WO2013071457A1 - Composition vétérinaire de ceftiofur et florfenicol à libération prolongée et contrôlée injectable dans des espèces animales, basée sur des microparticules de polyhydroxybutyrate hydroxyvalerate (phvb) - Google Patents

Composition vétérinaire de ceftiofur et florfenicol à libération prolongée et contrôlée injectable dans des espèces animales, basée sur des microparticules de polyhydroxybutyrate hydroxyvalerate (phvb) Download PDF

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Publication number
WO2013071457A1
WO2013071457A1 PCT/CL2012/000067 CL2012000067W WO2013071457A1 WO 2013071457 A1 WO2013071457 A1 WO 2013071457A1 CL 2012000067 W CL2012000067 W CL 2012000067W WO 2013071457 A1 WO2013071457 A1 WO 2013071457A1
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WO
WIPO (PCT)
Prior art keywords
microparticles
ceftiofur
phbv
florfenicol
composition according
Prior art date
Application number
PCT/CL2012/000067
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English (en)
Spanish (es)
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WO2013071457A4 (fr
Inventor
Cristian VILOS ORTIZ
Luis VELASQUEZ CUMPLIDO
Luis CONSTANDIL CORDOVA
Miguel RIOS RAMIREZ
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Universidad De Santiago De Chile
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Publication of WO2013071457A1 publication Critical patent/WO2013071457A1/fr
Publication of WO2013071457A4 publication Critical patent/WO2013071457A4/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/165—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/54—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one sulfur as the ring hetero atoms, e.g. sulthiame
    • A61K31/542—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one sulfur as the ring hetero atoms, e.g. sulthiame ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/545—Compounds containing 5-thia-1-azabicyclo [4.2.0] octane ring systems, i.e. compounds containing a ring system of the formula:, e.g. cephalosporins, cefaclor, or cephalexine
    • A61K31/546—Compounds containing 5-thia-1-azabicyclo [4.2.0] octane ring systems, i.e. compounds containing a ring system of the formula:, e.g. cephalosporins, cefaclor, or cephalexine containing further heterocyclic rings, e.g. cephalothin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/5005—Wall or coating material
    • A61K9/5021—Organic macromolecular compounds
    • A61K9/5031—Organic macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, poly(lactide-co-glycolide)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents

