of silicon dioxide representing special class of quantum dots (QD). and stable suspensions obtained in such a way for initiation of active form of oxygen in the human body in use in medical forms. Starting material: Initial stuff in the form of aggregates with size more than 0.5 micrometer is mixed with an aqueous solution of pharmaceutical inorganic acid, with subsequent direction to homogenizing for the first stage of mixing, after that the obtained aqueous suspension is subjected to thermal treatment and, then aqueous suspension is directed to the rotary rotor-type evaporator periodically for evaporation of inorganic acid with suspension expense trough the rotor-type evaporator no more than 25 l/min and then the obtained activated particles are mixed with water in hydrodynamical cavitation-wave cavitational homogenizer to quasi-with regulated pulsating wave mode until obtaining stable suspension of heterocrystal of titanium dioxide or particles of silicon dioxide with size less than 450 nm, and presence on the lattice surface up to 60-80% of electronically-excited triplet oxygen .sup.3+TO.sub.23O2 in the energy centers, namely, in the quantum dotszones of local overheating, ensuring heat synthesis catalytic activity for formation of active forms of oxygen in the living organism human body.
The surface of Stable suspension obtained by said method is characterized by distribution of activated crystals of titanium dioxide or with size up to 1 nm being 0.3 vol %, up to 20 nm being 5-40 vol %, particles with size up to 80 nm being 10-80 vol %, particles with size up to 150 nm being 5-30 vol %, particles with size up to 250 nm being 5-20 vol %, particles with size more than 250 nm-no more than 10 vol %, and distribution of activated particles of silicon dioxide with size 40-80 nm being 10-80 vol %, particles with size 80-150 nm being 10-80 vol %, particles with size 150-250 nm being less than 30 vol %, particles with size more than 250 nmno more than 15%. The surface of heterocrystals of titanium dioxide and particles of silicon dioxide has sorption ability, that is an important factor for use in medical forms, ensuring detoxication of an organism, elimination of hypoxia, antiviral effect of a medical agent, antipathogenous effect in the body of living organism and elimination of under oxidation processes in the human body, increasing induction of immune response of vertebrata.
Claims
1. The method of obtaining stable suspensions of heterocrystals of titanium dioxide or particles of silicon dioxide characterized by the fact that the starting material is in the form of aggregates with size more than 0.5 micrometer is mixed with an aqueous solution of pharmaceutically acceptable acid, with subsequent direction to homogenizing for the first stage of mixing, after that the obtained aqueous suspension is subjected to thermal treatment, then aqueous suspension is directed to the rotary rotor-type evaporator working under pressure lower than 100 kPa at temperature no more than 70 C. 70 C. for evaporation of pharmaceutically acceptable inorganic acid with suspension expense trough the rotor-type evaporator no more than 25 l/min and then the obtained activated particles are mixed with water in hydrodynamical cavitational homogenizer with regulated pulsating wave mode until obtaining stable suspension of heterocrystals of titanium dioxide or particles of silicon dioxide with size less than 450 nm, and presence on the lattice surface 60% up to 80% of electronically-excited triplet oxygen .sup.3+TO.sub.23O.sub.2 in the energy centers, namely, in the quantum dotszones of local overheating, ensuring catalytic activity for formation of active forms of oxygen in the human body of a living organism.
2. The method of obtaining stable suspensions of heterocrystals of titanium dioxide or and particles of silicon dioxide according to the claim 1, characterized in the distinction it that the obtained aqueous suspension of heterocrystals of titanium dioxide or particles of silicon dioxide is periodically directed for thermal treatment to the ultrasonic bath with ultrasonic frequency 20-90 kHz and keep it at temperature no more than 70 C. during no more than 2 hours.
3. The method of obtaining stable suspensions of heterocrystals of titanium dioxide or and particles of silicon dioxide according to the claim 1, characterized in the distinction that the obtained activated particles TiO.sub.2 or SiO.sub.2 are mixed with water in the hydrodynamical cavitational homogenizer with regulated pulsating wave mode until obtaining stable suspension of heterocrystals of titanium dioxide or particles of silicon dioxide from 0.0001-10 mass % concentration in suspension condition.
4. The method of obtaining stable suspensions of heterocrystals of titanium dioxide or and particles of silicon dioxide according to the claim 1, characterized in the distinction is that in the cavitational homogenizer a flow of mixed medium passes through the first block of preliminary mixing and further through the block of cavitational homogenizing, and then through the block with possibility of regulation in a flow of the ingredient dosing with subsequent periodical direction into the block of regulated output of homogenized product subjected to pulsation-wave homogenizing, at that the pulsation chamber of wave mixing is executed with regulated reflector of flow mixture, installed at the chamber output.
5. The method of obtaining stable suspensions of heterocrystals of titanium dioxide or and particles of silicon dioxide according to the claim 1, characterized in the distinction that activated crystals TiO.sub.2 or particles SiO.sub.2 are obtained with presence in structure of oxygen from 60% up to 80% in metastable electronically-excited third state triplet condition .sup.3+TO.sub.23O.sub.2, at that the particles TiO.sub.2 have Zeta-potential +30-+15 mV, and the particles SiO.sub.2 have Zeta-potential 20-15 mV and are characterized with presence of sorption properties.
6. Stable suspension of heterocrystals of titanium dioxide and particles of silicon dioxide obtained according to the claim 1 characterized by distribution of activated crystals of titanium dioxide with size up to 1 nm being 0.3 vol %, up to 20 nm being 5-40 vol %, particles with size up to 80 nm being 10-80 vol %, particles with size up to 150 nm being 5-30 vol %, particles with size up to 250 nm being 5-20 vol %, particles with size more than 250 nm-no more than 10 vol %, and distribution of activated particles of silicon dioxide with size 40-80 nm being 10-80 vol %, particles with size 80-150 nm being 10-80 vol %, particles with size 150-250 nm being less than 30 vol %, particles with size more than 250-450 nm-no more than 15%, and with presence on the lattice surface from 60% of up to 80% of electronically-excited third state oxygen .sup.3+TO.sub.23O2 in the energy centers, namely, in the quantum dots-zones of local overheating, ensuring catalytic activity for formation of Active Forms of Oxygen in the human body of living organism.
7. Use of stable suspensions of heterocrystals of titanium dioxide or and particles of silicon dioxide obtained according to the claim 6 in medical agents and in combination with other active pharmaceutical components, intravenously, intramuscularly, orally, nasally, vaginally, rectally, locally (ENT) use or topically, at that therapeutically important amount of an active substance of crystals or particles is for use: intravenous from 0.006 mg/ml, intramuscular from 0.01 mg/ml, subcutaneous from 0.1 mg/ml, orally from 0.01 mg/ml, nasally from 0.01 mg/ml, vaginally from 0.01 mg/ml, rectally from 0.01 mg/ml, local ENT use from 0.01 mg/ml, ocular use from 0.001 mg/ml, topical from 0.0003 mg/ml, as well as mucous, membranes, subcutaneous, intravascular, capillary, droplet, transmembrane and dermal, intracellular injection including locally into cell structures.
8. Use of stable suspensions of heterocrystals of titanium dioxide and particles of silicon dioxide according to the claim 6 as thermocatalystscatalysts of conversion of oxygen from triplet .sup.3+TO.sub.2.sup.3O.sub.2 into singlet .sup.1-3SO.sub.2 condition O2S1-3 condition at the expense of energy of thermal energy, including physiological temperature of from 36.6 C. in the human body and/or photonic hyperthermia and/or wave, and electromagnetic and resonant radiation, ensuring energy needed for conversion of oxygen from triplet into singlet condition for synthesis of active forms of oxygen in the human body.
9. Use of stable suspensions of heterocrystals of titanium dioxide and particles of silicon dioxide according to the claim 6, the distinction is that for induction of formation of active forms of oxygen in the inflammation focuses in presence of NAD+ enzyme of immune cells target delivery of said stable suspension is ensured into the areas of inflammation localization.
10. Use of stable suspensions of heterocrystals of titanium dioxide and particles of silicon dioxide according to the claim 6, characterized by the fact that antiviral effect of a medical agent is ensured.
11. Use of stable suspensions of heterocrystals of titanium dioxide and particles of silicon dioxide according to the claim 6, the distinction is that antipathogenous antiviral effect of a medical agent is ensured.
12. Use of stable suspensions of heterocrystals of titanium dioxide and particles of silicon dioxide according to the claim 6, the distinction is that sorption ability of the crystals and particles in a medical agent ensures detoxication of organism.
