ES2282025B1 - PROCESS OF OBTAINING AND BIOMEDICAL APPLICATIONS OF ALTERNATE POLYESTERS CONSTITUTED BY UNITS OF 4-HYDROXIBUTRIC ACID AND ALIFATIC HYDROXIACIDES. - Google Patents

Abstract

Process for obtaining and biomedical applications of alternating polyesters consisting of units of 4-hydroxybutyric acid and aliphatic hydroxy acids. The invention refers to a new synthesis process that is suitable for the preparation of copolyesters with an alternating sequence and that are constituted by units of 4-hydroxybutyric acid and different hydroxy acids of aliphatic nature. The hydroxy acids can be linear or have lateral methyl groups, such as lactic and 3-hydroxybutyric acids. All polymers can be described by the generic formula: - [O (CH {sub, 2}) {sub, 3} COOCHR (CH {sub, 2}) {sub, n-2} CO] x- where n varies from 2 to 10 and R can be a hydrogen atom or a methyl group. Some of these materials can be used in the biomedical sector as drug releasing systems, with glycolic, lactic and caprolactone derivatives being especially interesting. The degradability of the materials and their solubility in organic solvents that allow their preparation in the form of microspheres in which the required drug is included are key points.

Description

Obtaining process and biomedical applications of alternating polyesters consisting of acid units 4-hydroxybutyric and aliphatic hydroxy acids.

Technical sector

The present invention relates to a new synthesis process of acid-derived copolyesters 4-hydroxybutyric that allows its obtaining with high yields and molecular weights appropriate to your subsequent use The described process allows to synthesize these regular sequence polyesters in a very simple way to a very small number of stages is required.

Within this family of copolyesters it has special relevance the derivative of glycolic acid, material biodegradable and soluble in organic solvents that can used in biomedicine as a drug delivery system, in particular in the form of microspheres. Its thermal stability and its moderate melting point facilitates its thermal processing, guaranteeing at the same time its adaptation to the temperature of use.

State of the art

Biodegradable polymers have great interest for employment in the biomedical sector due to two factors paramount: a) the possibility that the material is removed from naturally once its function is finished, and b) the greatest severity of safety and biocompatibility requirements that biomaterials must present. The applications can vary widely from its use as surgical sutures bioabsorbable until used in tissue engineering or as drug releasing systems. In this last application, the polymeric materials are suitable, being practically impossible retain a drug in an array for an appreciable time constituted by a compound of low molecular weight. The agent active may vary from a growth factor of nature peptide to an antitumor agent, an antibiotic or a Thrombosis preventive compound.

It is interesting that a polymer to be used as a releasing system can be processed in the form of small microspheres, into which the active agent can be distributed evenly. Among the most common methods of preparation can be the extraction or evaporation of an organic phase that is finely dispersed in an aqueous phase (Jain, R., Shah, NH, Malick, AW, Rhodes, CT Drug Development and Industrial Pharmacy 28, 703 -727, 1998), the polymer and drug being dissolved in the first. The microspheres must have a porous structure and a controllable pore size in order to successfully perform their function as a matter transfer system with the surroundings.

Currently, polyhydroxyalkanoates constitute the main family of synthetic polymers used in biomedicine. Specifically, polyglycolide is the most commonly used material in clinical applications, especially as surgical sutures (Schmitt, EE, Polistina, RA, US Pat. 3,297,033, 1967). The polylactide has a lower degradation rate and a greater ease of processing, both factors that extend its applicability. For example, the rapid degradation of polyglycolide can generate an inflammatory response that makes tissue repair difficult when the implanted amount is high. On the other hand, it is difficult to prepare polyglycolide microspheres, suitable for use as drug-releasing matrices, due to their very limited solubility. Polycaprolactone is one of the most used materials as a releasing system due to its reduced glass transition temperature that gives it a rubbery nature, its low melting temperature, its low degradation rate and its ability to form alloys compatible with a wide variety of polymers (Pitt, CG, Marks, TA, Schindler, A., Baker, R. Controlled Release of Bioactive Materials , Academic Press Inc .: New York, 1980). Lactide, glycolide and caprolactone copolymers are also widely used as drug delivery systems and tissue engineering (Tamber, H., Johansen, P., Merkle, HP, Gander, B. Advanced Drug Delivery Reviews , 357, 57, 2005 ;).

