LIPOSOMES CONSISTING MAINLY OF CHOLESTEROL
The subject of the invention is a process for producing particles of bacterium-size, said particles can be obtained from cholesterol, suitable for stimulation of production of anti-cholesterol antibody, with the help of said process liposome particles of Micrococcus size (100-500 nm) from cholesterol of high purity degree can be produced.
In the state of the art there is no effective process for preventing atherosclerosis and there is no really effective medication for curing this disease either. Consequently atherosclerosis seems to belong to the group of incurable diseases.
During our research we observed an antibody in the human body, that reacted with cholesterol. (Horvath, I. Puskas, E. Horvath, Anna Balazs, E. Horvath, A.: Autoantibody or immunological defense against cholesterol, Orvosi Hetilap Medical Weekly 1994. 135.18.965-968) Searching the biological function of antibody we succeeded in producing a composition elaborated on experimental animals to induce production of antibody.
During our examinations for the verification of lues we could detect an antibody, an anticholesterol, reacting with cholesterol in seroconvert serums of non-lues origin, which observation led to the production of the composition in question. When seeking the biological function of the antibody we produced liposome antigens suitable for immunization and we realized, that production of anticholesterol antibody can be induced by them. (Horvath, Anna Narga, L. Horvath, I. Kόkai, M. Karadi, I. Romics, L. Fust, G.: Complement activation in human sera by liposomes made from cholesterol, Mol.Immunology, 1998, 35(6-7):102 (IF: 1,824) Anticholesterol antibody produced this way protected the experimental animals from atherosclerosis. (Horvath, I. Szende, B. Horvath, Anna Kocsis, I. Horvath, A.: Influence of cholesterol liposome immunisation and immunstimulation on rabbits atherosclerosis induced by a high cholesterol diet Med. Sci.Monit. 1998. 4(3):403-
407). During human tests a higher titre of anticholesterol antibody was observed in a sound constitution compared with organism of patients of atherosclerosis of the same age and gender. (Horvath, Anna Prohaszka, Z. Duba, J. Harczos, P. Horvath, I. Nallus, G. Romics, L. Fust, Gy. Karadi, I.: Comperative examination of anticholesterol antibody titre in patiens of atherosclerosis Magyar Belorvosi Archivum Archives of Hungarian Internists 1998/3:239)
According to data of textbooks it is not possible to produce liposome from pure cholesterol, because the crystal form of pure cholesterol prevents the formation of a lipoid membrane.
When working out the invention we set the aim to find a solution for producing particles suitable for stimulation of production of anti-cholesterol antibody.
When realizing the solution according to the invention we recognized, that the membranes of said particles are of Micrococcus size and said membranes comprise of cholesterol of high purity, the set aim can be achieved.
The invention relates to a process for producing particles of bacterium-size, said particles can be obtained from cholesterol, said particles are suitable for stimulation of production of anti-cholesterol antibody characterized by that, membranes of said particles are of Micrococcus size and said membranes comprise of cholesterol of high purity.
In a preferred application of the process according to the invention the structure of the membranes of the particles is retained stable in an electrolytic solution or in a lyophilized form. The particles preferably include different organic materials of great molecules and inorganic materials jointly or separately as well.
The particles preferably in themselves, with electrolytic solutions or filled with organic materials of great molecules are active from an immunological point of view and are suitable for immune-stimulation or immunization.
The particles produced during the process according to the invention are preferably suitable for intravenous injection into any animal (human being) whose blood contains cholesterol among natural circumstances. A preferable application of the particles produced during the process according to the invention 'vacant' particles induce production of anticholesterol antibody.
Another preferable application of the particles produced during the process according to the invention antibodies forming under influences of 'vacant' particles are suitable for prevention and treatment of diseases contracted due to deficiency of anticholesterol antibody or due to decline of antibody titre. Another preferable application of the particles produced during the process according to the invention is that antigen stimulating production of cholesterol effects hormones system substituted from cholesterol, and influences therapy.
The process according to the invention is set forth by the following:
Production of basic materials of the process according to the invention is as follows:
Purification of cholesterol:
Ordinary cholesterol is specially purified for the process:
1. We produce a saturated solution in an organic solvent at 56-90 °C, then it is crystallized by changing the temperature.
2. Crystals are collected on a sterile sieve, dried, then another saturated solution is prepared in another organic solvent at 56-90 °C.
3. It is crystallized by changing the temperature, then collected on an other sterile sieve the crystals are dissolved in a third organic solvent at 56-90 °C, and are crystallized again by changing the temperature.
4. Crystals are collected under sterile conditions, dissolved in an organic solvent at 56-90 °C, and are stored as a supersaturated solution at 5 °C.
Production of liposome:
Supersaturated solution is heated to the level of saturation, then an unsaturated solution is made.
1. We create conditions for producing liquid membranes arranged as crystals by changing the cholesterol in the unsaturated cholesterol solution with continuous, equal changing of the combination of polar and non-polar solutions. Transmitting physical energy of determined amplitude and energy it is transformed to particles having a characteristic structure that can be observed by a microscope.
2. In order to establish stabile particles it is treated for 1 to 10 minutes changing the rate of different solutions equally and continuously, increasing frequency of physical impact many times, then it is dosed to the required volumes after control by a microscope of dark visual field.
Characteristics of particles produced during the process according to the invention are as follows: iff
1. Their outer appearance on an ultramicroscope is similar to Micrococcus.
2. They settle relatively quickly when suspended in electrolytic solutions, and they retain their characteristics for years even at room temperature.
3. Particles can be dried by lyophilization,
4. They tend to agglutination.
5. Agglutinative features are decreased or eliminated in solutions of albumin and globulin.
The process according to the invention will be shown with the following example:
Example 1
20 grams of cholesterol is solved in 1000 ml ethanol and under pressure it is spread with a speed of lOOOul/sec on the 15 ° angle surface dented trough-like of metal sheet of determined silicium content or an acid-proof metal sheet or on the surface of a sheet formed as a pipe, which has equal ultramicro grooves parallel with the axis of the pipe. The three-dimensional sizes of the grooves of the surface parallel with the axis of the trough are controlled by a MULTISCOPE. The grooves can be 100-1000 nm deep and wide.
The sheet receiving the solution is exposed to physical impact with the help of an adjustable oscillator and the surface is treated with an electromagnetic radiation of 270-1080 nm wavelength from an adjustable monochromatic power-source during flowing of the fluid.
The monomolecular bits of membrane arranged by the oscillator and torn to films in the ultra micro grooves are entwined to globular formulas consistent with the 100- 500 nm diameter of the half of the wavelength by electromagnetic radiation. The bacterium-like particles produced by the above process are collected in a physiological solution under sterile conditions and are stored in the form of an 1 mg/ml suspension calculated on the dry-material content of the cholesterol at 5°C in one ml doses or in bigger volume, which can be dozed before use as required.
The advantage of the process according to the invention is that an effective medication for preventing and healing of atherosclerosis can be produced. The particles produced during the process according to the invention are suitable for the production of anticholesterol antibody by active immunization. In the form of intravenous injection the particles are suitable for prevention and treatment of diseases contracted due to deficiency of anticholesterol antibody or due to decline of antibody titre. In case of intravenous injection of the liposome particle suspension the stabilizing role is ensured by well-known additives and additives ensuring isotonic conditions.