EP4654950A1

EP4654950A1 - A method for the production of polyprenol nanoemulsion and the polyprenol nanoemulsion obtained

Info

Publication number: EP4654950A1
Application number: EP24735026.7A
Authority: EP
Inventors: Arturs RUBENS; Ilze Barene; Irena Daberte; Vadims Bartkevics; Juris Rubens; Viktor ROSHCHIN; Yury MARAKHOUSKI
Original assignee: Pharmlacrus Sia
Current assignee: Pharmlacrus Sia
Priority date: 2023-08-09
Filing date: 2024-05-20
Publication date: 2025-12-03
Also published as: WO2025034098A1

Abstract

A method for the production of a polyprenol nanoemulsion involving: - mixing of polyprenols with divalent alcohol in a mass ratio of 1:0.5-2; - the addition of an emulsifier containing polyethylene glycol sorbitan monooleate at a proportion of 50-100% of the mixture of polyprenols and divalent alcohol by mass; - mixing and heating of the resulting mixture for 5-15 minutes at a temperature of 70-80 °C; - dissolving NaCI in water, adding glucose, previously prepared phosphate buffer solution with the pH 6.88 and divalent alcohol, and mixing; - mixing and heating of the resulting aqueous mixture for 5-15 minutes at a temperature of 70-80 °C. A polyprenol nanoemulsion comprising by mass%: polyprenols 2.5-10; emulsifier that contains polyethylene glycol sorbitan monooleate 7-8; divalent alcohol 8-12; NaCI 0.9; glucose 5; phosphate buffer solution with pH 6.88 8-12; water, the rest.

Description

A method for the production of polyprenol nanoemulsion and the polyprenol nanoemulsion obtained

Technical Field

[001] The invention relates to the fields of medicine, veterinary and biopharmaceuticals, specifically to lipophilic biopolymers - acyclic polyisoprenoids (polyprenols) nanoemulsion and the method for their production.

Prior Art

[002] Polyprenols are naturally occurring long-chain isoprenoid alcohols with the general formula: H-(C5Hs)_n-OH, where n is the number of isoprene units. Polyprenols are usually of natural origin, and with known technologies, they can be obtained from pine or spruce (Pinus sylveslris Picea abies). ginkgo (Ginkgo bilobci). etc. green biomass. Polyprenols are non-toxic (median lethal dose LD50 is greater than 10,000 mg/kg), they have nonspecific multifunctional anti-inflammatory, immunomodulatory, anti -proliferative and cytotoxic properties, and they protect cells and tissues from various injury mechanisms. Several preclinical studies have shown that polyprenols can serve as a basis for therapeutic agents for treating several diseases and clinical conditions, including cancer, leukemia, stroke, myocardial infarction, rheumatoid arthritis, inflammatory bowel disease, pancreatitis, sepsis, and other medical purposes. Furthermore, nanoemulsions composed of zwaceae-derived polyprenol oils demonstrate long-term physical stability and stimulate in vitro cytokine production from human cells. Nanoemulsions derived from Pinaceae polyprenol oils improved pandemic influenza vaccine efficacy [1],

[003] Polyprenols have conspicuous lipophilic and hydrophobic properties and are poorly soluble in water. Therefore, they have low bioavailability, and pretreatment is required to use them to develop active medicinal products.

[004] It is known that nanodispersions (submicron dispersions) can improve the bioavailability of lipophilic, hydrophobic, water-insoluble components or natural biologically active substances. Such nanodispersions usually contain dispersed phase particles in solid or semi-solid form, while nanoemulsions are in the form of droplets (liquid), whose size does not exceed 500 nm. The droplet size of polyprenol in nanoemulsions should be in the range of 50-200 nm, which would give them the ability to cross various biological barriers and, as a result, ensure more effective use in pharmaceuticals [2],

[005] Particles of the dispersed polyprenol phase are dispersed in the dispersion medium. Nanodispersions are thermodynamically unstable systems that are characterized by aggregation (in the case of solid particles) and flocculation (in the case of droplets) of the particles of the dispersed phase, which in general creates instability of the nanodispersion and limits its storage time [3],

[006] Spontaneous emulsification methods based on water-soluble organic solvents such as ethanol, isopropanol, n-propanol, etc. are known - they are called self-emulsifying nanosystems. These self-emulsifying systems can contain surfactants in high concentrations (on the boundaries of toxicity), however, they are not stable enough and can degrade themselves under the influence of various factors (dilution, changes in electrolyte balance, etc.) [4],

