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1. WO2020112038 - PROCÉDÉ D'OBTENTION D'UN NANOPARTICULAIRE À ADDITIF D'ARGILE

Note: Texte fondé sur des processus automatiques de reconnaissance optique de caractères. Seule la version PDF a une valeur juridique

[ EN ]

CLAY ADDITIVE NANO-PARTICULATE OBTAINING METHOD

THE RELATED ART

The invention relates to clay additive nano-particulate obtaining method.

The invention particularly relates to a method for obtaining nano-particulates formed by means of making polylactide-co-glicolide (PLGA) mycelium stable by use of very low rates of sodium-montmorillonite (Na-MMT) in order to obtain a drug carrying system of self-assembly for use in intravenous (iv) applications for cancer treatment.

BACKGROUND OF THE RELATED ART

Ordinary development and death processes of cells in body continue in a certain and controlled order. However, growth and death in cancerous cells are out of such order and cells grow and start to reproduce in an uncontrolled way. One of the treatments applied commonly for treatment of cancerous cells is chemotherapy. Purpose in standard applications in chemotherapy treatment is to destroy or prevent growth of cancerous cells. In this line, chemotherapy drugs are administered to body through blood veins. The medicine taken into body via blood veins targets cells growing in uncontrolled way and kills them or prevents their growing.

Some disadvantages may occur during application of chemotherapy. Effective agents can be distributed to entire body through blood circulation and toxic effect may occur. Cancer medicine cannot progress into tumour area in adequate amount and its effect may decrease. In order to eliminate such disadvantages, obtaining effective results by use of lower dosage of medicine, administration of medicine to patient with bio-compatible composites and shortening treatment period can be provided by various treatment methods. Said treatment methods are drug delivery systems defined as“targeted cancer treatment”. Drug delivery systems can be administered orally as well as intravenously (iv) as preferred in cancer treatments in particular. Today among targeted cancer treatments, passive targeting and nano-biotechnological methods are the ones showing the most development.

Passive targeting method is provided by means of concentrating the drug on a point requiring effect of the used drug due to natural features and physiological structure of the tissue. Endothelial cells forming the blood vessels in tumour area are in more separated (about 200 nm) in comparison to those of healthy tissues. In passive targeting method drug delivery system is provided to penetrate into tumour area in passive way when passing through such areas. This effect is defined as the enhanced permeability and retention (EPR) effect. To ensure this effect the drug delivery system has to deliver the drug molecules to the target area without leakage and nano particulate sizes must be under 200 nm. However, targeted drug delivery systems in use today have problems in meeting these two requirements. For instance, drug molecules may leak from walls of liposome formulations or may form in various undesired sizes during formation. Prevention of them is provided by means of solutions adversely affecting current formulation toxicity and production process. Similarly, in another application, mostly toxic surfactants may need to be used in order to facilitate drug loading process and reduce intermediate surface stress and prevent formation of aggregate. In addition, the use of extruder is needed for control of size of forming liposome.

Nano-biotechnological methods are used in self-assembled (SA) drug delivery systems. Such systems are formed by means of combining amphiphilic materials consisting of hydrophilic or hydrophobe blocks in aqua ambience by exposing hydrophilic parts to water and avoiding hydrophobic parts from water. Delivery of drug molecules or interaction with such structure forming subject to feature of drug molecule to resolve in water, with hydrophilic or hydrophobic parts provides delivery. The constituted structures are generally in spherical form subject to concentration of amphiphilic material. When spherical forms are defined as supramolecular structure and the chains forming such structure as monomer, the structure of monomers provides formation of single or double layer supramolecular structures. Monomers in square or rectangular morphology constitute liposomes while triangle form monomers constitute mycelium. Formation of SA drug delivery systems obtained in any circumstances is subject to hydrophobic-hydrophobic attraction forces. And this case causes easy distribution particularly in capillaries having high pressure and areas near cardiac pump without delivery of drug to tumour areas by the nano-particulates. However, this disadvantageous case does not prevent use of said systems. Causes for such cases are high drug delivery capacity, having sizes under 200 nm, simple production technologies arising from easy formation in water and most importantly, existence of multiple bio-compatible and bio-degradable amphiphilic materials. Use of SA drug delivery systems in targeted cancer treatment is considerably common because of said causes on which various studies have been conducted to provide its development. In most of such studies functional groups to form cross-bonds of hydrophilic block are designed and such blocks are interconnected by means of reactions conducted under UV lights and cross-bonding chains. However, while performing such action, most of cross-bonding members have toxic features.

Bio-compatible PLGA is an amphiphilic material used in formation of SA drug delivery systems and used for delivery of many biological active agents from small drug molecules to peptide and proteins. Behaviour of this amphiphilic polymer is controlled by factors such as stereo-chemistry of lactic acid, crystal degree, lactic acid/glycolic acid rate and molecular weight etc. This amphiphilic material exposes the blocks insoluble in water to water and collects the parts disliking water, in the nucleic part and forms supramolecular aggregate similar to mycelium. Like other SA drug delivery systems, when PLGA mycelium is used as targeted cancer treatment agent, despite having multiple superior features, it bears the risk of distribution in blood flow and danger of freeing the delivered drug. In order to eliminate this disadvantage, many studies wrap mycelium formed by PLGA by Polyethylene Glycol (PEG) chains and try to provide environmental stability (Steric Stabilization) for the structure by use of water molecules wrapping PEG. Although this approach is logical, in the end it causes administration of more synthetic agents to body.