Definitions

  • the main field of application of the present invention is in the veterinary area, specifically to combat infectious diseases that preferably affect pigs, such as the Porcine Respiratory Syndrome (SRP).
  • SRP Porcine Respiratory Syndrome
  • the object of the present invention is the development of a composition formulated as injectable microparticles of prolonged release of Ceftiofur and Florfenicol based on the biodegradable and biocompatible polymer Polyhydroxybutyrate hydroxyvalerate (PHBV) for application in the treatment of infectious diseases in animal species.
  • PHBV Polyhydroxybutyrate hydroxyvalerate
  • Prolonged or sustained plasma release of antibiotics from microparticles (MPs) of biodegradable and biocompatible polymers presents an innovative strategy for the treatment of infectious diseases, especially against those that require the administration of multiple doses to maintain antibiotic concentrations within therapeutic range for long periods of time.
  • Poly 3-hydroxybutyrate-co-3-hydroxyvalerate (PHBV) polymer is a biodegradable and biocompatible polymer. It has been shown to be toxicologically safe, stable in the bloodstream, does not induce an immune response, and its degradation products are bioabsorbable and harmless to both humans and animals. Also, according to its physicochemical properties and its degradation mechanisms, they constitute an excellent matrix for the development of prolonged drug delivery systems.
  • Ceftiofur and Florfenicol have been described, due to their excellent antimicrobial activity against the causative pathogens of these diseases, and for presenting lower bacterial resistance.
  • Ceftiofur Broad spectrum cephalosporin active against gram positive and gram negative microorganisms. It is a beta-lactam antibiotic, which exerts a bactericidal effect by inhibiting the synthesis of the cell wall.
  • Florfenicol Injectable chemotherapeutic broad spectrum, used exclusively in veterinary medicine and specifically for the treatment of respiratory diseases in cattle and pigs. It is broad spectrum bacteriostatic and its mechanism of action is through the inhibition of protein synthesis. After injectable administration, Florfenicol presents a complicated pharmacokinetics and requires an administration of at least 20 mg / kg of weight per day for 5-7 days intramuscularly (IM) when the conventional release product is administered. This treatment, which is the one used Currently, it has the great disadvantage that as always happens in these cases, plasma fluctuations of the antibiotic reach lapses of subtherapeutic plasma levels that favor bacterial resistance to the drug. To this is added the stress produced in animals that hinders the successive administrations of the medication, which also makes it difficult to comply with the therapy. This results in an ineffective therapy, which does not solve the disease in the animal. As well as an increase in the probability of bacterial resistance.
  • the object of this patent application is the development of prolonged-release formulations of Ceftiofur and Florfenicol based on PHBV microparticles for application in the treatment of infectious diseases of animal species such as Porcine Respiratory Syndrome that affects cattle pig in all stages of development, especially piglets and during the transition and fattening phase.
  • the present invention corresponds to the development of an injectable formulation which, with the administration of a single dose, is effective in the treatment of respiratory infections in bovine and porcine animals. And additionally reduce the morbidity caused by stress induced by manipulation.
  • Biodegradable and biocompatible polymers In recent years, the use of biodegradable and biocompatible polymers has been proposed to solve various problems in the biomedical area. Numerous studies report its use in different areas such as tissue reconstruction, ligament regeneration, cell proliferation, development of antitumor strategies, fixation of surgical devices, medical devices and controlled drug release systems.
  • Biodegradable polymers can be of natural or synthetic origin, the latter being the most used in microencapsulation systems suitable for the development of pharmaceutical controlled release formulations .:
  • PHBV Poly (3 hydroxybutyrate-3-hydroxivalerate)
  • PHA polyhydroxyalkanoates
  • Their Physical-chemical properties and mechanical characteristics have made it an attractive source of research in the field of biomedicine, especially in controlled drug delivery systems and tissue engineering. Its application has been demonstrated in the preparation of matrices for tissue engineering, achieving fibroblast proliferation rates, similar to those used in conventional systems such as collagen sponges.
  • its use as a vehicle for the release of drugs specifically micro / nano injectable particles of Flubiophene and in the microencapsulation of Tetracycline, Diazepam and Rifampicin, has been reported.
  • the degradation mechanism of the microparticles formulated with polyesters occurs by hydrolysis, by a random cleavage of the ester bonds, where the degradation rate occurs depending on the monomeric structure, the molecular weight of the polymer or copolymer and its proportion. It has also been shown that the molecular weight of the polymer decreases before any observed mass loss, which indicates that a high hydrolysis rate precedes before soluble monomers or oligomers occur. On the other hand, it has been described during in vitro degradation tests that the binding sites with amorphous morphology in the lamellar structure of PLA and PLGA are the starting point of hydrolysis.
  • MPs of the same polymer produce a completely dissimilar release in different formulations with the same drug (case A: release of 40% of the encapsulated rifampin at 72 hours; case B and C: release of 90% and 20% of rifampicin at 10 days).
  • MPs formulated with different polymers and the same drug have completely different properties (size, encapsulation efficiency, release kinetics).
  • the general objective of this invention is the development of an injectable controlled release formulation for the administration of Ceftiofur and Florfenicol, using as a matrix microparticles of the polymer Polyhydroxybutyrate hydroxyvalerate (PHBV).
  • This formulation should have a release profile such that the administration of a dose thereof is equivalent to the administration of the drug in a conventional administration formulation, that is, multidose.
  • MPs microparticles
  • MPs microparticles
  • Figure 1. Shows the determination of the size of the PHBV microparticles with ceftiofur and florfenicol.
  • Figure 2. Shows the determination of the zeta potential of PHBV microparticles with ceftiofur and florfenicol.
  • Figure 3. Shows the determination of the Efficiency of Encapsulation of microparticles of PHBV with ceftiofur and florfenicol.
  • Figure 4. Shows the characterization of PHBV microparticles with ceftiofur and florfenicol by scanning electron microscopy.
  • Figure 5. Shows the determination of the release curve of PHBV microparticles with ceftiofur and florfenicol.
  • Figure 6. Shows the plasma concentrations of a drug administered in multiple doses (dotted line, blue) and a sustained release system (solid line, red).
