JPS6228125B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to submicroscopic particles formed from polymerized alkyl cyano-acrylates and containing biologically active substances, their preparation and applications.

Particles having a diameter of less than 500 nanometers and formed from polymerized substances and their preparation and use in particular as supports for biologically active substances are known. That is, Belgian patents No. 808034 and No. 839748 contain, for example, methyl methacrylate, butyl methacrylate,
Submicroscopic particles formed from polymerizable materials such as derivatives of acrylic acid or methacrylic acid, such as methacrylamide or mixtures of the same, are described. Submicroscopic particles formed by micellar polymerization of various monomers such as these have two properties. (1) Such particles may be fully or partially loaded with biological substances; (2) Such particles may, for example, be used for parenteral administration of the particles loaded with biologically active substances. It is possible to form colloidal aqueous solutions that allow

However, the polymers of acrylic acid or methacrylic acid described above for the preparation of submicroscopic particles containing biologically active substances are substantially stable, so that the particles remain in the tissue or cavity for a long time to which they are administered. remains unchanged over time. This thing is
This is particularly a disadvantage when administered parenterally to the human body.

The present invention uses alkyl-cyano-
The above drawbacks are improved by using an acrylate polymer. Such polymers, which are already used in surgery as tissue and hemostatic adhesives, are completely biodegradable.

In the electron microscope (Figures 1 and 2),
The submicroscopic particles according to the invention are in the form of balls with a highly dense, polymeric, multispheroidal network with a diameter of less than 500 nm, preferably less than 200 nm. The particles are formed by polymerization of alkyl-cyano-acrylates (alkyl means lower alkyl radicals having 1 to 4 carbon atoms, especially methyl radicals).

The particles according to the invention contain within their fibrous network a biologically active substance, which can be, for example, a pharmaceutical or diagnostic product for humans or veterinary animals. As a medicine, certain named chemical agents with pharmacological properties, such as methotrexate, actinomycin D
(actinomycin D), adriamycin, daunorubicin, bleomycin
and anti-mitotic or anti-tumor agents such as vincristine; antibiotics such as penicillin, cephalosporins and nalidixic acid; antibiotics of the aminoglycoside type and the virginiamycin family and hormonal agents, especially steroid hormones. It can be given. Such drugs can be particularly high molecular weight compounds such as insulin and heparin. Medicines also consist of biological products such as antigens, antibiotics, enzymes, proteins, viruses, or components found in viruses, bacteria, or cells. Submicroscopic particles according to the invention may also contain diagnostic products such as fluorescein and radioactive human serum albumin.

In human or veterinary medicine, the submicroscopic particles according to the invention can be used with suitable excipients to
It can be administered orally, subcutaneously, intradermally, intramuscularly or intravenously. Due to their ability to diffuse into tissues, the particles are therefore particularly suitable for general therapy.

The invention will be further explained with reference to the accompanying drawings.

FIG. 1 is a micrograph (scanning electron microscope) showing the morphological appearance of polyalkylcyanoacrylate nanoparticles.

FIG. 2 is a micrograph (electron microscope) showing the internal structure of polyalkylcyanoacrylate nanoparticles after cryogenic crushing.

FIG. 3 is a graph (% on the Y axis, time (hr) on the X axis) showing the kinetics that actinomycin D exhibits when dissociated from polyalkyl cyanoacrylate nanoparticles into serum fluid.

In this graph: (1) Curve A relates to polymethylcyanoacrylate.

(2) Curve B relates to a 2:1 mixture of polymethyl and polyethyl cyanoacrylate.

(3) Curve C relates to a 1:1 mixture of polymethyl and polyethyl cyanoacrylate.

(4) Curve D relates to a 1:2 mixture of polymethyl and polyethyl cyanoacrylate.

(5) Curve E relates to polyethylcyanoacrylate.