In recent years it has been disclosed that in addition to organic material of nanoparticulates used in cancer treatment, it can also be formulated by inorganic materials and this has provided widespread use of nanotechnological developments. It is particularly used as direct or auxiliary agent in formation of clay nano-particulates such as kaolinite, sepiolite and montmorillonite (MMT) of which crystal structure has been discovered. However, clay must be of feature allowing reduction to nano size in order to produce particulate of nano size by use of such materials. It has been proved that clay plays a reinforcing role in nanoparticulates to be prepared and helps formation of a spherical particle.

The application numbered US20060193787A1 discloses development of an orally taken drug delivery system by use of MMT. In said application, nanoparticle anticancer paclitaxel molecules prepared by use of PLGA and MMT were given and oral formulation use is suggested. In this study, Polyvinyl alcohol (PVA) polymer was used for exfoliation of clay and the size of particulate obtained as a result was 305 nm. The size of clay in this delivery composite is too high for intravenous applications. Furthermore, aggregate formation occurs in nanoparticles when centrifuge is used to remove PVA residue in production stage.

The application numbered TR 2017/1 1955 discloses a method of obtaining a bioactive component eluting nanocomposite and basically relates to an orally taken drug delivery system. The application discloses a method for obtaining a bioactive component eluting nanocomposite obtained by use of at least a bioactive component showing bioavailability without use of any organic solvent, a nano delivery agent of layer structure and obtained from an agent of delivery feature by centrifuging and homogenisation method and by use of

an additive enhancing interaction of bioactive component with nano agent. The most important part of the application of the bioactive component eluting nanocomposite is that it is applied after dissolution in a fluid.

In conclusion, the problems mentioned above and not solved in the light of related art have necessitated novelty in the related art.

BRIEF DESCRIPTION OF THE INVENTION

This present invention relates to a method for obtaining a clay additive nano-particulate for cancer treatment in order to eliminate above mentioned disadvantages and provide new advantages in the related art.

Main purpose of the invention is to disclose a method for obtaining a nano-particulate which can be administered intravenously (iv) for targeted cancer treatment.

Another purpose of the invention is to disclose a method for obtaining a nanoparticulate wherein dialysis membrane technique is used without need for use of any extra chemicals.

A further purpose of the invention is to disclose a method for obtaining a nano-particulate not containing any surfactant or any other similar toxic agents.

Another purpose of the invention is to disclose a method for obtaining a characteristic and smoothly dispersed nanoparticulate in sizes under 200 nm.

A further purpose of the invention is to disclose a method for obtaining nanoparticulate from a nano agent wherein layered structure of natural clay minerals is used as joining clips.

Another purpose of the invention is to disclose a method for obtaining nanoparticulate wherein nano agent size is under 100 nm.

Another purpose of the invention is to disclose a method for obtaining a nanoparticulate allowing placement of any drug molecules therein.

In order to achieve the purposes mentioned above and to be described below in the detailed description, the present inventions relate to a method for obtaining nano-particulates used in drug delivery system comprising procedure steps of:

a) reducing a delivery component into nano-delivery component by grinding in an attritor,

b) keeping obtained nano-delivery component in water,

c) dissolving an additive polymer and a drug in acetone and mixing with the nano-delivery component kept in water,

d) dialysing the solution comprising the nano-delivery component, the additive polymer and the drug to form nano-particulate in dialysis membrane against high volume water.

One characteristic of the method disclosed under the invention is that the obtained nanoparticulates comprising a nano-delivery component in range of 17.5 - 73 % and preferably 28.08 % by weight.

Another characteristic of the method disclosed under the invention is that the obtained nanoparticulates comprising an additive polymer in range of 27 - 79,5 % and preferably 70,22 % by weight.

Another characteristic of the method disclosed under the invention is that the obtained nanoparticulates comprising a drug in range of 0 - 3 % and preferably 1 ,7 % by weight.

Another characteristic of the method disclosed under the invention is that the procedure in said step (a) comprising steps of;

running attritor at preferably in range of 150-450 rpm so that grinder and liquid ambience is mixed thoroughly,

feeding the delivery component to preferably ¼ of attritor chamber, grinding in attritor at 2400 rpm at room temperature not less than 8 hours and preferably for 10 hours.

A further characteristic of the method disclosed under the invention is that said nano delivery component is average 30 nm in size.

Another characteristic of the method disclosed under the invention is that the process of keeping nano-delivery component in water in said step (b) lasts at least for 2 hours.

A further characteristic of the method disclosed under the invention is that obtained aqueous nano delivery component is preferably 20 ml and 0,1 % w/v.