[007] A method for obtaining a nanoemulsion of polyprenols by the inverse phase emulsification technique [5] is known, which includes dissolving polyprenols (10 g) in hexane (90 g), a mixture (17.5 g) of an emulsifier (in the ratio of sorbitan monooleate 51.4% and polyethylene glycol sorbitan monooleate 48.6%) gradually addition and stirring for 1 hour at 40°C to partially remove the hexane, and gradually addition of water by stirring until phase inversion occurs, where the polyprenol droplets pass from the hexane phase of the organic solvent to the water phase. Afterwards, the obtained mixture is heated at 60°C for 10-12 minutes at a stirring speed of 10,000-17,000 rpm. When stirring, the hexane is removed, and a polyprenol nanoemulsion in water is obtained with a polyprenol droplet size of 12.5-866 nm (with an average polyprenol droplet size of 90-290 nm). However, this known method has several drawbacks, as it is necessary to use different emulsifiers to stabilise the emulsion, the ratio of hydrophilic/hydrophobic groups must be meticulously adjusted to obtain the emulsion, and hexane is used as an organic solvent, which is difficult to dispose of, and technical solutions with high mixing efficiency are also required, which still results in particles with a very uneven distribution. Furthermore, the known method does not ensure the stability of the particles and does not foresee the further preparation of the polyprenol nanoemulsion to be used for medical purposes.

[008] An emulsion for the prevention of respiratory viral infection is known [6], which includes polyisoprenoids, non-ionic surfactants and water. Polyprenoids are used as polyisoprenoids in a known emulsion, in particular polyprenols from the Siberian spruce (Abies sibirica) green. The form of the finished emulsion is a water microemulsion with a turbidity parameter P, preferably not less than 2.0, with the percentage of the following components, % w/w: Siberian spruce polyisoprenoids - 0.01-5.0; non-ionic surfactant - 0.05-25; water - the rest up to 100%. The main disadvantage of the known emulsion is that large-sized (more than 500 nm) micellar structures are formed, which are not stable and can aggregate for a short time, use of liquids only topically (aerosol, etc.), possible irritating effect on mucous membranes (pH unknown).

Summary of the Invention

[009] The proposed invention solves the technological task of improving the homogeneity and stability of polyprenol nanoemulsion and ensuring its storage time of at least 24 months.

[010] The set goal is achieved by the proposed method of obtaining polyprenol nanoemulsion, which includes (i) mixing polyprenols with divalent alcohol in the weight ratio 0.5-2: 1; (ii) addition of an emulsifier containing polyethylene glycol sorbitan monooleate at 50-100% by weight of the mixture of polyprenols and divalent alcohol; (iii) mixing the resulting composition to obtain polyprenol emulsion and heating for 5-15 minutes at 70-80°C; (iv) gradually adding the obtained polyprenol emulsion to an aqueous solution to obtain a polyprenol nanoemulsion, wherein the preparation of the aqueous solution comprises the following steps: (a) dissolving NaCl in water, adding glucose, a preprepared phosphate buffer solution with pH of 6.5 to 7, and divalent alcohol and mixing up; (b) mixing and heating the obtained aqueous solution for 5-15 minutes at 70-80°C. The total weight of water in the obtained nanoemulsion is 55-70% of the weight of the polyprenol nanoemulsion. The divalent alcohol can be propylene glycol or another diol. The emulsifier can be polyethylene glycol sorbitan monooleate or another surface-active compound.

[011] The polyprenol nanoemulsion obtained by the proposed method contains, % w/w: polyprenols - 2.5-10; emulsifier containing polyethylene glycol sorbitan monooleate - 7-8; divalent alcohol - 8-12; NaCl - 0.9; glucose - 5; phosphate buffer solution with pH 6.5-7 - 8-12; water - the remaining weight. According to a preferred embodiment of the invention, the droplet diameter of the dispersed phase of the polyprenols is 50-200 nm. [012] The proposed method ensures the dissolution of isoprenols in a less harmful organic solvent - isopropanol or some divalent alcohol (this is a more environmentally, user- friendly, and safer process). Since it is not necessary to achieve inverse phase polyprenol emulsification, it is sufficient to add one emulsifier containing polyethylene glycol sorbitan monooleate, and it is not necessary to use different emulsifiers in a certain ratio.