Therefore, the formulation of submicroscopic particles according to the invention has a significant effect on the biological degradability of the polymerized material.
It relies on the gradual dissociation of biologically active substances. Since the biodegradability of alkyl polycyanoacrylates depends on the nature of the alkyl chain, the production of biodegradable polymerized states corresponding to established programs for dissociation of biologically active substances It is possible to choose things. The kinetics of degradation (degradation) of such nanoparticles within the serum as well as the dissociation of the adsorbed drug can be completely controlled and programmed according to the desired therapeutic effect. Such an objective can be achieved by using a suitable mixture of nanoparticles (Figure 3).

Other advantages of alkyl cyanoacrylates, compared to other acrylic derivatives traditionally used for the preparation of submicroscopic particles containing biologically active substances, are that they Exists. In fact, compared to other acrylic derivatives which require energy loading during polymerization which tends to compromise the stability of the adsorbed active substance, alkyl cyanoacrylates can be easily polymerized without such loading.

In the process according to the invention for the preparation of submicroscopic particles, the monomers are added to an aqueous solution of a surfactant, preferably a nonionic surfactant such as, for example, monolaurate of polyhydroxyethylated sorbitan. and subjected to vigorous agitation to form a micellar solution. The pH of the solution should therefore be such that pharmacologically acceptable acids such as hydrochloric, hydrobromic, hydroiodic, sulfuric, phosphoric, acetic, succinic or phosphoric acids are compatible with the medium components. 7, preferably 2, with such acids, as long as the
and 3. Alkyl-cyano-
The acrylate is polymerized at room temperature or even at temperatures below room temperature and the biologically active substance is added to the medium either before introduction of the monomer or after polymerization. The pH of the medium depends on the degree of polymerization and
When the biologically active substance is in an ionized state, both the degree of adsorption of the substance and the degree of adsorption of the substance are adjusted.

The polymerization process slows down when the PH decreases and adsorption of biologically active substances decreases when the substance is in a highly lipid-soluble form, i.e. in a non-ionized state or otherwise. It should be noted that the maximum is reached when the medium PH corresponds to the pKa value of the biologically active substance. As a practical matter, these two requirements are firstly set at a pH that allows control of the reaction, preferably between 2 and 3.
This can be achieved by carrying out the adsorption after the polymerization, with the PH value of the medium being adjusted, if necessary, to correspond to the pKa of the biologically active substance. .

The invention will be further illustrated by the following examples.

Example 1 In 45.5 ml of distilled water, 180 mg of monolaurate of polyhydroxyethylated sorbitan, hydrochloric acid (0.1N)
Dissolve 4.5 ml and 1 mg of ground actinomycin D and slowly add 0.83 ml of methyl-cyano-acrylate with vigorous stirring. Stirring is maintained for 30 minutes after the monomer addition is complete. The spontaneous polymerization makes the suspension initially opalescent and then gives it an opalescent appearance. The reaction medium was mixed with a few drops of normal caustic soda and phosphate buffer (30 ml of potassium phosphate (0.2M) and 30 ml of sodium hydroxide (0.2M)).
ml to 200 ml) buffer to pH 7 with 5 ml,
And centrifuge at 50000G centrifugal force.

Radiation measurements in the supernatant liquid and in the centrifugation residue (liquid scintillation) show that the amount of actinomycin D deposited on the particles corresponds to 90% of the amount used. It is possible to confirm that Examination of the particles by electron microscopy after cryo-fracture revealed that these particles were substantially spherical particles with a diameter of less than 200 nm.

Example 2 In 45.5 ml of distilled water, 180 mg of monolaurate of polyhydroxyethylated sorbitan, hydrochloric acid (0.1N)
4.5 ml and 1 mg of ground actinomycin D are dissolved and then 0.83 ml of ethyl cyanoacrylate is added with vigorous stirring. Stirring is continued for 1 hour after complete addition of the monomer. The reaction medium was passed through a fritted glass with pores having a diameter between 9 and 15 microns, and the solution was mixed with a few drops of 1N caustic soda and a phosphate buffer (potassium phosphate (0.2M ) 50ml and 30ml of sodium hydroxide (0.2M) to make 200ml)
The pH was adjusted to 7 using a method of water and centrifugation was performed at a centrifugal force of 50,000 G.