13. Use of stable suspensions of heterocrystals of titanium dioxide and particles of silicon dioxide according to the claim 6, the distinction is that it ensures in a medical agent elimination of under oxidation processes in the human body of living organism.
14. Use of stable suspensions of heterocrystals of titanium dioxide and particles of silicon dioxide according to the claim 6, the distinction is that induction of immune response of a vertebrate vertebrata by means of physical or chemical interaction with antigens is ensured.
15. The method of obtaining stable suspensions of heterocrystals of titanium dioxide or and particles of silicon dioxide according to the claim 2, characterized in the distinction is that in the cavitational homogenizer a flow of mixed medium passes through the first block of preliminary mixing and further through the block of cavitational homogenizing, and then through the block with possibility of regulation in a flow of the ingredient dosing with subsequent periodical direction into the block of regulated output of homogenized product subjected to pulsation-wave homogenizing, at that the pulsation chamber of wave mixing is executed with regulated reflector of flow mixture, installed at the chamber output.
16. The method of obtaining stable suspensions of heterocrystals of titanium dioxide or and particles of silicon dioxide according to the claim 3, characterized in the distinction is that in the cavitational homogenizer a flow of mixed medium passes through the first block of preliminary mixing and further through the block of cavitational homogenizing, and then through the block with possibility of regulation in a flow of the ingredient dosing with subsequent periodical direction into the block of regulated output of homogenized product subjected to pulsation-wave homogenizing, at that the pulsation chamber of wave mixing is executed with regulated reflector of flow mixture, installed at the chamber output.
17. The method of obtaining stable suspensions of heterocrystals of titanium dioxide or and particles of silicon dioxide according to the claim 4, characterized in the distinction that activated crystals TiO.sub.2 or particles SiO.sub.2 are obtained with presence in structure of oxygen from 60% up to 80% in metastable electronically-excited third state triplet condition .sup.3+TO.sub.23O.sub.2, at that the particles TiO.sub.2 have Zeta-potential +30-+15 mV, and the particles SiO.sub.2 have Zeta-potential 20-15 mV and are characterized with presence of sorption properties.
Description
EXAMPLES
[0160] Examples of obtaining stable suspension TiO.sub.2
Example 1
[0161] The original nano-titanium dioxide powder in the form of aggregates 0.5 micrometers in size (modification of anatase and rutile taken in a ratio of 10:90) is a white powder, which, when studied in stability in accordance with the requirements of the International Council on Harmonization (ICH), showed that over time, the nanoparticles agglomerate, that is, the highly dispersed state is lost and stability decreases. Product has a characteristic odor of butanol and isobutanol; moreover, due to the residual solvent content, the starting material does not meet pharmacopoeial requirements. Preparation of solution 500 ml 0.1 N initial solution of HCl is prepared of aqueous solution HCl.
Dispersion.
[0162] 100.0 g of TiO.sub.2 with water adsorbed (recalculated for dry mass) is weighed. Dispersion of TiO.sub.2 is executed in 300 ml 0.1 N solution of HCl.
Irradiation is Ultrasonic Bath
[0163] For preparation suspension is irradiated in ultrasonic bath working at frequency 20-90 kHz (bath temperature: 60 C., irradiation time: from 10-90 minutes).
[0164] During the process, the aqueous suspension is periodically sent to a hydrodynamic cavitation homogenizer.
[0165] Evaporation of dispersion medium.
[0166] Dispersion medium is steamed in vacuum-rotary evaporator.
[0167] During the evaporation of the dispersion medium, the final release of volatile hydrochloric acid vapor occurs, as well as the removal of residual amounts of volatile undesirable impurities (for example, butanol, isobutanol) to a level below the detection limit by gas chromatography (European Pharmacopoeia method).
Filtration
[0168] The dispersion is filtered through a filter fabric made of polyethylene terephthalate threads with a mesh size of 5 micrometers (Sefar Medifab). The filter is examined during process control: through a microscopic assessment, the integrity of the filter is checked by observing possible mechanical damage.
Evaporation of Dispersion Medium
[0169] Dispersion medium is evaporated in vacuum-rotary evaporator under pressure lower than 100 kPa at temperature no more than 70 C. In the process of evaporation of low pH dispersion medium, volatile vapors of hydrochloric acid are discharged meanwhile cleaning the surface of TiO.sub.2 nanoparticles, thereby activating as the chemical properties at the surface undergo significant changes. After evaporation of dispersion medium, dry activated powder TiO.sub.2 compliant with GMP and FDI requirements is obtained.
[0170] When releasing variations of aqueous solutions according to GMP requirements the powder of activated TiO.sub.2 crystals of the new QD class is directed into the hydrodynamical cavitational homogenizer, in which water is added for obtaining from 0.001% to 10% stable suspension TiO.sub.2 and expose to additional treatment during 10-90 minutes.
[0171] Stability in time for suspension obtained in such a way is from 24 months and more than 15 years without changes of optical properties. (
Example 2
[0172] Initial powder of nanosized titanium dioxide (modification of anatase and rutile taken in ratio 10:90) is white powder in the form of aggregates with size 0.5 micrometer with a characteristic smell of butanol and isobutanol, when studying stability in accordance with the requirements of the International Council for Harmonization (ICH), it was found that over time the nanoparticles agglomerate, that is, they lose their highly dispersed state and stability decreases. In addition, the odor of the solid material is undesirable, i.e. the product has a characteristic odor of butanol and isobutanol; moreover, due to the residual solvent content, the starting material does not meet pharmacopoeial requirements.
[0173] Preparation of solution500 ml 0.1 N initial solution of HCl is prepared of aqueous solution HCl.
Dispersion.
[0174] 100.0 g of TiO.sub.2 (recalculated for dry mass) is weighed. Dispersion of TiO.sub.2 is executed in 300 ml 0.1 N solution of HCl.
Irradiation is Ultrasonic Bath
[0175] For preparation suspension is irradiated in ultrasonic bath working at frequency 20-90 kHz (bath temperature: 60 C., irradiation time: 1.2 hours).
Filtration.
[0176] The dispersion is filtered through a filter fabric made of polyethylene terephthalate threads with a mesh size of 5 micrometers (Sefar Medifab). The filter is examined during the inspection process: through microscopic evaluation, the integrity of the filter is checked by observing possible mechanical damage.
Evaporation of Dispersion Medium.
[0177] The dispersed medium is evaporated in a vacuum rotary evaporator under a pressure below 100 kPa. at a temperature of not more than 70 C. During the evaporation of the pH of the dispersion medium, volatile vapors of hydrochloric acid are released meanwhile the surface of TiO.sub.2 nanoparticles is thereby activated as the chemical properties at the interface undergo significant changes. After evaporation of the dispersion medium, dry activated TiO.sub.2 powder is obtained. In addition, evaporation helps remove residual amounts of volatile unwanted impurities (e.g. butanol, isobutanol) to levels below the detection limit using gas chromatography (European Pharmacopoeia method) In the manufacturing of various options for aqueous dispersions in accordance with GMP requirements, activated crystal powder TiO.sub.2 of the new QD class is loaded into the hydrodynamical cavitational homogenizer, in which water is added for obtaining from 0.01% to 10% stable suspension TiO.sub.2 and expose to additional treatment during from 10 to 90 minutes (1 minutes from 10 liters of treated suspension, where content depends on the specified indices, i.e. 0.001-10%).
[0178] Stability in time for suspension obtained in such a way is at least 24 months. During not less than 15 years changes in particle size distribution were not indicated.
[0179] Example of obtaining stable suspension SiO.sub.2
Example 3
[0180] Silicon dioxide nanopowder was investigated in a stability study in accordance with the International Council for Harmonisation (ICH) requirements. During the study it was found that the nanoparticles agglomerate, i.e. lose their highly dispersed state and reduce stability, resulting in non-compliance with the particle size stability requirements. 255 g of SiO.sub.2 powder is dispersed in 10 l of water adding 0.0001 N of HCl solution. Then dissolved nano-powder is placed into a reaction vessel with volume of 5000 ml, where dispersion is executed under pressure 900 kPa-1200 kPa with speed 10 l/min.
Irradiation is Ultrasonic Bath.
[0181] For preparation suspension is irradiated in ultrasonic bath working at frequency 20-90 kHz (bath temperature: 60 C., irradiation time: 1.2 hours).