Although poly (3-hydroxybutyrate) occurs naturally, its biomedical use is restricted due to its very slow degradation rate in the human body. Copolymers of 3-hydroxybutyric acid and 4-hydroxybutyric acid can also occur naturally (Choi, MH, Yoon, SC, Lenz, RW, Applied and Environmental Microbiology , 1570, 65, 1999) although for the moment their applications are still poorly studied as well. which occurs with the homopolymer of 4-hydroxybutyric acid.

Synthetic polyhydroxyalkanoates mentioned above are usually prepared by mass thermal polymerization by opening glycolide ring, lactide or caprolactone. Naturally occurring polyhydroxyalkanoates they are produced intracellularly by different microorganisms, accumulating in granular form as a source of coal and reserve energetic The composition of polymers and copolymers depends of fermentation conditions and monomers employees.

Recently, a process has been developed for the synthesis of polyesters derived from α-hydroxy acids, mainly polyglycolic and polylactic (Epple, M., Herzberg, O., Journal of Biomedical Materials Research , 43, 83-88, 1998; Herzberg, O ., Epple, M., European Journal of Inorganic Chemistry , 6, 1395-1406, 2001). This can be considered alternative to ring opening polymerization although the molecular weights obtained are very low. The process is based on the thermal polymerization of metal salts of halogenated derivatives of glycolic and lactic acids.

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The method has been tried to extend, although without success, the preparation of polyesters derived from? -Hydroxy acids. On the contrary, the process has been satisfactory when different types of regular polyesteramides are synthesized, including glycolic acid units (Vera, M., Rodríguez-Galán, A., Puiggalí, J., Macromolecular Rapid Communications, 25 , 812-817 , 2004).

The process described here allows us to prepare regular sequence copolyesters and which include acid units 4-hydroxybutyric, also guaranteeing a weight adequate molecular. The synthesis of these copolyesters is achieved starting from a representative monomer of the sequence and having as terminals a halogen atom and a metal salt.

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Description of the invention

The present invention relates to a new Polyester preparation method consisting of units of 4-hydroxybutyric acid and various hydroxy acids aliphatic The process is based on a polymerization of salts Chlorinated derivatives metal. The products obtained are novel by its regular sequence, being particularly interesting the derivative of glycolic acid for its potentials applications in the biomedical field.

The products are semi-crystalline and are characterized by its degradability and its low temperature of glass transition Its solubility in organic solvents allows that can be prepared in the form of microspheres by the method of evaporation of biphasic mixtures.

Polyesters are characterized by the repetitive unit - [O (CH 2) 3 COOCHR (CH 2) n-2 CO] -, being more interesting those with R = H and values 2 and 6 of n . These correspond to derivatives of glycolic acid and ε-caprolactone which are the most frequent units of the polymers currently used in biomedicine. The comonomers are
? -hydroxy acids when R corresponds to a hydrogen atom. The comonomers corresponding to lactic acids ( n = 2 and R = CH 3), 3-hydroxybutyric acid ( n = 3 and R = CH 3) and 4-hydroxyvaleric acid ( n = 4 and R = CH_) are also considered {3}).

The attached scheme illustrates the procedure that has been used to obtain the mentioned alternating polyesters.

To get the regular sequence is done react the 4-chlorobutyryl chloride with the appropriate hydroxy acid in an organic solvent The product to be recovered by evaporation dissolved or suspended in water, and neutralizes with a metal hydroxide (preferably sodium, potassium or cesium) up to a pH close to 10. The corresponding salt recover from the reaction medium and after washing properly it constitutes the monomer that is used in the polymerizations.

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The product is usually highly hygroscopic because what should be taken special care in removing moisture so much if you want to store as if you want to polymerize, avoiding this way possible further degradations.

The polymerizations have the driving force as formation of metallic chloride, when new ester bonds are established between the monomers. The melting temperature of the monomer, the kinetics of the condensation reaction and the optimum temperature of polymerization depend on the metal salts used. In In general, it is observed that both the melting temperature and the temperature of polymerization decrease with cation size. The reaction has preferably place in a liquid state when the polymerization temperature and having the final polymer a low melting point. However, the reaction can start in a state solid in a first stage where the reaction is carried out at reduced temperature. Proper orientation of the reactive groups in the Crystalline solid allows a prepolymer to form quickly. The second stage, carried out at a higher temperature, takes place exclusively in liquid phase, allowing stirring (magnetic or mechanical) of the medium.