Radiation measurements (liquid scintillation) in the liquid floating on the surface and in the centrifugation residue show that the amount of actinomycin D attached to the particles is approximately equal to the amount used.
It is possible to confirm that it corresponds to 85%.
In a scanning electron microscope, the particles can be observed to have the same structure and dimensions as the particles obtained in Example 1.

Example 3 300 mg of monolaurate of polyhydroxyethylated sorbitan and 1 mg of actinomycin D are dissolved in 30 ml of hydrochloric acid (0.1N) and then the process of Example 1 is followed with 0.5 ml of methyl-cyano-acrylate. Under these conditions, particles comparable to those obtained in Example 1 are obtained. The diameter of the particles is generally between 300nm and 500nm.

Example 4 In 30 ml of distilled water, dissolve 3 ml of hydrochloric acid (0.1N) and 50, 100, 200, 300 and 700 mg each of morelaurate of polyhydroxyethylated sorbitan. Gradually and with vigorous stirring, add 0.5 ml of methyl cyanoacrylate, then add Example 1
Continue the process. At the end of each step, particles are obtained which are morphologically and granulometrically equivalent to the particles obtained in Example 1.

Example 5 The process of Example 1 is carried out without the addition of actinomycin D. Under these conditions, electron microscopy after cryofracture reveals active substance-free particles with an internal structure consisting of a network of somewhat spherical, very dense polymers with a diameter smaller than 200 nm and a large specific surface area. can get. These nanospheres have no outer covering and therefore cannot belong to nanocapsules. (Figure 2) Example 6 80 mg of polyhydroxyethylated sorbitan morelaurate and 4.5 ml of hydrochloric acid (0.1N) were dissolved in 45.5 ml of distilled water, and then methyl-cyano-
0.83 ml of acrylate is added gradually and with vigorous stirring and stirring is continued for 30 minutes after complete addition of the monomer. This reaction medium was mixed with a few drops of normal caustic soda and a phosphate buffer (50 ml of potassium phosphate (0.2M) and 30 ml of sodium hydroxide (0.2N)).
ml to 200 ml), add 1 mg of ground actinomycin D with stirring, continue stirring for 30 minutes, and then centrifuge at 50000 G centrifugal force.

Measurements in the liquid floating on the surface and in the centrifugation residue (liquid scintillation) show that the amount of actinomycin D attached to the particles is 66% of the amount used.
It turns out that it corresponds to %.

Example 7 In 50 ml of distilled water, 250 mg morelaurate of polyhydroxyethylated sorbitan, hydrochloric acid (0.1 N) 5
ml and 10 mg of fluorescein are dissolved, then 0.6 ml of ethyl-cyano-acrylate is slowly added with stirring, stirring is continued for 30 minutes after complete addition of the monomer, then the suspension is dissolved by addition of distilled water.
Dilute to 200 ml and then centrifuge the reaction medium at a centrifugal force of 50000 G. Fluorophotometric measurements of fluorescein in the surface liquid and centrifugation residue show that the amount of fluorescein attached to the particles corresponds to approximately 65-70% of the amount used.

Example 8 In 45 ml of distilled water, 200 mg of monolaurate of polyhydroxyethylated sorbitan, 5 ml of hydrochloric acid (0.1 N)
ml and methotrexate (methotrexate) 5
mg is dissolved and then 0.83 ml of methyl-cyano-acrylate is added gradually with vigorous stirring and stirring is continued for 30 minutes after complete addition of the monomer. 20 ml of this reaction medium is mixed with a few drops of normal caustic soda and 50 ml of phosphate buffer (potassium phosphate (0.2M)).
and sodium hydroxide (0.2N) (30 ml to 200 ml) to bring the suspension to 25 ml. The reaction medium is then
Centrifuged at 50000g.