[0182] Suspension of SiO.sub.2 particles is obtained with particles size in 95.0 number %<250 nm. Content of silicon dioxide is 50 mg/ml.
[0183] Reactionary mixture is filtered through a mesh filter 10 micrometer (Sefar Medifab).
[0184] After filtration all the volume of suspension is pumped into a glass container and crimped thoroughly.
[0185] Obtained suspension is placed into an autoclave at temperature 121 C., exposure time: 30 min.
Evaporation of Dispersion Medium.
[0186] Dispersion medium is evaporated in vacuum-rotary evaporator under pressure lower than 10 kPa at temperature not more than 70 C.
[0187] In the process of evaporation of low pH dispersion medium volatile vapors of hydrochloric acid and other impurities are discharged. Meanwhile, surface of SiO.sub.2 particles is cleaned and activated, as chemical properties at the surface undergo significant changes. After evaporation of dispersion medium, dry activated powder SiO.sub.2 compliant with GMP and FDI requirements is obtained.
[0188] When releasing variations of aqueous solutions according to GMP requirements circulation of mixed aqueous mixture of silicon dioxide of the new QD class through a suspending device in the hydrodynamical cavitational homogenizer with input pressure 900 kPa-1200 kPa during up to 90 minutes at room temperature.
[0189] Appearancewhite or gray-white suspension, shaken up translucent, but orange tinted, easily resuspended.
[0190] Size of particles EP 2.9.31 min 90.0 number %<250 nm. Content of silicon dioxide: 50 mg/ml, pH of the product: 5.0-7.5, dynamic viscosity: 0.94-1.14 mPas.
[0191] Stability in time for suspension obtained in such a way is at least 24 months. During not less than 15 years changes in particle size distribution were not indicated (
Example 4
Calculation of the Presence of Oxygen on the TiO.sub.2 Crystals and SiO.sub.2 Particles Surface.
[0192] The particle size distribution (of TiO.sub.2 or SiO.sub.2) was determined by dynamic light scattering (photon correlation spectroscopy) by using a Malvern Zetasizer Nano ZS instrument applied with a laser source emitting laser beam of wavelength =633 nm at 25 C. in aqueous dispersions diluted to 0.6 mg/mL concentration. The instrument provides intensity, volume and number weighted distribution profiles.
[0193] In order to obtain the surface-weighted size distribution in case of spherical crystals or particles for evaluation of sorption features related to different energetic states of dissolved oxygen, from the volume-weighted distribution, the following formula was used:
wherein [0194] A.sub.iS(%) is the area corresponding to fraction i of spherical crystals or particles expressed in % of total area; [0195] V.sub.iS(%) is the volume corresponding to fraction i of spherical crystals or particles expressed in % of total volume; [0196] D.sub.i is the diameter of fraction i crystals or particulates.
[0197] However, for non-spherical crystals or particles of the new Quantum Dots (QD) class according to present invention different calculation method for the surface-weighted size distribution is required, i.e. using the following formula to have insight into the sorption properties with relevance to different O.sub.2 species according to present invention:
wherein [0198] A.sub.iQD(%) is the area corresponding to fraction i of non-spherical crystals or particles expressed in % of total area; [0199] V.sub.iQD(%) is the volume corresponding to fraction i of non-spherical crystals or particles expressed in % of total volume; [0200] F.sub.i is the individual shape factor (non-sphericity parameter) corresponding to fraction i [0201] D.sub.i is the diameter of fraction i crystals or particles.
[0202] Exemplary calculations for a known nanosized TiO.sub.2 disclosed in PCT patent application WO2008068062 in comparison with the TiO.sub.2 crystals of the new quantum dots QD class according to present invention are shown in Table 8, which shows conversion of surface-weighted size distribution from volume-weighted distribution
[0203] Spherical crystals having AiS (%) distribution profile as shown in Table 8 according to WO2008068062 represents 5 micromol/m2 adsorptive capacity and 90 m2/g specific surface area. 1 g of such nanosized TiO.sub.2 hence possesses 450 micromol/g adsorptive capacity which is equal to 0.0144 g 02/g TiO.sub.2 (1.44% by weight). Non-spherical crystals of the special QD class according to present invention show significantly higher AiQD (%) at smaller particulate size population (i.e. 20 nm) which allows adsorbing more reactive form of oxygen in the amount of about 60-80% is in electronically excited triplet state.
[0204] The TiO.sub.2 crystals of the QD class according to the present invention are a group of irregular, prismatic shape with rectangular faces, sometimes with pointed ends, and in the case of QD class SiO.sub.2 they have a crystalline structure and form interfaces and, as in the case of QD class TiO.sub.2, have the same narrow contact zones with each other, i.e. the (secondary) nanodispersed particles are formed by agglomeration already after the preparation of the suspension, the stability of which is indicated in Tables 2 and 4. The increased surface area due to the nonsphericality allows to increase the adsorption capacity for different excited states of oxygen.
[0205] Taking into account the new structure of crystals and particles of the QD class, containing on their surface many energy centres in the form of exciton semiconductor channel outlets, these outlets correspond to the location of oxygen molecules already in the electronically excited (EE) state, since they are included in the crystal lattice structure-which appear as a special class of quantum dots (QD), where in the structures of oxide heterocrystals oxygen molecules occupy from 60-80% and are in the electronically excited triplet state (.sup.3+TO.sub.2).
[0206] The present invention relates to stable suspensions of titanium dioxide or silicon dioxide heterocrystals, methods of obtaining said quasi-stable suspensions and use provide more effective delivery of biologically active agents into the bloodstream of a subject, accordingly distributed throughout the body as conclusively demonstrated in Table 6, sufficient for causing a specified biological reaction, that is, a therapeutically effective amount of a therapeutic agent is meant. What is meant is a pharmaceutically active substance sufficient to produce an acceptable biological response when administered. Correspondingly, TiO.sub.2 or SiO.sub.2 suspensions may be delivered with a help of usual micro-fluidized spray, hydrogel, topical products, aerosol or liquid. Delivery may be executed by parenteral, intrathecal, intravenous, intravascular, droplet, transmembrane and dermal, intracellular administration including locally into cell structures through mucous membranes, and also due to their structures and range limits provide new models-forms of administration or every other generally recognized method of medical agent's delivery.
[0207] A peculiarity of TiO.sub.2 crystals and SiO.sub.2 particles is that on their surface an acceptor, i.e. O.sub.2, is initially in excited triplet triplet third stage (.sup.3+TO.sub.2) condition, with regulated possibility of catalysis in AFO, even in the cases where physiological body temperature is sufficient.
[0208] Heterogeneous crystals of TiO.sub.2 or SiO.sub.2 particles are characterized by presence on the surface of oxygen, O.sub.2, which occupies from 60 to 80% of structure have biological activity, solution by thermal synthesis of AFO, achieved for the first time due to the human body temperature 36.6 C. itself, where it creates a therapeutic energy storage (days, weeks), which ensures a continuous, permanent process of AFO synthesis in the body.
[0209] Medical forms on the base of titanium dioxide crystals or silicon dioxide particles, as catalyst of synthesis of active oxygen (AFO), firstly, selectively penetrate into the pathogenic cells of organism, targeting the areas of inflammation, with infectious and non-infectious nature (see Examples 7, 8), in particular, the cells using a ferment NADP-H for AFO formation, phagocytes and macrophages, causing in them numerous cytomorphologic changes (vacuoles in cytoplasm, fragmentation of membrane, abnormality of mitosis), that launches apoptosis or necrosis type of death of pathogenic cells (because the pathogenic cells do not have effective antioxidant ferments). Active oxygen, AFO become firstly claimed in the pathologies in focus (Examples 5,6,7,8,9), where oxygen necessity increases many times.
[0210] NADP-H oxydases membrane-bound enzymatic complex, inversed into extracellular space of plasmatic membrane, and also in membranes of phagosomes, used by neutrophilic leukocytes (immune cells, white blood corpuscles) for absorption of microorganisms is executed with the immune response. (See Example 8).
[0211] NADP-H oxidase (NOX) is one of the main sources of cellular active forms of oxygen (AFO) and is formed by the way of catalysis.
[0212] Accordingly, when oxygen (O.sub.2), on crystal lattices is initially in the excited state .sup.3+TO.sub.2 in cases of form, topical use in the form of patches, impregnations for tampons, pads, diapers, tapes, bandages, etc., which have antimicrobial and antiseptic effects, anti-inflammatory, neuroprotective and immuno-normalizing effects, including adsorption effects, as well as mucosal forms of application, thereby creating a new modality of effective therapy.