  • Dotted lines parallel to the X axis represent the therapeutic window and asterisks represent dose ranges.
  • Figure 7. Shows a diagram of the experimental configuration used.
  • PHBV microparticles with ceftiofur and florfenicol was performed using the double emulsion method (w / o / w) evaporation.
  • 15 mg of Ceftiofur or Florfenicol dissolved in methanol, and 300 ⁇ of double distilled water ( ⁇ ) were added to a solution of 50 mg / ml of PHBV in dichloromethane (oi).
  • This mixture was emulsified with a homogenizer at 35,000 rpm for 30 seconds (w ⁇ o).
  • a 5% w / v polyvinyl alcohol solution (w 2 ) was added and homogenized at 35,000 rpm for 30 seconds.
  • the present invention is directed to a prolonged release composition of ceftiofur based on polymeric microparticles.
  • Ceftiofur is a first-line antibiotic for a wide range of infectious pathologies of different animal species, therefore, the development of these pharmaceutical forms would generate a high impact on the animal industry.
  • the low commercial cost of the PHBV polymer makes it feasible for industrial scaling.
  • the MPs formulated by the double emulsion-evaporation method of the solvent have a hydrophilic center.
  • Ceftiofur and Florfenicol have a higher affinity for polar solvents such as methanol, which means that a higher percentage of the antibiotic will be effectively encapsulated inside the MPs.
  • the encapsulation efficiency (EE%) describes the percentage of the drug encapsulated in the microparticles.
  • EE% the percentage of the drug encapsulated in the microparticles.
  • UV-visible stectroscopy was used, where the wavelengths described in the literature 292 nm for Ceftiofur and 228 nm for Florfenicol were used as reference, see Figure 3.
  • the plaque inhibition halos method was used. Experimentally, a lawn planting was carried out with the Escher ⁇ chia coli bacteria (ATCC 25922) on Muller Hinton agar. Then, 15 pL of a microparticle suspension (2 mg / ml) was inoculated, the controls used correspond to microparticles without antibiotics and pure antibiotics. Plates were read within 24 hours of inoculation.
  • the results of the sizes of the PHBV microparticles with Ceftiofur and Florfenicol were in the range of 1-3 pm, and did not show significant differences, see Figure 1.
  • the surface charge, that is, the zeta potential obtained from the microparticles of PHBV was -8.43 ⁇ 2.6 mV for those containing Ceftiofur, and -11.0 ⁇ 2.6 mV for those containing Florfenicol, see Figure 2.
  • the particle size is suitable for an injectable pharmaceutical form because, when administered intramuscularly, it will remain at the injection site without passing into the central circulation.
  • the surface charge of the microparticles is negative, which generates electrostatic repulsion interactions that prevent their aggregation.
  • the morphology of the microparticles describes particles of spherical shape and with porosity or roughness on their surface, which according to the literature would facilitate the entry of water, and therefore the diffusion of the drug.
  • the in vitro release profile shows a system capable of prolonged release for at least 7 days, suggesting that its application in infectious diseases with a reduced amount of dose.
  • a second polymer with emulsifying and stabilizing properties is added to the emulsion made, which on the one hand will give the microparticles a certain stiffness and, on the other, a negative surface charge that will prevent agglomeration, for which 2 ml of a 1% w / v solution of polyvinyl alcohol (PVA) very slowly, and again emulsified with a homogenizer or Ultra-Turrax tissue disruptor at a speed of 35,000 rpm for 40 seconds.
  • PVA polyvinyl alcohol
  • the newly synthesized microparticles contain dichloromethane residues, to remove this organic solvent the formulation is placed in a volume of 40 ml of water and is kept in an orbital shaker at 120 rpm for 12 hours, at which stage the microparticles become solid and the solvent evaporates. After 12 hours, the microparticles are centrifuged at 5,000 rpm for 10 minutes to precipitate them, then the supernatant is removed and water is added again to be resuspended by ultrasound in a bath sonicator. This procedure is performed at least three times to completely remove remains of non-encapsulated antibiotic and polymer.
  • microparticles are resuspended in a final volume of 1 ml, then frozen for 8 hours at -80 ° C and lyophilized for 24 hours.
  • the product obtained after the lyophilization process is a powder that is made up of polymeric microparticles with encapsulated antibiotic.
  • Example 2 A formulation of PHBV microparticles with encapsulated ceftiofur of a regulated size corresponding to 10 ⁇ was prepared. According to the studies carried out in the development of the microparticle synthesis method, the size can be regulated by the factors: emulsification speed, emulsification time and polymer concentration. In this example we modify the variables emulsion time and concentration of the polymers to obtain the microparticles of the desired size.
  • microparticles were washed, they were resuspended in a final volume of 1 ml and frozen for 8 hours at -80 ° C and lyophilized for 24 hours.
  • the product obtained corresponded to a white powder formed by microparticles containing the encapsulated ceftiofur.
  • the pharmaceutical formulation obtained was tested in vivo in an animal model of pigs.
  • the formulation described above was suspended in water for injection and administered at a concentration dose of 10 mg / kg to 6 pigs.
  • the ceftiofur of the biodegradable pharmaceutical composition was released in a significant immediate amount over the course of hours after the injection, and then showed a constant and significant release over a period of at least 12 days with a 90% release.
  • Example 3 A formulation of PHBV microparticles with encapsulated florfenicol of a regulated size corresponding to 1 ⁇ was prepared, for this purpose and as described in example 2, in this case the concentration of the polymers and the emulsification time were modified to obtain the desired size
  • the average size of the microparticles obtained was 1 ⁇ 0.6 micrometers and their descriptive statistics values were:
  • a formulation of PHBV microparticles with encapsulated ceftiofur of a regulated size corresponding to 5 ⁇ was prepared for this and as described in example 2, and in this case the time and intensity of emulsification was modified to obtain the desired size .
  • microparticles were washed, they were resuspended in a final volume of 1 ml and frozen for 8 hours at -80 ° C and lyophilized for 24 hours.
  • the product obtained corresponded to a white powder formed by microparticles containing the encapsulated ceftiofur.
  • the average size of the microparticles obtained was 5 + 1, 2 micrometers and their descriptive statistics values were: Minimum 3.8 micrometers
  • the pharmaceutical formulation obtained was tested in vivo in an animal model of cattle.
  • the formulation described above was suspended in water for injection and administered at a concentration dose of 12 mg / kg to 6 cows.
  • the ceftiofur of the biodegradable pharmaceutical composition was released in an important immediate amount within hours after injection, and then showed a constant and significant release over a period of at least 5 days with a release of 78%.