Fluorophotometric measurements of methotrexate in the surface flotation liquid and centrifugation residue show that the amount of methotrexate attached to the particles corresponds to approximately 25% (±50%) of the amount used.

Example 9 In 50 ml of distilled water, 250 mg monolaurate of polyhydroxyethylated sorbitan, 5 ml of hydrochloric acid (0.1 N)
ml and daunorubicin (10 mg), then methyl-cyano-acrylate
0.6 ml are added gradually with vigorous stirring (stirring is continued for 30 minutes after complete addition of the monomer), the reaction medium is then adjusted to 100 ml by the addition of distilled water, and the suspension is dissolved in sodium carbonate ( Buffer to pH 9 by adding a few drops of borate buffer (boric acid/potassium chloride mixture (0.2M) 50
ml and 21ml of sodium hydroxide (0.2N) to 200ml
Adjust the volume to 200 ml (prepared above) and centrifuge at 50,000 G.

By fluorometric measurements of daunorubicin in the surface flotation fluid and centrifugation residue, it is possible to determine that the amount of daunorubicin attached to the particles corresponds to approximately 85% of the amount used.

Example 10 200 mg of monolaurate of polyhydroxyethylated sorbitan, 5 ml of hydrochloric acid (0.1N) and 5 mg of actinomycin D are dissolved in 50 ml of apyrogenic distilled water and methylated with vigorous stirring.
0.83 ml of cyano-acrylate was added and the medium was brought to a pH of 7 with a few drops of normal caustic soda and a phosphate buffer (50 ml of potassium phosphate (0.2M) and 30 ml of sodium hydroxide (0.2N) adjusted to 200 ml). The reaction medium was then sterilized, strained and placed in glass bottles, each containing actinomycin.
Dispense at the rate of 0.5 mg of D, seal the glass bottle,
Store away from light. These bottles contain unit dosages suitable for their administration after reconstitution in an isotonic diluent of a type suitable for either topical parenteral administration or intravenous injection.

Example 11 Dissolve 180 mg of polyhydroxyethylated sorbitan and 4.5 ml of hydrochloric acid (0.1N) in 45.5 ml of distilled water, add 800 mg of amylose, and heat this suspension to about 80°C to completely dissolve amylose. The solution is then cooled and filtered.
Mix 50ml of propylene glycol with this liquid,
Then 0.5 ml of butyl-cyano-acrylate was added with stirring, and after the addition of the monomers was complete, 4 ml of butyl-cyano-acrylate was added with stirring.
Continue stirring for an hour and then mix this medium with a few drops of specified caustic soda and phosphate buffer (50 ml of potassium phosphate (0.2M) and 30 ml of sodium hydroxide (0.2N)).
ml to 200 ml) Buffer to pH 7 with 10 ml.

Example 12 180 mg of monolaurate of polyhydroxyethylated sorbitan and 4.5 ml of hydrochloric acid (0.1N) are dissolved in 45.5 ml of distilled water, and 0.83 ml of alkyl-cyano-acrylate (methyl or ethyl) is gradually added with vigorous stirring. and this stirring was continued for 1 hour after the addition of said monomers was completed, and then insulin 250 UI was dissolved in the suspension of nanoparticles produced;
After 1 hour of contact, the reaction medium was mixed with a few drops of normal caustic soda and a phosphate buffer (50 ml of potassium phosphate (0.2M) and 30 ml of sodium hydroxide (0.2N)).
ml to 200ml), buffered to PH7, 50000G
Centrifuge with centrifugal force.

Radioimmunological measurements of insulin in the surface fluid and centrifugation residue showed that the amount of insulin attached to the nanoparticles was 80% of the amount used in the case of polymethylcyanoacrylate nanoparticles.
%, and in the case of nanoparticles of polyethyl cyanoacrylate, it is possible to quantify that it corresponds to 75% of the amount used.

After subcutaneous administration of the latter to male WHISTAR R mice, which had been rendered diabetic by intramembranous injection of alloxan, a blood sugar-reducing effect was obtained 24 hours later.