[0213] The mechanism of action where TiO.sub.2 crystals or/and SiO.sub.2 particles are present in the composition of a medical product is that when electronically excited O.sub.2 interacts on the surface of crystals or particles with the membrane cellular enzyme complex NADPH, AFO is formed, providing energy exchange in the body. The energy returned to the organism, passing through the nerve impulses, is converted into an action potential. The action potential at the presynaptic synapse depolarises the presynaptic membrane, causing a rapid current of Ca.sup.2+ ions. The temporary increase in the concentration of Ca.sup.2+ ions stimulates the fusion of the synaptic vesicle membrane with the plasma membrane and causes the release of the mediator, for exampleserotonin, into the synaptic cleft. The interaction of mediator-serotonin in the synaptic cleft with its chemoreceptor in the postsynaptic membrane changes the membrane permeability for (Na.sup.+) ions, which provides normalisation of serotonin receptor function, restoring the serotonin balance of the organism.
[0214] Efficiency of using stable suspensions of TiO.sub.2 heterocrystals and SiO.sub.2 particles for adjuvant therapy of cancer diseases is also experimentally proven in combination with cytostatic agents (Doxorubicin, Lenalidomide (Revlimid), Nivolumab, Ibrutinib).
[0215] A positive result was demonstrated on the model of metastasis into spleen and liver, primary tumor significantly reduces after intra-abdominal administration, after administration together with cytostatic agent. In addition, a tendency to decrease of metastatic nodes was observed.
[0216] The experiments have proven (see Examples 5-8) the use of stable suspension of heterocrystals of titanium dioxide or silicon dioxide particles, for formulation of medical agents, used intravenously, intramuscularly, orally, nasally, vaginally, rectally, locally (ENT) use or topically, at that therapeutically important amount of an active substance is for intravenous use from 0.006 mg/ml, intramuscular use from 0.01 mg/ml, orally from 0.01 mg/ml, nasally from 0.01 mg/ml, vaginally from 0.01 mg/ml, rectally from 0.01 mg/ml, local ENT use from 0.01 mg/ml, ocular use from 0.001 mg/ml, and also medical articles of topical use in the form of hydrocolloidal medical plasters from 0.0003 mg/g. (patent WO2023/214200).
[0217] Usually, therapeutically effective amount of a medical means is meant. The term therapeutically effective amount means that pharmaceutically efficient amount is considered in relation of, for example, pharmaceutical preparations. Pharmaceutically efficient amount is amount of a medical remedy or pharmaceutically active substance that is sufficient for achieving acceptable biological reaction in its use.
[0218] A medical remedy may be executed in the form of rectal-vaginal suppositories, gel, ointment, liquid.
[0219] THE FOLLOWING EXAMPLES OF EFFECTIVE IMPACT NOT ONLY ON THE DISEASE (PATHOLOGY) EXAMPLE, BUT ILLUSTRATE A WIDE RANGE OF POSSIBILITIES OF EFFECTIVE THERAPY OF VARIOUS NOSOLOGIES OF MEDICAL PRODUCT APPLICATION FOR ELIMINATION OF UNDEROXIDATION PROCESSES IN LIVING ORGANISM DUE TO AFO SYNTHESIS.
Example 5
5.1. In Vitro Studies Related to Alzheimer's Disease with the Use of SiO.sub.2 Particles of the New QD Class (Adam-QD/S)
In Vitro Studies
Study Number QD-2023-1
[0220] The purpose of this in vitro interaction study is to explore the mechanism of action of the SiO.sub.2 particles to exert its therapeutic effect via a the peroxidase-mimetic catalytic effect of the particles using 3,3,5,5 Tetramethylbenzidine (TMB) reagent, which plays the role of the reducing agent (hydrogen donor) in the reduction of H.sub.2O.sub.2 to water.
[0221] Test samples: Representative SiO.sub.2 particles of the QD class characterized by volume and surface weighted distribution per
[0222] Blank sample was prepared analogously without SiO.sub.2.
[0223] The Test and Blank samples were transferred separately into a 50 ml beaker, placed them in a 39-40 C. water bath and stirred them in air at a speed of 500 RPM. The spectrophotometric analysis of the samples was performed with an Agilent 8453 spectrophotometer, applying a quartz cuvette with an edge length of 1 cm, at a wavelength of 650 nm. The absorbances of the Test samples were measured without dilution, directly against the Blank sample.
[0224] The results showed that in the presence of nanoparticulate SiO.sub.2 the rate of oxidation of TMB (3,3,5,5 tetramethylbenzidine) was significantly enhanced in concentration dependent manner in the range of 0.024-0.06 mg/mL (30-60-fold). At pH 4, the rate of oxidation approximately tripled compared to what was observed at pH 3.
5.2. In Vitro Studies Related to Alzheimer's Disease with the Use of TiO.sub.2 Crystals of the New QD Class (Adam-QD/T)
Study Number QD-2023-1
[0225] The purpose of this in vitro interaction study is to explore the mechanism of action of the TiO.sub.2 crystals to exert its therapeutic effect via the peroxidase-mimetic catalytic effect of the crystals using 3,3,5,5 Tetramethylbenzidine (TMB) reagent, which plays the role of the reducing agent (hydrogen donor) in the reduction of H.sub.2O.sub.2 to water.
[0226] Test samples: Representative TiO.sub.2 crystals characterized by volume and surface weighted distribution per
[0227] Blank sample was prepared analogously without TiO.sub.2.
[0228] The Test and Blank samples were transferred separately into a 50 ml beaker, placed them in a 39-40 C. water bath and stirred them in air at a speed of 500 RPM. The spectrophotometric analysis of the samples was performed with an Agilent 8453 spectrophotometer, applying a quartz cuvette with an edge length of 1 cm, at a wavelength of 650 nm. The absorbance of the Test samples was measured without dilution, directly against the Blank sample.
[0229] The results showed that in the presence of TiO.sub.2 crystals the rate of oxidation of TMB (3,3,5,5 Tetramethylbenzidine) was significantly enhanced in concentration dependent manner in the range of 0.024-0.06 mg/mL (25-50-fold). At pH 4, the rate of oxidation approximately doubled compared to what was observed at pH 3.
5.3. In Vitro Studies (Yeast Cell Study) in the Anti-Aging and Oxidative Stress by Using TiO.SUB.2 .Crystals of the New QD Class (ADAM-QD/T)
Study Number QD-2024-1
[0230] The purpose of this in vitro interaction study is to explore the mechanism of action of the TiO.sub.2 crystals, how it exerts its therapeutic effect. It is postulated that these crystals prevent oxidative stress by deactivating oxidative agents such as H.sub.2O.sub.2. To achieve the aim, a biorelevant study on pH- and concentration dependence of redox catalytic activity i.e. peroxidase-mimetic effect of the corresponding TiO.sub.2 crystals was modelled using a model dye, MTT Formazan (1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan) which reagent plays the role of the reducing agent (hydrogen donor) in the reduction of H.sub.2O.sub.2 to water. Peroxidases are important enzymes in antiaging and combat oxidative stress.
[0231] MTT is the most commonly used colorimetric assay for assessing cell metabolic activity usually used to measure cytotoxicity (loss of viable cells) or cytostatic activity of potential medicinal agents. NADPH-dependent cellular oxidoreductase enzymes reflect the number of viable cells present. These enzymes are capable of reducing the MTT dye which has a purple colour which enables the recordation of the kinetics photometrically.
[0232] Representative TiO.sub.2 crystals characterized by volume and surface weighted distribution per
[0233] Liquid samples were prepared containing constant amount of MTT formazan, set to target pH, containing various concentrations of nanosized TiO.sub.2 (0.006-0.12 mg/mL), hydrated live yeast cells (0.023 mg/mL on dry cell basis) and fixed amount of H.sub.2O.sub.2 (in 1.5 M concentration). The samples were stirred in a dark cabinet thermostated close to body temperature. The samples were repeatedly analyzed with 30 min frequency in a total of four hours interval with Agilent 8453 spectrophotometer.