Abstract

La présente invention concerne une composition vétérinaire injectable à libération prolongée comprenant des microparticules qui contiennent un polymère biodégradable biocompatible choisi de Poli-3-hydroxybutirate co-3-hydroxyvalerate (PHBV), lequel encapsule un agent antimicrobien choisi de Ceftiofur ou Florfenicol ou un mélange de ceux-ci. L'invention concerne également un procédé de préparation.
PCT/CL2012/000067 2011-11-18 2012-11-15 Composition vétérinaire de ceftiofur et florfenicol à libération prolongée et contrôlée injectable dans des espèces animales, basée sur des microparticules de polyhydroxybutyrate hydroxyvalerate (phvb) WO2013071457A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CL2901-2011 2011-11-18
CL2011002901A CL2011002901A1 (es) 2011-11-18 2011-11-18 Composicion veterinaria inyectable de liberacion prolongada y dosis unica que comprende microparticulas de polihidroxibutirato co-3-hidroxivalerato (phbv), un agente antimicrobiano seleccionado de ceftiofur o florfenicol y agua para inyeccion; procedimiento de preparacion; util para tratar infecciones en animales.

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WO2013071457A1 true WO2013071457A1 (fr) 2013-05-23
WO2013071457A4 WO2013071457A4 (fr) 2013-07-18

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113730652A (zh) * 2021-09-01 2021-12-03 北京大清生物技术股份有限公司 一种注射用混合凝胶及其制备方法和应用

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0293172A2 (fr) * 1987-05-29 1988-11-30 Ici Australia Operations Proprietary Limited Paricules polymériques
CN101756909A (zh) * 2010-03-18 2010-06-30 青岛康地恩药业有限公司 肺靶向头孢噻呋微球及制备方法
CN101849913A (zh) * 2009-03-30 2010-10-06 北京大北农科技集团股份有限公司 一种兽用氟苯尼考肺靶向明胶微球的制备方法

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0293172A2 (fr) * 1987-05-29 1988-11-30 Ici Australia Operations Proprietary Limited Paricules polymériques
CN101849913A (zh) * 2009-03-30 2010-10-06 北京大北农科技集团股份有限公司 一种兽用氟苯尼考肺靶向明胶微球的制备方法
CN101756909A (zh) * 2010-03-18 2010-06-30 青岛康地恩药业有限公司 肺靶向头孢噻呋微球及制备方法

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113730652A (zh) * 2021-09-01 2021-12-03 北京大清生物技术股份有限公司 一种注射用混合凝胶及其制备方法和应用

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CL2011002901A1 (es) 2012-07-06
WO2013071457A4 (fr) 2013-07-18

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