ES2322008B1 - PROCESS OF OBTAINING AND BIOMEDICAL APPLICATIONS OF ALTERNATE POLYESTERS CONSTITUTED BY UNITS OF GLYCOLIC ACID AND ALIFATIC HYDROXIACIDES. - Google Patents

Abstract

Obtaining process and biomedical applications of alternating polyesters consisting of acid units glycolic and aliphatic hydroxy acids.

The invention refers to a new process of synthesis that is suitable for the preparation of copolyesters with an alternating sequence and that are constituted by units of glycolic acid and various hydroxy acids of nature aliphatic

The hydroxy acids can be linear or present lateral methyl groups, as is the case with acid 3-hydroxybutyric.

All polymers can be described by the generic formula:

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- [O (CH 2) COOCHR (CH 2) n-2 CO] x -

where n varies from 3 to 10 and R it can be a hydrogen atom or a group methyl.

Some of these materials can be used in the biomedical sector as drug releasing systems, being derivatives of glycolic acids, 3-hydroxybutyric and 6-hydroxyhexanoic especially interesting.

The degradability of the materials, their biocompatibility and their solubility in solvents volatile organic that allows its preparation in the form of microspheres in which the required drug is included.

Description

Obtaining process and biomedical applications of alternating polyesters consisting of acid units glycolic and aliphatic hydroxy acids.

Technical sector

The present invention relates to a new synthesis process of glycolic acid derived copolyesters and hydroxy acids of an aliphatic nature that allow obtaining them with high yields and molecular weights appropriate to its subsequent use The described process allows to synthesize these regular sequence copolyesters in a very simple way by a very small number of stages is required.

In general, polyesters constitute the group of polymers of greater application in the field of biomedicine, covering applications as diverse as surgical sutures bioabsorbable, bone fixations, dosing systems drugs, or temporary scaffolding. The function that these materials they have to comply by being in contact with the physiological environment is maintain its properties and biocompatibility, during the period of time in which they are used. Once this time has elapsed, the  degradation products generated due to the resorption process Biodegradative should be removed from the body.

Within the family of polyesters are precisely polymers consisting of acid units glycolic / glycolide the most used for applications previously indicated. In this environment, they also have a high relevance homopolymers and / or copolymers derived from acid 6-hydroxyhexanoic / caprolactone, acid 4-hydroxybutanoic / butyrolactone and acid 3-hydroxybutanoic. The present invention allows prepare a family of degradable and biocompatible copolyesters with properties that can be modulated based on the number of methylene groups of the selected hydroxy acid.

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 you have finished your function and, b) the greatest severity of safety and biocompatibility requirements that They must present the biomaterials. Applications may vary. widely since its use as bioabsorbable surgical sutures until used in tissue engineering or as systems drug releasers In this last application, the materials Polymers are suitable, being virtually impossible to retain for a considerable time a drug in a matrix constituted for a compound of low molecular weight. The active agent can vary from a growth factor of a peptide nature to an antitumor agent, an antibiotic or a preventive compound of thrombosis

It is important 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 surrounding environment.

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 a bioabsorbable surgical suture (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) is produced by biotechnological processes, its biomedical use is restricted due to its excessively 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 as occurs with the homopolymer of 4-hydroxybutyric acid.

The naturally occurring polyhydroxyalkanoates 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 on the fermentation conditions and the monomers used. He rest of the synthetic polyhydroxyalkanoates for medical uses usually prepared by thermal polymerization in maca by ring opening of glycolide, lactide or caprolactone.

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). The latter can be considered as an 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, to the preparation of polyesters derived from ? -hydroxy acids.

On the contrary, the process has been satisfactory when applied to the synthesis of several types of regular polyesteramides that include glycolic acid units (Vera, M., Rodríguez-Galan, A., Puiggalí, J. Macromolecular Rapid Communications , 25, 812-817, 2004). Also, it has been possible to apply the method for the preparation of alternating copolyesters consisting of 4-hydroxybutyric acid and various aliphatic hydroxy acids (Martínez-Palau, M., Franco, L., Ramis, X., Puiggalí, J. Macromolecular Chemistry & Physics , 207, 90 2006). In this case, the starting monomer has a terminal derived from 4-chlorobutyryl chloride. Its reactivity is reduced, so the kinetics of thermal polycondensation are slow. The process, however, has proven effective in obtaining a series of aliphatic copolyesters that have in common the presence of 4-hydroxybutyrate units. Recently a patent has been applied for its synthesis and its applications in the field of biomedicine (Martínez Palau, M., Franco, L., Rodriguez Galán, A., Puiggalí, J., Spanish Patent with application number P200502217) .