[0234] In 25 ml screw cap scintillation vials, 8.00 ml 96 V % ethanol, 4 ml TiO.sub.2 crystals suspension (set to different concentration), 3.00 ml of 0.25 mg/ml ethanolic MTT formazan solution, finally purified water, finally hydrated live yeast cells (0.023 mg/mL on dry cell basis) were added in this order. After optional pH setting with 1% Na.sub.2HPO.sub.4, 4 ml 30% H.sub.2O.sub.2 reagent was added. The kinetic curves obtained during the study (i.e. the decreasing concentration of MTT formazan) of the oxidative process were recorded spectrophotometrically.
[0235] It was found that under the used experimental conditions, TiO.sub.2 crystals can facilitate the reduction of H.sub.2O.sub.2 by catalyzation of a biologically relevant electron transfer reaction. Deactivation of H.sub.2O.sub.2 is well known to have therapeutic relevance and also may be desirable in various life science applications. The model reaction represents peroxidase-mimetic effect, wherein MTT formazan is the hydrogen donor hence it reveals the mechanism how TiO.sub.2 crystals combat oxidative stress.
[0236] TiO.sub.2 crystal was found effective in the concentration range 0.006-0.12 mg/ml in concentration dependent manner reaching maximum activity at 0.06 mg/ml. The effect of TiO.sub.2 has been well demonstrated at pH 6 and 8 (the latter value is mitochondrial pH).
5.4. In Vivo Studies Related to Alzheimer's Disease with the use of SiO.sub.2 Particles of the New QD Class (ADAM-QD/S)
Study Number: 9160
[0237] Efficacy of SiO.sub.2 particles after intramuscular dosing of 5FAD mice.
[0238] The aim of this study was to test the efficacy of SiO.sub.2 particles after intramuscular dosing of 5FAD mice. A total of 12 female transgenic 5FAD mice at an age of 7 months, as well as 6 age- and sex-matched wildtype littermates were used for the study. After finishing behavioral testing, when the animals reached an age of 10 months, all mice were euthanized. Cerebrospinal fluid and terminal blood were collected. Brains were dissected after transcardial perfusion with saline and hemisected at midline. The left hemibrain was further dissected in hippocampus, cortex and rest, all parts were weighed and snap frozen on dry ice for biochemical analysis. The right hemibrain was post-fixed by immersion in 4% PFA, and cryo-protected in sucrose/PBS before embedding and freezing in cryomolds for histological evaluations. NF-L levels were quantified in plasma and CSF samples from all animals (18 samples) using the NF-Light ELISA. Measurements were performed in duplicates. Hemibrains from 6 animals per group (total of 30 hemi brains) dedicated for histological analysis were embedded in sagittal orientation in OCT medium and 10 m cryosections were collected. Sections were collected from 12 levels of the brain and 5 sections per level, a total of 60 sections per animal. Amyloid--positive plaques, tau phosphorylation (Ser202, Thr205) and neuroinflammation (astrocytosis and astrogliosis) were evaluated using immunofluorescence labeling on a uniform systematic random set of five sections per mouse in a quadruple staining:
[0239] Representative SiO.sub.2 particles characterized by volume and surface weighted distribution per
[0244] All sections were counterstained with the nuclear dye DAPI.
Outcome:
[0245] General: All animals tolerated the treatment. Neither the treatment route nor treatment with the test item led to unusual occurrences. The mean body weight of all groups increased over time as expected, whereas non-transgenic animals showed a typically higher body weight compared to transgenic animals without reaching significance at all times. Behavior: Statistical significances could be detected in learning curves throughout all evaluated parameters in these two groups.
[0246] Treatment with SiO.sub.2 particles significantly increased the mean size of detected objects in the cortex; this indicates that SiO.sub.2 particles reduces the formation of new (small) plaques as shown by Amyloid signal detection. B.5FAD vehicle group analysis showed 1605 m.sup.2 cortex object sizes, whereas SiO.sub.2 particles treated group analysis resulted in 1805 m.sup.2 cortex object sizes.
5.5. In Vivo Studies Related to Alzheimer's Disease with the Use of TiO.sub.2 Particles of the New QD Class (Adam-QD/T)
Study Number 0430
[0247] The aim of the study is to examine the effect of titanium dioxide crystals on cytotoxicity and mitochondrial activity in neural cells upon H.sub.2O.sub.2 lesion in two different cell systems, i.e. cortical glutamatergic neurons, microglia and astrocytes.
[0248] Dysfunction of cortical glutamatergic neurons may be relevant to neurological diseases, such as Alzheimer's disease.
[0249] Microglial degeneration and death have been reported in research on schizophrenia and Alzheimer's disease.
[0250] Clinically significant pathologies involving astrocytes include astrogliosis and astrocytopathy associated with multiple sclerosis, anti-AQP4+ neuromyelitis optica, Rasmussen's encephalitis, Alexander disease, and amyotrophic lateral sclerosis. Studies have shown that astrocytes may be implied in neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, Huntington's disease, Stuttering and amyotrophic lateral sclerosis.
[0251] The study involved determination the EC50 of H2O2 for four read-outs: mitochondrial activity, cytotoxicity, mitochondrial singlet oxygen imaging and ATP assay. Instrument used for quantitation: Incucyte Live-cell imaging system.
[0252] Overall, neurons were found vulnerable to H.sub.2O.sub.2 of 100-1000 M concentration. TiO.sub.2 crystals were able to reduce cell death and to increase mitochondrial health for cortical glutamatergic neurons and microglia. For astrocytes, temporary increased mitochondrial activity could be observed after lesion addition lasting for 10 hours.
[0253] Representative TiO.sub.2 crystals characterized by volume and surface weighted distribution per Example 4 was used for the studies.
[0254] For astrocytes, the total intensity (expressed as OCUm.sup.2/image) as shown by the TMRM test in the presence of 100 M H.sub.2O.sub.2 and 6 g/mL, 60 g/mL, 600 g/mL of TiO.sub.2 crystals was 1.5-2 times higher than the lesion control.
[0255] The cortical glutamatergic neurons, lesion of 300 M H.sub.2O.sub.2 was detrimental, however, in the presence of 6-600 g/mL TiO.sub.2 crystals the total intensity 1.0-1.5 million OCU x m.sup.2/image remained in the TMRM test.
[0256] For microglia, lesion of 1000 M H.sub.2O.sub.2 was caused the TMRM signal from 4 million to 500000 OCUm.sup.2/image within 20 hours incubation, however, in the presence of 6-600 g/mL TiO.sub.2 crystals the total intensity of 2-7 million OCUm.sup.2/image remained in the TMRM test.
[0257] The results clearly indicate the neuroprotective effect exerted by the applied TiO.sub.2 QD class crystals against cellular damage caused by aggressive, H.sub.2O.sub.2 lesion. Oxidative stress was highly reduced owing to the presence of TiO.sub.2 which offers notable potential in therapeutic or preventive applications for neurodegenerative conditions. Hindering the oxidative potential of H.sub.2O.sub.2 is the mechanism of action manifested in anti-Alzheimer action of TiO.sub.2.
Example 6
6.1. In Vitro Studies Related to Osteoarthritis with the Use of TiO.sub.2 Crystals of the New QD Class (Adam-QD/T)
Study Number: L-03
[0258] The objective of the study was to effects of TiO.sub.2 crystals on differentiation of mouse osteoblasts and differentiation and activity of human osteoclasts in vitro.
[0259] Osteoblasts and osteoclasts are specialized cells that play crucial role in bone development and regeneration. Osteoblasts form new bones and add growth to existing bone tissue. Osteoclasts remove old and damaged bone tissue so it can be replaced with new, healthier cells created by osteoblasts. Both cells differentiate (develop) from special stem cells. The below study is aimed to model how the TiO.sub.2 test item is able to cause alteration of metabolic pathways i.e. eliminating underoxidized metabolic byproducts from bone tissue causing osteoarthritic conditions. Abnormal joint tissue metabolism is associated with the formation of unwanted byproducts that are produced abundantly in osteoarthritic articular tissues, their levels are higher in osteoarthritic condition compared to normal and when the oxidative homeostasis of articular cartilage becomes unbalanced.