Example 13 180 mg monolaurate of polyhydroxyethylated sorbitan, hydrochloric acid (0.1 N) in 45.5 ml distilled water.
4.5ml and tritiated vinblastine 17.5
Dissolve mg of ethyl cyano-
0.83 ml of acrylate is added, stirring is continued for 1 hour after the addition of the monomer, and the reaction medium is mixed with a few drops of normal caustic soda and a phosphate buffer (50 ml of potassium phosphate (0.2M) and sodium hydroxide (0.2N)). The mixture was buffered to PH7 using 5 ml (30 ml was adjusted to 200 ml) and centrifuged at 50,000 G centrifugal force.

By radioactivity measurements in the surface flotation fluid and centrifugation residue (liquid scintillation) it is possible to quantify that the amount of vinblastine attached to the particles corresponds to approximately 70% of the amount used before polymerization.

When vinthrustine is combined with polyethylcyanoacrylate nanoparticles, the distribution of this drug within the body is severely limited after its intravenous administration to male WHISTAR R mice. Tissue concentrations significantly higher than those of the pure product are observed in the liver, spleen, lung and muscle.

Example 14 180 mg monolaurate of polyhydroxyethylated sorbitan, hydrochloric acid (0.1 N) in 45.5 ml distilled water.
4.5 ml and 40 mg of Levamisol chlorohydrate were dissolved and 0.750 ml of methyl-cyano-acrylate was added gradually with vigorous stirring, this stirring continued for 30 minutes after the addition of the monomers was completed, and then the reaction The medium is a few drops of normal caustic soda and 5 ml of phosphate buffer.
After 24 hours, the suspension was buffered to pH 7 by
Centrifuge with centrifugal force.

By spectrophotometric measurements (212 nm) of levamisole in the surface flotation liquid and centrifugation residue, it is possible to quantify that the amount of active ingredient attached to the nanoparticles corresponds to 25% of the amount contained.

Example 15 180 mg monolaurate of polyhydroxyethylated sorbitan, hydrochloric acid (0.1 N) in 45.5 ml distilled water.
4.5 ml and 50 mg of Potassium V penicillin are dissolved, 0.800 ml of methyl-cyano-acrylate is slowly added with vigorous stirring, stirring is continued for 30 minutes after complete addition of the monomers, and the reaction medium is then dissolved in a few drops of normal caustic soda and phosphate buffer 5
ml to pH 7 and centrifuge the suspension at 50,000 G centrifugal force.

Spectrophotometric measurements in the surface-floating liquid (after decomposition of penicillin by imidazole and reaction with _HgCl2 ,
325 nm), it can be determined that the amount of active ingredient attached to the nanoparticles corresponds to approximately 50% of the amount contained.

[Brief explanation of the drawing]

FIG. 1 is a photomicrograph (electron microscopy scan) showing the morphological appearance of polyalkylcyanoacrylate nanoparticles. FIG. 2 is a micrograph (electron microscope) showing the internal structure of polyalkylcyanoacrylate nanoparticles after cryogenic crushing. FIG. 3 is a graph showing the kinetics (% on the Y axis, time (hr) on the X axis) when actinomycin D is dissociated from polyalkyl cyanoacrylate nanoparticles into serum fluid.

Claims (1)