The process described here allows to prepare copolyesters of regular sequence and which in this case include glycolic acid units, also guaranteeing a suitable molecular weight. The synthesis of these copolyesters is achieved using a representative monomer of the sequence that is prepared from 2-chloroacetyl chloride. The monomer is further characterized by the presence of a halogen atom and a metal salt as terminals. The resulting monomer is much more reactive than the one studied previously (Martínez-Palau, M., Franco, L., Ramis, X., Puiggalí, J. Macromolecular Chemistry & Physics , 207, 90 2006), derived from 4- chloride chlorobutyryl In this way, the new process is distinguished from the process applied to obtain alternating copolyesters derived from 4-hydroxybutyric acid, firstly by the different family of copolyesters that are produced and secondly by the greater reactivity of the new system that affects in milder reaction conditions and in an increase in the molecular sizes of the resulting polymers.

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Description of the invention

The present invention relates to a new Polyester preparation method consisting of units of glycolic acid and various aliphatic hydroxy acids. The process is based on a polymerization of metal salts of chlorinated derivatives. The products obtained are novel by their regular sequence, the acid derivative being particularly interesting 6-hydroxyhexanoic by its potential applications in the biomedical field.

The products are semi-crystalline and are characterized by its degradability, its low temperature of glass transition, and its reduced cytotoxicity. Its solubility in Organic solvents allow them to be prepared in the form of microspheres by the evaporation method of biphasic mixtures.

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

The attached scheme illustrates the procedure that has been used to obtain the mentioned alternating polyesters. The process described is not suitable for 4-hydroxybutyric acid derivative (n = 4 and R = H) due to its high tendency to cycle, giving rise to to butyrolactone, in the course of the synthesis.

To obtain these copolyesters with sequence regular the chloride is reacted from 2-Chloroacetyl with the appropriate hydroxy acid in a organic solvent The product that is recovered by distillation in a ball oven, dissolves or is suspended in water, and neutralize with a metallic bicarbonate (preferably sodium, potassium or cesium) to a pH close to 8. The corresponding salt is 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 hygroscopic so it special care must be taken to remove moisture whether want to store as if you want to polymerize, avoiding this form possible later 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 also having the final polymer a low melting point However, the reaction can be initiated in solid state in a first stage where the process is carried out to reduced temperature The proper orientation of the groups reagents in the crystalline solid allows it to form quickly a prepolymer In this case, a second stage can be carried out higher temperature that occurs exclusively in the liquid phase and allows agitation (magnetic or mechanical) of the medium.

Polymerization advances in most of the cases up to appropriate molecular weights for subsequent Applications. Intrinsic viscosities measured in acid dichloroacetic acid at 25 ° C may be greater than 1.0 dL / g. The monomers that should polymerize at the highest temperatures, they may in some cases suffer some degradation in times of elevated reaction that limits molecular size.

The polymers are obtained in the form of a composite with a polymer matrix that occludes the metal salt. This can be removed by a simple wash with water giving rise to a material of porous nature. It is also possible to partially dissolve the reaction product, for example by dichloromethane, and to precipitate 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 a volatile 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 describe a specific process of obtaining the polymer with metal chloride formation as the driving force and the preparation of polymer microspheres with the incorporation of a drug. Additionally, the degradability of the material, its suitability as a liberating system and its reduced toxicity

1. Synthesis of acid 6- (2-Chloroacetoxy) Hexanoic

Chloroacetyl Chloride Dissolves (7.2 ml, 69 mmol) in 50 mL of anhydrous chloroform. This solution is added. slowly to a 250 mL flask containing 50 mL of a solution of 6-hydroxyhexanoic acid (76 mmol, 1.1 eq) in chloroform. The addition is carried out over a period of 1 h under vigorous stirring and keeping the flask at 0 ° C by water and salt bath. To neutralize hydrochloric acid gases produced during the reaction a gas outlet tube is used sodium hydroxide filler. Subsequently, the reaction mixture it is kept under stirring at room temperature for another three hours and a water wash is performed using a funnel extraction. After evaporating the organic phase an oil is obtained that it is purified in a glass oven for vacuum distillation in a tube of balls. Finally, a colorless oil is obtained with a 50% yield corresponding to the desired product.

Infrared: 3047 (OH, acid), 2941 and 2869 (CH2), 1737 (C = O, ester), 1703 (C = O, acid), 1243 and 1173 (C-O, ester) cm -1.

1 H NMR (CDCl 3, TMS): δ = 4.20 (triplet, 2H, COOCH 2), 4.06 (singlet, 2H, ClCH 2 COO), 2.38 (triplet, 2H, C H 2 COOH), 1.69 (multiplet, 4H, C H 2 CH 2 C H 2 CH 2 COOH), 1.44 (multiplet, 2H, C H 2 CH_ {2 CH2COOH) ppm.