[0260] In the osteoblast differentiation assay, KS483 mouse osteoprogenitor cells were cultured for 8 days, after which the formed osteoblasts were quantitated by measuring the amount of intracellular alkaline phosphatase (ALP) activity. Bone morphogenetic protein 2 (BMP-2) was included in the study as a reference compound that stimulates osteoblast differentiation and bone formation to demonstrate that the culture system works as expected. Test compound and the reference compound BMP-2 were added at day 0 (when cells are seeded) and at day 4 concomitant with medium was refreshment. The osteoclast differentiation assay was performed using human bone marrow-derived CD34+ osteoclast precursor cells. The cells were cultured on bovine bone slices for 7 days in conditions favoring osteoclast differentiation and allowed to differentiate into bone-resorbing osteoclasts. Denosumab was included in the study as a reference compound to demonstrate that the test system can detect inhibition of osteoclast differentiation. The test compound and the reference compound denosumab were added at day 0 (when cells were seeded) and at day 3 concomitant with medium refreshment. Tartrate-resistant acid phosphatase 5b (TRACP 5b) activity was measured in the culture medium collected at day 7 as an index of the number of osteoclasts formed during the differentiation period. For the osteoclast activity assay, human bone marrow derived CD34+ osteoclast precursor cells were cultured on bovine bone slices for 10 days in conditions favoring osteoclast differentiation. Odanacatib was included in the study as a reference compound to demonstrate that the test system can detect inhibition of osteoclast activity. The test compound and the reference compound odanacatib were added into the culture medium at day 7, and the formed osteoclasts were allowed to resorb bone for 3 days. C-terminal crosslinked telopeptides of type I collagen (CTX-I) was measured in the culture medium at day 10 as an index of bone resorption. The tests were performed in 96-well plates containing a baseline group with vehicle and a control group including the reference compound (BMP-2/denosumab/odanacatib). Seven concentrations of the test compound were included.
[0261] TiO.sub.2 products of the QD class decreased mouse osteoblast differentiation significantly at 50-1000 g/ml concentration range (from 90 to 75%) while 0.05-5 g/ml concentration also caused decrease (from 15 to 10%).
[0262] TiO.sub.2 products of the QD class decreased human osteoclast differentiation significantly (TRACP 5b) at 50-1000 g/ml concentration range (from 99 to 59%) while 5 ng/ml-5 g/ml also caused decrease (from 11 to 19%).
[0263] TiO.sub.2 products of the QD class decreased human osteoclast resorption activity significantly at 1000 g/ml (by 73%) and while the effect is moderate at 5 ng/ml to 500 g/ml concentration (by 27 to 11%).
[0264] Instrument used: VICTOR2 Multilabel Counter (PerkinElmer, Waltham, MA, USA).
[0265] The results show that the differentiation of both cells can be modulated by the presence of TiO.sub.2 crystals of the QD class (ADAM-QD/T) which has promising potential in treatment of osteoarthritic conditions with involvement of eliminating/preventing the formation of underoxidized metabolic byproducts before their early stages of occurrence.
6.2. In Vitro Studies Related to Osteoarthritis with the Use of SiO.sub.2 Particles of the New QD Class (Adam-QD/S)
Study Number: L-02
[0266] Osteoblasts and osteoclasts are specialized cells that play crucial role in bone development and regeneration. Osteoblasts form new bones and add growth to existing bone tissue. Osteoclasts remove old and damaged bone tissue so it can be replaced with new, healthier cells created by osteoblasts. Both cells differentiate (develop) from special stem cells. The below study is aimed to model how the SiO.sub.2 test item is able to cause alteration of metabolic pathways i.e. eliminating underoxidized metabolic byproducts from bone tissue causing osteoarthritic conditions. Abnormal joint tissue metabolism is associated with the formation of unwanted byproducts that are produced abundantly in osteoarthritic articular tissues, their levels are higher in osteoarthritic condition compared to normal and when the oxidative homeostasis of articular cartilage becomes unbalanced.
[0267] Instrument used: VICTOR2 Multilabel Counter (PerkinElmer, Waltham, MA, USA).
[0268] SiO.sub.2 particles decreased osteoblast differentiation significantly (ALP specific activity) at 5-1000 g/ml concentration (by 80 to 52%), however the 5 ng/ml caused 15% increase compared to the baseline.
[0269] SiO.sub.2 products of the QD class decreased osteoclast differentiation significantly (TRACP 5b) at 50 ng/ml and 0.5-1000 g/ml concentration (by 100 to 19%) and the 5 ng/ml decreased by 14% compared to the baseline.
[0270] SiO.sub.2 products of the QD class decreased osteoclast resorption activity significantly (CTX-I) at 50-1000 g/ml concentration (by 96 to 55%), and it caused 28% decrease in 50 to 5 ng/mL compared to the baseline.
[0271] The results show that the differentiation of both cells can be modulated by the presence of SiO.sub.2 particles of the QD class (ADAM-QD/S) which has promising potential in treatment of osteoarthritic conditions with involvement of eliminating/preventing the formation of underoxidized metabolic byproducts.
6.3. IN VIVO Studies Relating to Osteoarthritic Changes with the Use of SiO.sub.2 Particles of the New QD Class (ADAM-QD/S)
[0272] The effects of SiO.sub.2 particles of the QD class on osteoarthritic changes induced by unilateral surgical medial meniscal tear and medial collateral ligament transection in rats
[0273] The objective of this study is to investigate impact of the SiO.sub.2 particles on osteoarthritic pain and histological changes in knee joints exhibiting unilateral medial meniscal tear and medial collateral ligament transection in young adult male rats.
[0274] Each group contained 12 male Lewis rats that were three months of age at the beginning of in-life phase of the study. Before the in-life phase, the rats were trained for pain measurement devices (hind paw weight distribution and paw withdrawal threshold.
[0275] Instrument of analysis: CatWalk XT computer-assisted method of locomotor analysis (Noldus Information Technology, Wageningen, The Netherlands).
[0276] Test item in the form of SiO.sub.2 suspension of the QD class was administered invasively (into the tight muscle-musculus quadriceps femorisof the right operated hind limb and also, non-invasively, via a form of hydrocolloidal medical plaster. Upon invasive application, the vehicle solution was administered into the same area of the non-operated left hind leg to avoid treatment-induced differences in pain measurement between the legs. Upon non-invasive application, a medical article of topical use in the form of hydrocolloidal medical plasters containing nanosized SiO.sub.2 was applied on the right hind leg of a separate group of test animals for 19 days. Control animals had unloaded (blank) plasters adhered to their right hind leg. Both groups had unloaded (blank) plasters adhered to their left hind legs also to avoid false conclusion on their modified movement pattern.
[0277] The test compound dosing suspension was 0.64 mg/ml, and dosed intramuscularly in thigh muscle of the right leg as 0.045 mg/kg and dosing volume 0.070 ml/kg.
[0278] Static mechanical allodynia was determined using filament response data measured as paw withdrawal threshold. After the operations and invasive treatment period with SiO.sub.2 suspension, the test compound significantly reduced statistic mechanical allodynia on study days 7, 13 and 19. SiO.sub.2 product of the QD class (ADAM-QD/S) has demonstrated positive effects on reducing static mechanical allodynia and static knee joint discomfort/pain in the test system. Compared to the vehicle control, the pain tolerability threshold increased twofold after 7 days followed by treatment with SiO.sub.2 product of the QD class (ADAM-QD/S) and threefold after 13 and 19 days post treatment.
[0279] Throughout the non-invasive treatment period, it was also found that the SiO.sub.2 particles taking effect from the plaster matrix, also have shown significantly reduced statistic mechanical allodynia on the study days (7th, 13th and 19th day). Even in non-invasive application, the SiO.sub.2 product has demonstrated positive effects on reducing static mechanical allodynia and static knee joint discomfort for the test animals. Compared to the unloaded plaster control, the pain tolerability threshold increased twofold after 7 days followed by non-invasive treatment with SiO.sub.2 particles and threefold after 13 and 19 days post treatment.
[0280] The results show that administration of SiO.sub.2 products of the QD class (ADAM-QD/S) regardless of invasive or non-invasive application helps alleviation of the symptoms of osteoarthritis and hence reduces pain upon walking. It is postulated that the mechanism involves eliminating/preventing the formation of underoxidized metabolic byproducts via processes by analogous modulation of redox catalysis as shown in Example 5.
6.4. IN VIVO Studies Relating to Osteoarthritic Changes with the Use of TiO.sub.2 Crystals of the New QD Class (ADAM-QD/T)
[0281] The effects of TiO.sub.2 crystals on osteoarthritic changes induced by unilateral surgical medial meniscal tear and medial collateral ligament transection in rats.
[0282] The objective of this study is to investigate the impact of TiO.sub.2 crystals on osteoarthritic pain and histological changes in knee joints exhibiting unilateral medial meniscal tear and medial collateral ligament transection in young adult male rats.