Polymerization advances in most of the cases up to molecular weights that can be considered suitable for subsequent applications. Viscosities intrinsic measurements in dichloroacetic acid at 25 ° C can reach be greater than 0.5 dL / g. Sodium salts should polymerize to the highest temperatures, being able in some cases to occur some degradation at high reaction times that limits the molecular size

The polymers are obtained in the form of composite with a polymer matrix that occludes the metal salt. This can be removed by a simple wash with water resulting in a porous nature material. You can also proceed to the partial dissolution of the reaction product, for example by dichloromethane, and the precipitation of the polymer with methanol.

The solubility of these polyesters in organic solvents allow microspheres of the same by the evaporation method of biphasic mixtures. The microspheres can incorporate different drugs and therefore be used as dosing systems. A solution of polymer and the appropriate drug in an organic solvent such as Dichloromethane is dispersed finely in an aqueous phase, keeping a vigorous mechanical agitation. A surfactant is also added such as polyvinyl alcohol and sodium chloride. Agitation is maintains until the complete evaporation of the organic phase, recovering the polymer by centrifugation. For applications dosing, screening is done and the fraction is selected with diameters between about 45 and 80 µm since it Consider the most appropriate. The process allows to obtain microspheres uniform size, rough surface and controllable porosity.

Description of a preferred embodiment

The following examples are not intended to be limitations, but to describe a process of obtaining the polymer with formation of potassium chloride as a driving force and the preparation of Polymer microspheres with the incorporation of a drug. Additionally the degradability of the material is demonstrated and illustrates its suitability as a liberating system.

1. Synthesis of the potassium salt of the carboxymethyl ester of 4-chlorobutanoic acid

Using an addition funnel, 12.5 are added cm3 (0.11 mol) of 4-chlorobutyryl chloride dissolved in 20 cm 3 of anhydrous chloroform at 100 cm 3 of a glycolic acid solution (8.4 g, 0.11 mol) in chloroform anhydrous contained in a flask cooled to 0 ° C by means of a bath of water and salt. The addition is carried out slowly over a period of one hour under vigorous agitation. To neutralize the gases of hydrochloric acid produced during the reaction a tube is used sodium hydroxide filler. Subsequently, the reaction mixture it is kept under stirring at room temperature for three hours more and a water wash is carried out by means of a funnel of extraction. After evaporating the organic phase an oil is obtained colorless with a yield of 75% corresponding to carboxymethyl 4-chlorobutanoic acid ester. This oil is subsequently dissolved in water and neutralized with hydroxide potassium (1 M), keeping the medium at 0 ° C. When the pH reaches a value between 10 and 11, the solution is frozen and lyophilized. After washing abundantly with isobutanol the salt is obtained with a yield 80%

Infrared (KBr, ν, cm -1): 2962 and 2926 (CH 2), 1728 (CO, ester), 1611 (CO, salt), 1210 and 1184 (C-O-C, ester).

1 H NMR (DMSO, 300 MHz): δ 4.03 (singlet, 2H, OCH 2 CO), 3.67 (triplet, 2H, ClCH 2), 2.34 (triplet, 2H, CH 2) COO), 1.92 (quintuple, 2H, C H 2 CH 2 COO) ppm.

13 C NMR (DMSO, 300 MHz): δ 171.95 (COO), 169.28 (COOK), 64.10 (O C H 2 CO), 44.74 (ClCH 2), 31.24
( C H 2 COO), 28.03 ( C H 2 CH 2 COO) ppm.

2. Synthesis of the alternating polyester consisting of units of 4-hydroxybutyric acid and glycolic acid (poly (4HB- alt -glc))

In a flask with agitation and under a inert atmosphere of nitrogen the previous salt is introduced crushed and previously dried in a vacuum desiccator. In a first phase the flask is heated to a temperature of 60 ° C for 2.5 h and then at 100 ° C until the liquefaction of the reaction medium. In a second phase it heats up the flask to a temperature of 150 ° C for 8 h. After cooling until room temperature, a white solid mass is obtained that corresponds to the mixture of polymer and potassium chloride. Polymer dissolved in dichloromethane, separated from chloride by centrifugation, recovered by precipitation with methanol and washed abundantly with water, methanol and ethyl ether. Viscosity intrinsic (measured in dichloroacetic acid at 25 ° C) is greater than 0.5 dL / g while the weight average molecular weight determined by CPG it is greater than 20,000 g / mol. The performance of polymerization is close to 30%.

Infrared (KBr, cm -1): 2969 and 2853 (CH2), 1738 (CO, ester), 1243 and 1156 (C-O-C, ester).