13 C NMR (CDCl 3, TMS): δ = 179.6 (COOH), 167.4 (ClCH 2 C OO), 65.9 (COO C H 2), 40.8 (Cl C H 2 }) 33.7 ( C H 2 CO
OH), 28.1 (COOCH 2 C H 2), 25.2 ( C H 2 CH 2 COOH), 24.1 ( C H 2 CH 2 CH 2 COOH) ppm.

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2. Synthesis of sodium, potassium and cesium salts of 6- (2-Chloroacetoxy) hexanoic acid

These compounds will be prepared by neutralization of an aqueous acid solution 6- (2-Chloroacetoxy) hexanoic acid with the corresponding 1M solution of sodium bicarbonate, potassium or Césico The solution is maintained at a temperature of 0 ° C for The whole process of addition. When a pH of 8 is reached the mixture reaction is frozen and lyophilized. The sodium and potassium salts are Purify by repeated washing with ethyl acetate. The Final monomers are stored in a vacuum desiccator. High reactivity of some products may cause a certain amount of oligomers during the recovery process and / or storage. The indicated returns have been calculated including the amounts of prepolymer formed, while the spectroscopic data correspond only to salts organic synthesized.

Sodium salt : The final yield is close to 97%.

Infrared: 2937 and 2855 (CH2), 1732 (C = O, ester), 1560 (C = O, salt), 1293 and 1263 (C-O, salt), 1189 and 1166 (C-O, ester) cm -1.

1 H NMR (DMSO): δ = 4.32 (singlet, 2H, ClCH 2 COO), 4.02 (triplet, 2H, COOCH 2), 1.87 (triplet, 2H, CH 2 COO
Na), 1.45 (multiplet, 4H, C H 2 CH 2 C H 2 CH 2 COONa), 1.26 (multiplet, 2H, C H 2 CH 2 CH 2 2} COONa) ppm.

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Potassium salt : The final yield is close to 75%.

Infrared: 2929 and 2859 (CH2), 1734 (C = O, ester), 1561 (C = O, salt), 1295 and 1267 (C-O, salt), 1192 and 1165 (C-O, ester) cm -1.

1 H NMR (DMSO): δ = 4.32 (singlet, 2H, ClCH 2 COO), 4.02 (triplet, 2H, COOCH 2), 1.91 (triplet, 2H, CH 2)
COOK), 1.51 (multiplet, 4H, C H 2 CH 2 C H 2 CH 2 COOK), 1.29 (multiplet, 2H, C H 2 CH 2 CH 2 2} COOK) ppm.

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Cesium salt : The final yield is practically quantitative.

Infrared: 2940 and 2864 (CH2), 1739 (C = O, ester), 1559 (C = O, salt), 1286 (C-O, salt), 1190 and 1149 (C-O, ester) cm -1.

1 H NMR (DMSO): δ = 4.31 (singlet, 2H, ClCH 2 COO), 4.01 (triplet, 2H, COOCH 2), 1.83 (triplet, 2H, CH 2 COO
Cs), 1.50 (multiplet, 4H, C H 2 CH 2 C H 2 CH 2 COOCs), 1.27 (multiplet, 2H, C H 2 CH 2 CH 2 2} COOCs) ppm.

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3. Polymerization: synthesis of the alternating polyester consisting of units of glycolic acid and 6-hydroxyhexanoic acid (poly (Glc- alt- 6HH)) from the potassium salt of 6- (2-chloroacetoxy) hexanoic acid

In a reaction tube provided with stirring, the potassium salt of the crushed 6- (2-chloroacetoxy) hexanoic acid previously dried in a vacuum desiccator is introduced. In a first phase the flask is heated under vacuum to a temperature of 70 ° C for 2 h and in a second phase the flask is heated to a temperature of 100 ° C for 2 h. After cooling to room temperature, a white solid mass is obtained that corresponds to the polymer and potassium chloride mixture. The polymer is dissolved in dichloromethane, separated from the chloride by centrifugation, recovered by precipitation with methanol and washed thoroughly with water, methanol and ethyl ether. The intrinsic viscosity (measured in dichloroacetic acid at 25 ° C) corresponds to 1.05 dL / g while the weight average molecular weight determined by GPC is greater than 44,000 g / mol. The polymerization yield is close to
80%

Infrared: 2941 and 2871 (CH2), 1735 (C = O, ester), 1148 (C-O, ester) cm -1.

1 H NMR (DMSO): δ = 4.58 (singlet, 2H, OCH 2 COO), 4.02 (triplet, 2H, OCH 2), 2.32 (triplet, 2H, CH 2 COO) , 1.49 (multiplet, 4H, C H 2 CH 2 C H 2 CH 2 COO), 1.28 (multiplet, 2H, C H 2 CH 2 CH 2 COO) ppm.