[013] According to the invention, if a polyprenol nanoemulsion intended for injections is prepared, when preparing an aqueous solution, such an amount of NaCl and glucose are dissolved in water that the mass fraction of NaCl in the obtained polyprenol nanoemulsion is 0.9% and the mass fraction of glucose is 5%.

[014] The mass fraction of polyprenols is mostly 2.5-10% of the mass of the polyprenol nanoemulsion. When the mass fraction of polyprenol in the nanoemulsion is less than 2.5%, it is not possible to ensure sufficient pharmaceutical activity. When the mass fraction of polyprenols in the nanoemulsion is greater than 10%, due to the too high content of dispersed phase droplets, their flocculation can occur, which can reduce the stability of the polyprenol nanoemulsion, reducing its storage time.

[015] The polyprenol emulsion in divalent alcohol is gradually added to the aqueous solution until the polyprenol emulsion in the divalent alcohol makes up 10-25% of the weight of the polyprenol nanoemulsion.

Examples of implementation of the invention

Example 1

[016] Polyprenol emulsion in divalent alcohol - propylene glycol was prepared as follows.

2.5 g of polyprenol obtained from conifer green by the method described in patent LV15612 [7] was mixed with 5.0 g of propylene glycol; 7.5 g of an emulsifier containing polyethylene glycol sorbitan monooleate was added, mixed and heated for 10 minutes at 70 °C. 15 g of polyprenol emulsion was obtained.

[017] The aqueous solution was prepared as follows. 0.9 g of NaCl was dissolved in 64.1 g of water suitable for injections, 5 g of glucose, 10 g of pre-prepared phosphate buffer solution pH=6.88 and 5 g of divalent alcohol were added, mixed and heated for 10 minutes at 75 °C. 85 g of aqueous solution was obtained. (Phosphate buffer solution was prepared by taking 0.340 g KH2PO4 and 0.355 g Na2HPO4, mixing with water for injections up to 100.0 ml, pH = 6.88). [018] Polyprenol emulsion in propylene glycol was gradually added to the aqueous solution, obtaining 100 g of polyprenol nanoemulsion.

[019] The obtained nanoemulsion of polyprenols contains, % w/w: polyprenol s 2.5; an emulsifier containing polyethylene glycol sorbitan monooleate 7.5; propylene glycol 10;

NaCl 0.9; glucose 5; phosphate buffer solution with pH=6.88 10.0 water 64.1.

[020] The prepared polyprenol nanoemulsion is filled in ampoules; they are melted and sterilised for 25 minutes in an autoclave at a pressure of 0.5 atm.

[021] The obtained polyprenol nanoemulsion is homogeneous, yellowish-white, opalescent. The storage time of polyprenol nanoemulsion at indoor temperature is at least 24 months, without any signs of precipitation or phase separation.

Example 2

[022] Polyprenol emulsion in divalent alcohol - propylene glycol was prepared as follows. 5.0 g of polyprenols obtained from conifer green by the method described in patent LV15612 were mixed with 5.0 g of propylene glycol, 7.5 g of an emulsifier containing polyethylene glycol sorbitan monooleate was added, mixed and heated for 12 minutes at 70°C. 17.5 g of polyprenol emulsion was obtained.

[023] The aqueous solution was prepared as follows. 0.9 g of NaCl was dissolved in 61.6 g of water suitable for injections, 5 g of glucose, 10 g of pre-prepared phosphate buffer solution of pH=6.88 and 5 g of divalent alcohol were added, mixed and heated for 10 minutes at 75°C. 82.5 g aqueous solution was obtained. (Phosphate buffer solution was prepared by taking 0.340 g KH2PO4 and 0.355 g Na2HPO4, mixing with water for injections up to 100.0 ml, pH = 6.88).

[024] Polyprenol emulsion in propylene glycol was gradually added to the aqueous solution, obtaining 100 g of polyprenol nanoemulsion.

[025] The obtained nanoemulsion of polyprenols contains, % w/w: polyprenols 5.0; an emulsifier containing polyethylene glycol sorbitan monooleate 7.5; propylene glycol 10;

NaCl 0.9; glucose 5; phosphate buffer solution with pH=6.88 10 water 61.6.

[026] The prepared polyprenol nanoemulsion is filled in ampoules; they are melted and sterilised for 25 minutes in an autoclave at a pressure of 0.5 atm.