1 H NMR (CHCl 3, TMS, 300 MHz): δ 4.62 (singlet, 2H, OCH 2 CO), 4.24 (triplet, 2H, OCH 2), 2.52 (triplet, 2H , CH 2 COO), 2.03 (quintuple, 2H, C H 2 CH 2 COO) ppm.

13 C NMR (CHCl 3, TMS, 300 MHz): δ 172.00 (CO, butyric), 167.63 (CO, glycolic), 64.15 (OCH 2), 60.64
(COO C H 2 COO), 30.72 (CH 2 COO), 23.75 ( C H 2 CH 2 COO) ppm.

Differential thermal scanning analysis shows that the polymer has a near melting temperature at 110 ° C and a glass transition temperature of -15 ° C. He thermogravimetric analysis indicates that the polymer can be processed from the molten state to the start temperature of decomposition above 250 ° C.

3. Preparation of poly (4HB- alt -glc) microspheres that include the active agent

The polymer microspheres are prepared by the evaporation method of a mixture consisting of a phase Organic and an aqueous phase. 2 g of polymer dissolve in 45 cm 3 of dichloromethane, adding the appropriate amount of drug. The resulting solution is dispersed in 500 cm 3 of a aqueous solution containing 4 g of polyvinyl alcohol (degree of hydrolysis 87-89%, weight average molecular weight of 13000-23000 g / mol) and 5 g of sodium chloride, the addition is done while mechanically stirring at a speed of 1,300 rpm. The biphasic mixture is kept under stirring until All dichloromethane evaporates. The microspheres are separated by centrifugation, as after successive washing with water. Only after the last washing stage is the product by filtration.

The granulometric analysis performed using sieves with pore sizes of 1000, 500, 250, 125, 100, 80 and
45 µm shows a bimodal curve with a significant fraction of microspheres with diameters between 45 and 80 µm which is considered the most suitable for use as a releasing system. The observation by scanning microscopy reveals that the particles have a spherical shape and a rough surface.

4. Degradation of poly (4HB- alt -glc)

The microspheres of poly (4HB- alt -glc) are hydrolytically degraded under physiological conditions provided by a phosphate buffer of pH 7.4 and a temperature of 37 ° C. Thus, after 10 and 35 days of exposure, a drop in the average molecular weight by weight is observed from an initial value of 20,000 g / mol to final values of 16,700 and 8,900 g / mol, respectively. Under these conditions, degradation takes place at a moderate speed.

The polymer is easily degraded by the action of enzymes with esterase activity such as Pseudomonas Cepacia lipase. A weight loss of 10% is achieved after 20 days of exposure in a medium at 37 ° C and pH 7.4 containing the enzyme (0.1 mg / cm 3), while the molecular weight is reduced to a value of 10,000 g / mol after only fourteen days of exposure.

5. Triclosan release from poly microspheres (4HB- alt -glc)

The following figure illustrates the kinetics of release of the triclosan drug in a cellular medium (medium of Eagle modified by Dulbecco containing 7.5% (weight / volume) of sodium bicarbonate and 10% (volume / volume) of bovine serum at pH 7.42). It is observed that approximately 80% of the initial charge is releases to the environment after about 50 hours of exposure, time that is insufficient to produce a marked hydrolytic degradation of polymer.

Claims (5)

1. A method of preparing polyesters biodegradable alternating sequence by polycondensation thermal of the metal salt of an acid-derived chloro acid 4-chlorobutyric acid and an aliphatic hydroxy acid. 2 2. A method for preparing biodegradable polyesters according to claim number 1, wherein the aliphatic hydroxy acid is linear and is characterized by a variable number n from 2 to 10. 3 3. A method for preparing biodegradable polyesters according to claim number 1, wherein the aliphatic hydroxy acid has a lateral methyl group (R = CH 3) and a variable number n from 2 to 4. 4 4. A drug dosing matrix constituted by a biocompatible polyester with a structure chemistry where acid units alternate 4-hydroxybutyric acid and a hydroxy acid, whose unit repetitive corresponds to the generic formula: 5 where n varies from 2 to 10, and where R can correspond to a hydrogen atom or a methyl group. 5. Microspheres usable as systems drug releasing and consisting of polyesters where alternating units of 4-hydroxybutyric acid and a any of the following units: glycolic acid, acid lactic acid, 3-hydroxybutyric acid and acid 6-hydroxyhexanoic.