13 C NMR (DMSO): δ = 172.59 (CO, 6HH), 168.01 (CO, Glc), 64.62 (OCH 2), 60.67 (COO C H 2 COO), 33.07 ( C H_ {2} COO), 27.82 (OCH 2 CH 2), 24.76 ( C H 2 CH 2 COO), 24.12 ( C H 2 CH 2 CH 2 COO) ppm .

Differential thermal scanning analysis shows that the polymer has a near melting temperature at 63 ° C and a glass transition temperature of -37 ° C. He thermogravimetric analysis indicates that the polymer can be processed from the molten state to the start temperature of decomposition above 300 ° C.

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4. Preparation of poly (Glc- alt -6HH) 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 will be separated by filtration, as after successive washing with water.

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

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5. Degradation of poly (Glc- alt -6HH)

Poly (Gl- alt- 6HH) microspheres are hydrolytically degraded under simulated physiological conditions by a phosphate buffer of pH 7.4 and a temperature of 37 ° C. Thus, after 7 and 25 days of exposure, a drop in the average molecular weight by weight is observed from an initial value of 44,000 g / mol to final values of 30,000 and 20,000 g / mol, respectively. Under these conditions, degradation takes place at a moderate speed.

Degradation studies conducted with 200 µm thick polymer films corroborate that the sample is susceptible to hydrolytic attack although the process is slow. In this way, after 120 days of exposure a 6% weight loss and a molecular weight drop in weight to a value of 15,000 g / mol. In accelerated conditions produced by an increase in the temperature of the medium, up to a value of 50 ° C, the above changes are observed after only 28 days of exposition.

The polymer is easily degraded by the action of enzymes such as Pseudomonas cepacia lipase and proteinase K. The complete degradation of 200 µm thick films exposed to these media is observed after 5 and 10 days, respectively.

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6. Triclosan release from poly (Glc- alt -6HH) and poly (Glc- alt -4HB) microspheres

The following figure illustrates the release kinetics of the triclosan drug in a cellular medium (Dulbecco-modified Eagle medium containing 7.5% (weight / volume) of sodium bicarbonate and 10% (volume / volume) of bovine fetal serum at pH 7.4) for the microspheres of two representative polyesters of the family considered, specifically those incorporating units of 6-hydroxyhexanoic acid (poly (Gl- alt- 6HH)) or 4-hydroxybutyric acid (poly (Gl- alt -4HB)) as comonomers. It is observed, a different behavior that is in accordance with the characteristics of the corresponding polymers. In this way, triclosan is released more slowly in the polymer of a more hydrophobic nature (poly (Gl- alt- 6HH)). Specifically, after 50 hours of exposure, 80% of the initial charge is released in the medium when poly (Gl- alt -4HB) is used as a polymer matrix, while 110 hours are required to release 62% when poly is used (Glc- alt -6HH). These times are clearly insufficient to produce a marked hydrolytic degradation of the corresponding polymers. The present invention consequently allows to prepare a family of materials with different release kinetics.

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7. Biocompatibility and cell growth assays performed with poly (Glc- alt -6HH)

Cytotoxicity tests were performed in according to ISO-140320, evaluating the toxicity of the products released by the material after its incubation for 50 days in a cell culture medium (DMEM, half Eagle modified by Dulbecco containing 7.5% (weight / volume) of sodium bicarbonate and 10% (volume / volume) of fetal bovine serum at pH 7.4). Extraction was performed at 37 ° C in humid atmosphere with 5% CO2 and 95% air. The cells used in both cytotoxicity and adhesion tests were L-929 mouse fibroblasts (EACC, Sigma-Aldrich, UK). The tests performed demonstrate low toxicity of extracts and adequate cell proliferation and adhesion on the polymer.

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Description of the figures

Figure 1. SEM micrographs showing the morphology microspheres of poly (Glc- alt -6HH) loaded with triclosan.

Figure 2. Triclosan release in a medium of cell culture from two representative polymers of the Serie.

Figure 3. Optical micrograph showing cell proliferation on a poly (Glc- alt- 6HH) film after 72 exposure. It can be seen how L-929 cells remain perfectly attached forming monolayer on the new material. 100x magnification

Claims (5)

1. A method of preparing polyesters biodegradable alternating sequence by polycondensation thermal of the metal salt of an acid-derived chloro acid glycolic and an aliphatic hydroxy acid.

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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 3 to 10.

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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 3 to 4.

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4. A drug dosing matrix constituted by a biocompatible polyester with a structure chemistry where glycolic acid units alternate and a hydroxy acid, whose repetitive unit corresponds to the formula generic:

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where n varies from 3 to 10, and where R can correspond to a hydrogen atom or a methyl group. The case of n = 4 is excluded when R corresponds to a hydrogen atom. 5. Microspheres usable as systems drug releasing and consisting of polyesters where alternate glycolic acid units and one of the following units: 3-hydroxybutyric acid and acid 6-hydroxyhexanoic.