[0283] Each group contained 12 male Lewis rats that were three months of age at the beginning of in-life phase of the study. Before the in-life phase, the rats were trained for pain measurement devices (hind paw weight distribution and paw withdrawal threshold.
[0284] Test item in the form of TiO.sub.2 suspension was administered into the tight muscle (musculus quadriceps femoris) of the right hind limb (operated); Vehicle solution was administered into the same area of the non-operated left hind leg to avoid treatment-induced differences in pain measurement between the legs.
[0285] The test compound dosing suspension was 0.56 mg/ml, and dosed intramuscularly in thigh muscle of the right leg as 0.045 mg/kg and dosing volume 0.080 ml/kg.
[0286] Instrument of analysis: CatWalk XT computer-assisted method of locomotor analysis (Noldus Information Technology, Wageningen, The Netherlands).
[0287] The treatment with TiO.sub.2 product significantly increased (by 13% difference in group average) weight bearing of the right hind limb, as well the guarding index (difference between weight bearing of the left heathy and right operated hind limb) compared to the vehicle-treated group on study day 19. In accordance there was a statistical trend of the weight bearing of the left hind limb being lower in the TiO.sub.2 product group compared to the vehicle group similarly on study day 19.
[0288] Compared to the vehicle control, the pain tolerability threshold increased by 20% after 7 day (post treatment with TiO.sub.2 product of the QD class (ADAM-QD/S)), whereas by 30% on day 13 and by 40% on the 19th day post treatment.
[0289] The results show that administration of nanoparticulate TiO.sub.2 helps alleviation of the symptoms of osteoarthritis and hence reduces pain upon walking.
[0290] The results show that administration of TiO.sub.2 products helps alleviation of the symptoms of osteoarthritis and hence reduces pain upon walking. Mechanism of TiO.sub.2 products of the QD class involves eliminating and preventing the formation of underoxidized metabolic byproducts via processes by analogous modulation of redox catalysis as shown in Example 5.
[0291] ON THE FOLLOWING EXAMPLES OF EFFECTIVE IMPACT NOT ONLY ON THE DISEASE (PATHOLOGY) EXAMPLE, WHICH ILLUSTRATES A WIDE RANGE OF PROVIDING ANTIVIRAL EFFECT OF THE MEDICINAL PRODUCT FOR DIFFERENT PATHOGENS.
Example 7
7.1. In Vitro Studies Related to SARS-COV2 Infections with the Use of SiO.sub.2 Particles of the New QD Class (Adam-QD/S)
[0292] Testing the antiviral efficacy of SiO.sub.2 particles against SARS-COV-2 in vitro Study number: 3025
[0293] A potential antiviral drug, SiO.sub.2 suspension of the QD class was evaluated in vitro for its antiviral property against SARS-COV-2.
[0294] To test for antiviral activity of SiO.sub.2 particles, virus infection and replication after the presence of the compound was evaluated. Before performing the above-mentioned assay, SARS-COV-2 was titrated on the Vero TMPRSS2 cells to determine the relevant titre. The compound was freshly diluted before each experiment in culture medium. To assess the potential antiviral activity against SARS-COV-2, the compound in six concentrations was added to VERO TMPRSS2 cells for 3 hrs. Hereafter, the SARS-COV-2 in three concentrations were added to the cells.
[0295] Representative SiO.sub.2 suspensions of the QD class characterized by volume and surface weighted distribution per Example 4 were used for the studies.
[0296] Experimental set 1: After two days, cell monolayers were washed and stained for the presence of SARS-COV-2 antigen with an immunoperoxidase monolayer assay. Plates were read microscopically and judged for the presence of virus and immunostaining was scored in a semi-quantitative way: : no cells; +: 25 cells; ++: >25 cells. In addition, spots in the individual wells were counted using an AID ELISPOT reader.
[0297] Experimental set 2: After two days, cells and supernatants were harvested and analysed for (the presence of cytotoxicity by LDH release.
[0298] SARS-COV-2 reproduction in VERO TMPRSS2 cells was as expected in the non-treated groups, whereas hIFN was able to inhibit viral reproduction at the concentrations of 750 and 1000 U/ml at all three SARS-COV-2 concentrations used. The NIBSC reference serum inhibited SARS-COV-2 reproduction in a dose dependent manner at Multiplicity of infection of 0.005.
[0299] SiO.sub.2 particles at concentrations of 50 g/ml and 500 g/ml inhibited the multiplication of SARS-COV-2, indicating that the compound has antiviral properties against SARS-COV-2.
[0300] The LDH cytotoxicity test showed a slight increase of the LDH activity in the supernatant of the SiO.sub.2 particles treated group at a concentration of 500 g/ml. This finding can be evaluated as a slight cytotoxicity effect.
[0301] The compound SiO.sub.2 particles at concentrations 50 g/ml and 500 g/ml showed the inhibition of the reproduction of SARS-COV-2 (by reducing the mean SARS-CoV2-positive spots per M96-well from 600 to 230 and 130, respectively) which explicitly show that the compound has an antiviral property against SARS-COV-2.
[0302] THE FOLLOWING EXAMPLES ILLUSTRATE PROVIDING INDUCTION OF AN IMMUNE RESPONSE IN VERTEBRATES BY PHYSICAL OR CHEMICAL INTERACTIONS WITH ANTIGENS.
7.2. In Vivo Studies Related to SARS-Cov2 Infections with the Use of TiO.sub.2 Crystals and SiO.sub.2 Particles of the New QD Class (Adam-QD)
Study Number: 23201
[0303] The aim of the study was to evaluate the adjuvant capacity of TiO.sub.2 crystals and SiO.sub.2 particles of the QD class using ovalbumin as a prototypical antigen and to compare them with a formulation consisting of an equivalent formulation of alum (Hydroxygel). This proof of concept study was aimed to study the capacity of the formulations to induce an antibody response, assess the immunization regimen for adverse effects on the animals (assessed by changes in weight) and to determine the predominant isotypes induced by each formulation.
Methods:
[0304] Representative TiO.sub.2 and SiO.sub.2 products characterized by volume and surface weighted distribution per Example 4 was used for the studies.
[0305] Eight-week-old C57Bl/6 (B6) mice were immunized and received a boost under the same conditions in the same month.
[0306] The groups of mice were (8 mice per group): [0307] 1. Ovalbumin (10 g)+phosphate buffered saline (PBS) [0308] 2. Ovalbumin (10 g)+alum (1:1) [0309] 3. Ovalbumin (10 g)+QD product TiO.sub.2 (1:1) [0310] 4. Ovalbumin (10 g)+QD product SiO.sub.2 (1:1)
[0311] The mice were weighted at days 0, 1, 2, 3 and 14, 15, 16, 17, 18. The overall condition of the mice was checked visually every day, including weekends.
[0312] Antibody titers were measured by capture ELISA. Anti-ovalbumin IgM was measured in d0, d7, d14 and d28 sera. Total anti-ovalbumin IgG was measured in d0, d14 and d28 sera. Anti-ovalbumin IgG1, IgG2b, IgG2c and IgG3 were measured in d28 sera. None of the immunizations resulted in major changes in the weight of the mice. Evaluation of the adjuvant capacity of both of TiO.sub.2 or SiO.sub.2 QD products showed that both are capable of inducing a response similar to that demonstrated for the alum preparation. Among the differences observed between TiO.sub.2 and SiO.sub.2 products, the rapid induction of IgM response seems to indicate a stronger ability to activate B cells for the latter, which corresponds to a slight increase in the IgG3 isotype, which is the earliest, T-independent isotype exhibited in the B-cell response.
[0313] Overall, this study clearly demonstrates the adjuvant activity of TiO.sub.2 and SiO.sub.2 products against the prototypical antigen, ovalbumin, when using a specific formulation (1:1) and immunization route (intramuscular) increasing IgG titres fourfold at the end of the study compared to the control measurement.
[0314] THE FOLLOWING EXAMPLES ILLUSTRATE THE EFFECT OF THE QD PRODUCTS ON LIFESPAN.
Example 8
In Vivo Studies Related to Lifespan
Autophagy InductionStudy No.: 2023794
[0315] Autophagy is essential for maintaining healthy cellular functioning and exhibits a gradual decrease in its capacity during aging which is associated with the incidence of various age-associated degenerative diseases, such cancer, neurodegeneration, tissue atrophy and fibrosis, is linked to defective autophagy. Abnormal cellular metabolism by aging is associated with the formation of unwanted byproducts leading to accumulation. The metabolic byproduct levels are higher at the end of lifespan and also when the oxidative homeostasis becomes unbalanced.