Another characteristic of the method disclosed under the invention is that the solution comprising the additive polymer and the drug in said step (c) is preferably 5 ml.

A further characteristic of the method disclosed under the invention is that the dialysis membrane operation in said step (d) is conducted at preferably 37 °C and for at least 24 hours.

A further characteristic of the method disclosed under the invention is that said nano particulate size is preferably 120 nm.

Another characteristic of the method disclosed under the invention is that said nano delivery component is a clay mineral having layered structure and is preferably natural Na-MMT from Tokat Re§adiye region in Turkey.

A further characteristic of the method disclosed under the invention is that said additive polymer is preferably PLGA.

A further characteristic of the method disclosed under the invention is that the obtained nanoparticulates in SA drug delivery system can be administered intravenously.

In order to make the embodiment and additional members being subject of the present invention as well as the advantages clearer for better understanding, it should be assessed with reference to the following described figures.

DETAILED DESCRIPTION OF THE INVENTION

In this detailed description, method for obtaining clay additive nanoparticulate being subject of this invention has been disclosed solely for the purpose of better understanding of the subject and with samples described in a manner not causing any restrictive effect.

Relating to a nano drug delivery system administrable intravenously in cancer treatment, the invention is basically a nanoparticulate composed of a layered nano delivery, an additive polymer and a drug. Method for obtaining said nanoparticulate is achieved in preferably 3 stages. In this line, first of all, delivery component is reduced to nano size by use of mechanical-physical nanoparticle production processes. Then an additive polymer dissolved in acetone, and a drug are added to the solvent consisting of delivery agent reduced to nano size. Lastly, by use of dialysis membrane technique, the obtained mixture is transformed into nanoparticulates which are appropriate for use in drug delivery system and can be administered intravenously.

Procedure steps of method for obtaining nanoparticles are detailed as described below. Firstly, the delivery component is reduced into under micrometer size in wet medium by use of attritor device of preferably model“Union Process 01 -HD”. This device also performs cutting and milling operations in addition to dispersion and grinding processes. For conduct of the procedure, firstly device is operated at low revolutions per minute (150-450 rpm). After grinder and liquid pulp are thoroughly mixed, chamber of the device is fed with delivery component sample in rate of ¼. Then the device is operated at 2400 rpm. The procedure is performed at preferably room temperature. Cooling or heating may cause failure of delivery component so the cooling water was constantly run in process. Following a grinding process not less than 8 hours and preferably 10 hours, nano delivery components of average 30 nm in size are obtained. Nano delivery component is waited in water for preferably at least 2 hours. This procedure is required for maximum interaction between the drug to be obtained in the next step and additive polymer mixer. For preparation nano particulate, additive polymer and drug are dissolved in acetone at room temperature during the next stage too. The volume of the solution is preferably 5 ml. This solution is also mixed with the nano delivery component waited in the water. Aqueous nano delivery component added into the mixture is preferably 20 ml and 0,1% w/v. As the final operation, total solution composed by all components is dialyzed against high volume water in dialysis membrane. Pure water in the external medium is constantly changed during dialysis operation. The operation is conducted at preferably 37 °C and for at least 24 hours. At the end of operation, nanoparticulates are obtained. The obtained nanoparticulates’ sizes are preferably 120 nm. Table 1 given below shows usable range and preferred quantities by weight of raw materials of clay additive nanoparticulate embodiment.

Table 1. Raw materials used in clay additive nanoparticulate embodiment


Any clay mineral having layered structures may be used in the nanoparticulate embodiment being subject-matter of the invention. The absolute condition is that the clay mineral must be bio-compatible and be reducible to nano size. Preferably Na-MMT is used as delivery component. Any additive polymers having SA characteristic in wet medium can be used in nanoparticulate embodiment. The additive polymer used here has to have amphiphilic characteristic, of which one end dissolves in water and the other end dissolves in oil and also has bio-compatible characteristic. As additive polymer preferably PLGA is used. Use of any drug molecules is possible. Particularly, with nanoparticulate embodiment being subject of the invention, the solubility of drugs in water can be enhanced. Moreover, delivery of drug molecules soluble in water is also possible.

Reduction of Na-MMT to nano size by mechanical-physical methods prevents increase in size of nano particulate when it interacts with PLGA during next stages. In addition, in spherical particulate composed of nano Na-MMT and PLGA, stronger particle formation is provided thanks to interconnecting of PLGA chains. This case prevents disperse of nano delivery agent and releasing drug in points such as strokes and fine liver veins. PLGA is bio compatible and bio-degradable polymer. It is referred to as“Gold Standard” by FDA: When entering into interaction with Na-MMT, toxicity of nano-particulate is prevented.

Nanoparticulates obtained by use of method disclosed under the invention can be structured in sizes allowing intravenous administration in targeted cancer treatment applications. Since no chemicals is used during obtaining nanoparticulate process, a nano-particle not containing surfactant or similar toxic agent can be obtained. The invention provides obtaining a nano-particle wherein any drug can be placed and can offer use in many various treatments with SA drug delivery system.