[027] The obtained polyprenol nanoemulsion is homogeneous, yellowish-white, opalescent. The storage time of polyprenol nanoemulsion at indoor temperature is at least 24 months, without any signs of precipitation or phase separation.

Example 3

[028] A polyprenol emulsion in divalent alcohol - propylene glycol was prepared as follows. 10.0 g of polyprenol obtained from conifer green by the method described in patent LV15612 was mixed with 5.0 g of propylene glycol, 7.5 g of an emulsifier containing polyethylene glycol sorbitan monooleate was added, mixed and heated for 15 minutes at 80° C. 22.5 g of polyprenol emulsion was obtained.

[029] The aqueous solution was prepared as follows: 0.9 g of NaCl was dissolved in 56.6 g of water suitable for injections, 5 g of glucose, 10 g of pre-prepared phosphate buffer solution pH=6.88, and 5 g of divalent alcohol were added, mixed and heated for 10 minutes at 75°C. 82.5 g of aqueous solution was obtained. (Phosphate buffer solution was prepared by taking 0.34 g KH2PO4 and 0.355 g Na2HPO4, mixed with water for injection up to 100.0 ml, pH = 6.88).

[031] The obtained nanoemulsion of polyprenols contains, % w/w: polyprenol s 10 an emulsifier containing polyethylene glycol sorbitan monooleate 7.5; propylene glycol 10;

NaCl 0.9; glucose 5; phosphate buffer solution with pH=6.88 10 water 56.6. [032] The prepared polyprenol nanoemulsion is filled into ampoules, they are melted and sterilised for 25 minutes in an autoclave at a pressure of 0.5 atm.

[033] The obtained polyprenol nanoemulsion is homogeneous, yellowish-white, opalescent. The storage time of polyprenol nanoemulsion at indoor temperature is at least 24 months, without any signs of precipitation or phase separation.

Example 4

[034] A polyprenol emulsion in divalent alcohol - propylene glycol was prepared as follows. 2.5 g of polyprenol obtained from conifer green by the method described in patent LV15612 was mixed with 4.0 g of propylene glycol, 7.0 g of an emulsifier containing polyethylene glycol sorbitan monooleate was added, mixed and heated for 15 minutes at 80°C. 13.5 g of polyprenol emulsion was obtained.

[035] The aqueous solution was prepared as follows. 0.9 g of NaCl was dissolved in 68.6 g of water suitable for injections, 5 g of glucose, 8 g of pre-prepared phosphate buffer solution pH=7 and 4 g of divalent alcohol were added, mixed and heated for 10 minutes at 75°C. 86.5 g of aqueous solution was obtained.

[036] Polyprenol emulsion in propylene glycol was gradually added to the aqueous solution, obtaining 100 g of polyprenol nanoemulsion.

[037] The obtained nanoemulsion of polyprenols contains, % w/w: polyprenol s 2.5 an emulsifier containing polyethylene glycol sorbitan monooleate 7.0; propylene glycol 8;

NaCl 0.9; glucose 5; phosphate buffer solution with pH=7 8 water 68.6.

[038] The prepared polyprenol nanoemulsion is filled in ampoules, they are melted and sterilised for 25 minutes in an autoclave at a pressure of 0.5 atm.

[039] The obtained polyprenol nanoemulsion is homogeneous, yellowish-white, opalescent. The storage time of polyprenol nanoemulsion at indoor temperature is at least 24 months, without any signs of precipitation or phase separation. Example 5

[040] A polyprenol emulsion in divalent alcohol - propylene glycol was prepared as follows. 10.0 g of polyprenol obtained from conifer green by the method described in patent LV15612 was mixed with 6.0 g of propylene glycol, 8.0 g of an emulsifier containing polyethylene glycol sorbitan monooleate was added, mixed and heated for 15 minutes at 80°C. 24.0 g of polyprenol emulsion was obtained.

[041] The aqueous solution was prepared as follows. 0.9 g of NaCl was dissolved in 52.1 g of water suitable for injections, 5 g of glucose, 12 g of pre-prepared phosphate buffer solution with pH=6.5 and 6 g of divalent alcohol were added, mixed and heated for 10 minutes at 75°C. 76 g of aqueous solution was obtained.

[042] Polyprenol emulsion in propylene glycol was gradually added to the aqueous solution, obtaining 100 g of polyprenol nanoemulsion.