[0316] A tractable model tissue for studying the activity of autophagy is the fat body of the L3 stage Drosophila larva. In this organ at this stage, the basal level of autophagy is almost at an undetectable level. When a compound or environmental factor induces the process, fat body cells display an obvious amount of autophagic structures, autophagosomes and autolysosomes.
[0317] After 3 hours of treating the animals by a SiO.sub.2 product of the QD class, fat bodies were isolated from 5 larvae, then examined by a Zeiss AxioImager M2 epifluorescent microscope equipped with a ApoTome 2 semi-confocal setup. Magnification 400 was used. For preparing pictures, the program Zeiss AxioVison 4.82 was applied. Two different reporters were used, GFP-p62/Ref (2) P and 3mCherry-Atg8a. p62/Ref (2) P is a substrate of autophagy, so its level inversely correlates with the capacity of the process, whereas Atg8a serves as a key marker of autophagy which labels the membrane of early and late autophagic structures, autophagosomes and autolysosomes, respectively.
[0318] SiO.sub.2 products of the QD class were tested on a model obtained from the fat body of L3 stage larvae of Drosophila melanogaster (fruit flya popular in vivo model for autophagy research), after the larvae were fed on a special culture medium containing nanoparticulate SiO.sub.2 based on the developed methodology.
[0319] Using 3mCherry-Atg8a marker, SiO.sub.2 product showed an autophagy-inducing effect at a concentration of 1.2 mg/ml and at a concentration of 0.6 mg/ml in a concentration-dependent manner. Atg8a marker-positive autophagy structures were detected as red dots in Drosophila larval fat body. Nuclei were labeled with Hotchst dye (blue). The active substance of the QD class SiO.sub.2 (ADAM-QD/S) significantly increases the amount of autophagic structures. The control value 21 (area ratio of 3mCherry-Atg8a-positive structures) increased to 41 and 72 by use of QD products SiO.sub.2 in 0.6 mg/ml concentration and in 1.2 mg/ml concentration, respectively.
[0320] The above results indicate that SiO.sub.2 of the QD class has a significant potential in medical application as a potent autophagy-inducing agent which may find its use as a therapeutic substance to alleviate symptoms of age-related degenerative illnesses. Analogously to the previous examples, it is a well-founded explanation to the found effect that the mechanism involves eliminating and, importantly, preventing the formation of underoxidized metabolic byproducts via processes by analogous modulation of redox catalysis as shown in Example 5.
Example 9
In Vitro Studies on Tumour Cell Lines: Antitumour Effects with the Use of TiO.sub.2 Crystals of the New QD Class (ADAM-QD/T)
9.1. In Vitro Studies on Tumor Cell Lines:
[0321] 1. Estimation on changes of cell viability the proportion of apoptotic and necrotic cells in different cell cultures: [0322] HT-29 Human colon adenocarcinoma cell line. [0323] ZR-75.1 Her-2 expressing human breast adenocarcinoma cell line.
[0324] In these studies cells (510.sup.4) were seeded in 6-well plates and cultured in RPMI-1640 medium containing 10% fetal bovine serum. The cultures incubated for 2 hours in CO.sub.2 thermostate at 37 C. After the preincubation period the medium was removed, the cells were washed with physiological saline (21 ml 0.9% NaCl) and were exposed to TiO.sub.2 suspension of the QD class at concentrations of 60 and 120 g/ml for 60 min. After removal of the test compounds, fresh RPMI 1640 medium containing 10% FBS was added and cultures were incubated for 24 h at 37 C. in a humidified thermostat (5% CO.sub.2). At the end of the post-treatment period, the medium and culture supernatants containing released cells were removed and stored for cell counting. Cells in monolayer were released using trypsin EDTA. Cells obtained from both supernatants and monolayers were stained with propidium iodide and processed on a flow cytometer (FACS, Becton Dickinson, USA) Cells in the sub-G1 region were scored as apoptotic cells.
[0325] Results: In short-term studies (i.e., treatment before the onset of mitotic phase), the number of cells in TiO.sub.2-treated cultures decreased slightly. At the same time, a large proportion of the cell population exhibited markers of apoptosis (10% in the control study, but 28% and more than 55% in the presence of TiO.sub.2 of the QD class at concentrations of 60 and 120 g/ml, respectively).
[0326] 2. Measurement of cell kinetic parameters
[0327] To measure cell kinetic parameters, i.e. cells in DNA presynthetic phase (G1), DNA synthetic phase(S), premitotic or mitotic phase (G2/M), HT1080 fibrosarcoma cells were cultured as described above. After treatment for 48 hours, tumour cells were removed from the plates, stained with propidium iodide and cell cycle phases were analysed on a flow cytometer. (Becton-Dickinson USA).
[0328] Results: Treatment with the TiO.sub.2 product of the QD class at a concentration of 120 g/ml induced cell accumulation in the synthetic S-phase of DNA. Compared to the control value of 19.0%, this proportion increased to 26.8% in the presence of TiO.sub.2 at a concentration of 120 g/ml).
9.2. In Vivo Study of the Effect Against Human Tumours in Preclinical Models of HT-1080 Human Fibrosarcoma and ZR-75.1 Breast Adenocarcinoma with Transplantation into Nude Mice with the Use of TiO.sub.2 Crystals of the New QD Class
Experimental Animals:
[0329] C57Bl/6 male mice for transplanting Colon-38 and B-16 melanoma tumor as well as HT1080 human fibrosarcoma cell line and nude mice (SCID, C17/Icr, Charles River Laboratories, Germany) for studying human tumors were used.
[0330] To investigate the effect of TiO.sub.2 crystals of the QD class against human tumors in preclinical models HT-1080 human fibrosarcoma and ZR-75.1 mammary adenocarcinoma were transplanted in nude mice. HT-1080 tumor cells (510.sup.4) were transplanted subcutaneously at the foot pad of the male nude mice. Upon reaching a palpable size (100 mg) treatment was started at 180 microgram nanosized TiO.sub.2 dose, three times in every three days. ZR-75.1 mammary adenocarcinoma cells (510.sup.5) were transplanted in female nude mice at the mammary gland lines. Treatment was started when the tumor reached a palpable size, six times for three weeks. The change of the tumor volumes was determined as described above in case of murine tumors.
[0331] Results: The measurements of the life-span of the tumor bearing nude mice revealed a significant prolongation in the survival time: [0332] 37 days mean survival compared to 32 days recorded for the control group in the Colon-38 transplant group [0333] 28 days mean survival compared to 24 days recorded for the control group in the B-16 melanoma transplant group [0334] 25 days mean survival compared to 15 days recorded for the control group in the HT1080 human fibrosarcoma transplant group
[0335] The results of the in vivo experiments of Example 9.2. confirmed the in vitro data of Example 9.1. that the aqueous solution of TiO.sub.2 crystals of the QD class has therapeutic potency in the test animals bearing human tumours.
INDUSTRIAL APPLICABILITY
[0336] Used elaborated stable suspensions in composition of medical remedies have the following advantages: [0337] It is catalyst of energy-exchange reactions of the ROS synthesis in the body; [0338] Absence of general toxicity; [0339] Absence of allergenic properties, i.e. hypoallergen; [0340] Absence of harmful toxicological compounds; [0341] Heterogenous suspension structures are photocatalysts having catalytic properties; [0342] Have anti-pathogenous antiviral activity, preventing penetration of virus into the cells and promotes destruction of viruses with lipid membrane due to damaging of the latter. [0343] It is possible to effectively influence onto pathogens. [0344] Being a thermal catalyst for the synthesis of ROS, separately and/or in synergy It is possible to use high light powers (5 or more W/sq.Math.cm.) It is possible to accelerate process of influence (up to 1-3 min). [0345] Absence of toxic action of photocatalyst with/without photodynamic therapy (PDT); [0346] Use of stable suspensions, obtained by the elaborated method, ensures antiviral therapeutic effect, antipathogen therapeutic effect, sorption capacity of crystals and particles ensures detoxication of organism, ensures elimination of underoxidation processes of in the body of living organism. [0347] Provides the possibility of developing formulations with other active ingredients as well as forms of application (page 48, publication EP4272745A1).
[0348]