[Scope of Claims] 1 Biologically degradable, formed by the weight of micelles of alkyl cyanoacrylates having an alkyl group of 1 to 4 carbon atoms in a surfactant solution with a pH of 7 or less, Biologically degradable high-density ultrafine particles having a diameter of 500 nm or less, comprising a spherical, dense and fibrous polymer network, in which a biologically active substance is dispersed. 2. Biologically degradable high-density ultrafine particles according to claim 1, which have a diameter of 200 nm or less. 3. Biologically degradable high-density ultrafine particles according to claim 1 or 2, characterized in that they are formed by polymerization of methylcyanoacrylate. 4. Biologically degradable high-density ultrafine particles according to claim 1 or 2, characterized in that they are formed by polymerization of ethyl cyanoacrylate. 5. Biologically degradable high-density ultrafine particles according to claim 1 or 2, characterized in that they are formed by polymerization of butyl cyanoacrylate. 6 consisting in a mixture of particles formed from different alkyl cyanoacrylates, thereby having within said mixture biologically degradable particles with different biological degradability. Biologically degradable high-density ultrafine particles according to claim 1 or 2. 7 Biologically active substances include substances with anti-fission, anti-tumor, antibiotic or hormonal properties, viruses, viral components, bacterial components, cellular components,
Claims 1 to 6, characterized in that they are products of antigens, allergens, or enzymes.
2. Biologically degradable high-density ultrafine particles according to any one of paragraphs. 8. Biologically degradable high-density ultrafine particles according to any one of claims 1 to 6, characterized in that the biologically active substance is a diagnostic product. 9 Adjust the pH value to lower than 7 with a formulatable acid,
a step of preparing an aqueous solution in which a surfactant is dissolved; a step of adding an alkyl cyanoacrylate having an alkyl group having 1 to 4 carbon atoms to the aqueous solution while stirring the aqueous solution; stirring until substantially all of the alkyl polycyanoacrylate has polymerized into biologically degradable particles comprising the alkyl polycyanoacrylate, and adding a biologically active substance to the reaction solution either before or after the polymerization. a biologically active substance having a diameter of 500 nm or less and containing a biologically active substance; A method for preparing high-density ultrafine particles that can be biodegraded. 10. Preparing an aqueous or colloidal solution containing a biologically active substance and a surfactant, adjusted to a pH value lower than 7 with a formulatable acid, and containing 1 to 4 carbon atoms in the aqueous or colloidal solution.
a step of adding an alkyl cyanoacrylate having alkyl groups with stirring, and a step of stirring until substantially all of the alkyl cyanoacrylate introduced into the reaction solution is polymerized into ultrafine particles containing alkyl polycyanoacrylate. The method according to claim 9, characterized in that: 11. A step of preparing an aqueous solution containing a surfactant and adjusted to a pH value lower than 7 with a formulatable acid, adding an alkylcyanoacrylate having an alkyl group having 1 to 4 carbon atoms to the aqueous solution with stirring. a further step of stirring until substantially all of the alkyl cyanoacrylate introduced into the reaction solution is polymerized into ultrafine particles containing alkyl polycyanoacrylate, and adding a biologically active substance to the resulting suspension of ultrafine particles. 10. The method according to claim 9, comprising the step of adding. 12 Biologically active substances have anti-nuclear, anti-tumor, antibiotic or hormonal properties, viruses, viral components, bacterial components, cellular components,
12. The method according to claim 9 or 11, characterized in that it is a product of an antigen, an allergen or an enzyme. 13. A method according to any of claims 9 or 11, characterized in that the biologically active substance is a diagnostic product. 14 The aqueous solution or colloidal solution, before adding the alkyl cyanoacrylate to the solution,
The method according to any one of claims 9 to 13, characterized in that the pH value is adjusted to a range of 2 to 3. 15. After the suspension of biologically degradable high-density ultrafine particles containing the biologically active substance is formed, the pH value of the suspension is adjusted to a value of about 7. The method according to any one of claims 9 to 14. 16 After the suspension of ultrafine particles is formed,
15. The method according to any one of claims 9 to 14, characterized in that the pH value of the suspension is adjusted to a value corresponding to the PKa of the biologically active substance. 17. The method according to any one of claims 9 to 16, wherein the alkyl cyanoacrylate is methyl cyanoacrylate. 18. The method according to any one of claims 9 to 16, wherein the alkyl cyanoacrylate is ethyl cyanoacrylate. 19. The method according to any one of claims 9 to 16, wherein the alkyl cyanoacrylate is butyl cyanoacrylate. 20. A method according to any of claims 9 to 19, characterized in that the biologically degradable high-density ultrafine particles have a diameter of less than 200 nm.