[043] The obtained nanoemulsion of polyprenols contains, % w/w: polyprenol s 10 an emulsifier containing polyethylene glycol sorbitan monooleate 8; propylene glycol 12;

NaCl 0.9; glucose 5; phosphate buffer solution with pH=6,5 12 water 52.1.

[044] The prepared polyprenol nanoemulsion is filled in ampoules, they are melted and sterilised for 25 minutes in an autoclave at a pressure of 0.5 atm.

[045] The obtained polyprenol nanoemulsion is homogeneous, yellowish-white, opalescent. The storage time of polyprenol nanoemulsion at indoor temperature is at least 24 months, without any signs of precipitation or phase separation.

[046] According to Examples 1-5 of the implementation of the invention the average droplet diameter of polyprenol nanoemulsion obtained is 50-200 nm [determined by dynamic light scattering equipment). [047] References

1. Fox, C. B et al., Vaccine adjuvant activity of emulsified oils from species of the

Pinaceae family, Phytomedicine,, 2019, 64, 152927,

2. Srivastav, A. K. et al., Lipid based drug delivery systems for oral, transdermal and parenteral delivery: Recent strategies for targeted delivery consistent with different clinical application, J. Drug Delivery Sci. Techno/., 2023, 86, 104526,

3. Ciuca, M. D. et al., Curcumin: Overview of Extraction Methods, Health Benefits, and Encapsulation and Delivery Losing Microemulsions and Nanoemulsions, Int. J. Mol. Sci., 2023, 24(10), 8874, https://doi.org/10.3390/ijms24108874

4. Boateng, I. D., Polyprenols in Ginkgo biloba; a review of their chemistry (synthesis of polyprenols and their derivatives), extraction, purification, and bioactivities, Food Chem., 2023, 418,, 136006, https://doi.Org/10.1016/i.foodchem.2023.136006

5. Tao, R. et al. Polyprenols of Ginkgo biloba enhance antibacterial activity of five classes of antibiotics. Biomed Res. Int., 2016, 2016, 4191938,

6. RU 2 189 231 C.

7. LV 15612 B.

Claims

1. A method for obtaining a polyprenol nanoemulsion comprising the preparation of a polyprenol emulsion in an organic solvent by adding an emulsifier containing a polyethylene glycol sorbitan monooleate; heating and mixing the obtained mixture with water; characterised in that the preparation of polyprenol nanoemulsion emulsion further comprises:

(i) mixing polyprenols with divalent alcohols in a weight ratio of 0.5-2: 1;

(ii) addition of an emulsifier containing polyethylene glycol sorbitan monooleate at 50- 100% by weight of the mixture of polyprenols and divalent alcohol;

(iii) mixing the resulting composition to obtain polyprenol emulsion and heating for 5-15 minutes at 70-80°C;

(iv) gradually adding the resulting polyprenol emulsion to an aqueous solution to obtain a polyprenol nanoemulsion, wherein the preparation of the aqueous solution comprises the following steps: (a) dissolving NaCl in water, adding glucose, a phosphate buffer solution with pH of 6.5 to 7, and divalent alcohol, and mixing; (b) mixing and heating the resulting aqueous solution for 5-15 minutes at 70-80°C.

2. The method according to claim 1, characterized in that in step (iv)(a), such an amount of NaCl and glucose dissolved in water that the mass fraction of NaCl in the resulting polyprenol nanoemulsion is 0.9% and the mass fraction of glucose is 5%

3. The method according to claim 1 or 2, characterized in that the mass fraction of polyprenols in the obtained polyprenol nanoemulsion is 2.5-10% of the weight of polyprenol nanoemulsion.

4. The method according to claim 1 or 2, characterized in that the polyprenol emulsion in the divalent alcohol comprises 10-25% of the weight of the polyprenol nanoemulsion.

5. The method according to claim 1 or 2, characterized in the total weight of water makes up 55-70% of the weight of polyprenol nanoemulsion.

6. A polyprenol nanoemulsion obtained by the method of claims 1-5, containing, w/w%: polyprenols 2.5-10; an emulsifier containing polyethylene glycol sorbitan monooleate 7-8; a di val ent al cohol 8-12; NaCl 0.9; glucose 5; phosphate buffer solution with pH 6.5-7 8-12; water the rest.

7. The polyprenol nanoemulsion according to claim 6, characterized in that the divalent alcohol is propylene glycol.

8. The polyprenol nanoemulsion according to claim 6 or 7, characterized in that the droplet diameter of the dispersed phase of polyprenols is 50-200 nm.