RO128361B1 - Collagen biomaterials with zeolite and essential oils for treating skin infections and process for preparing the same - Google Patents

Abstract

The invention relates to a collagen biomaterial for treating the skin infections produced by the bacteria S. aureus and P. aeruginosa and to a process for preparing the same. According to the invention, the biomaterial consists of a natural polymer - fibrillar collagen type I - as gel, with native triple helix structure, a natural tribomechanically activated zeolite - clinoptilolite, an Origanum or Thymbra spicata L. essential oil and a cross-linking agent. According to the invention, the process consists in treating the zeolite with essential oil, the resulting suspension is added to the collagen gel, is homogenized, the pH is adjusted to 7.4 and there is added distilled water until the final composition contains 1.2% collagen as dry substance and then it is cross-linked, after which the resulting composition as suspension is frozen immediately, is dried by lyophilization, there resulting a spongy collagen biomaterial.

Description

The invention relates to a composition of collagenous sponge biomaterials, based on natural zeolite and essential oils with antibacterial action against Staphilococcus aureus and Pseudomonas aeruginosa, used as a natural treatment for skin infections, and to a process for obtaining it.

Staphylococcus aureus is recognized as one of the most important pathogenic bacteria that seriously contributes to the worldwide problem of hospital infections. S. aureus, especially methicillin-resistant S. aureus (MRSA) is a major nosocomial pathogen (requiring hospitalization), which causes morbidity and mortality worldwide. Antimicrobial drugs resistant to staphylococci have raised a serious problem worldwide. For example, the incidences of MRSA have increased, and drugs such as penicillins, cephalosporins have been reported to be ineffective in the treatment of staphylococcal infections. In essence, many MRSA strains have acquired resistance to both beta-lactams and aminoglycosides.

Pseudomonas aeruginosa, the primary human pathogen of the genus Pseudomonas, is widespread in nature. It may colonize in healthy people without causing disease, but it is also a significant opportunistic pathogen, and a major cause of nosocomial infections. P. aeruginosa is a regular cause of nosocomial pneumonia, nosocomial urinary tract infections, surgical infections, severe burn infections and infections of patients undergoing chemotherapy for neoplastic disease, or antibiotic therapy.

Although P. aeruginosa is rare in normal human flora, it is frequently isolated in hospitalized patients. P. aeruginosa is one of the most important microorganisms that cause clinical problems, due to its high resistance to antimicrobial agents. The widespread occurrence of antibiotic-resistant P. aeruginosa strains is a growing concern in hospitals. Unfortunately, there are no specific measures to prevent pseudomonal nosocomial infections. Despite the availability of a variety of effective antimicrobial agents, P. aeruginosa frequently exhibits resistance to multiple antimicrobial agents. Serious infection due to strains of P. aeruginosa, which has resistance to all antipicrobial antimicrobials, is an increasingly serious problem.

Medicines for the treatment of P. aeruginosa infections are aminoglycoside antibiotics, such as tobramycin and gentamicin. They are active against Pseudomonas and various penicillins, including carbenicillin, ticarcilin, piperacillin, mezlocillin and azlocillin. In addition, generation III cephalosporins are widely used in the treatment of pseudomonas infections, but recently increased resistance to antipseudomone antibiotics such as third generation cephalosporins has been observed. For these reasons, new drugs and therapies are continuously being investigated.

In recent centuries, medicines for infections have been systemically administered. The major disadvantage of systemic administration is the even distribution throughout the body, which produces undesirable toxic side effects on healthy organs. In order to reduce toxicity, the concentration of the drug should be as low as possible, but this may result in insufficient amount of drug in the diseased organs. The solution to solve this problem is the local application of drugs on the affected area. Topical drug delivery systems are used to produce local effects on the surface of the skin or mucous membranes (topical action). The molecules of active substance penetrate into the stratum corneum, diffuse to the target tissues in the vicinity of the place of application, and thus the therapeutic effect occurs. To achieve the topical effects of locally administered products, so far, different drug delivery systems have been used, liquids (emulsions, solutions, suspensions), solids (powders, matrices, membranes) or semi-rigid forms (gels, hydrogels).

In severe wound infections, systemic drug administration may lead to insufficient drug concentration in the affected tissue, or to side effects due to drugs and systemic toxicity. A successful solution to this problem 3 has been found in the local application of medicines, therefore, delivery systems of drugs based on a support and a drug (antibiotic / antiseptic) 5 have been developed to control the infection.

Most of the medical devices used in local drug delivery have 7 polymeric support, the ideal vehicle for release, due to its biodegradability. Polymers are classified as natural (proteins, polysaccharides and lipids) and synthetic (biodegradable and non-biodegradable). Due to its excellent biocompatibility and biodegradability, well-known structure and biological characteristics, collagen is an appropriate support for drug delivery, offering the advantage of a natural biomaterial with hemostatic and wound healing properties. In today's medicine, infections and lesions of the skin and soft tissues that need local treatment are mostly cured with antibacterial drugs, which kill or inhibit the development of bacteria, fungi and viruses. Collagen-based drug delivery systems for local administration have incorporated drugs such as tetra- 17 cyclin, doxycycline, rolitetracycline, minocycline, metronidazole, ceftazidime, cefotaxim, gentamicin, amicacin, tobramycin, vancomycin, rosorlastine, ros chloroflavin, pile- 19 carpentry. In addition to drugs, growth factors, hormones, genes and liposomes have been used for local treatment under different conditions. 21

Although many researchers are investigating potential strategies that could be an alternative to antibiotic therapy for the treatment of local infections, bacteria have become increasingly resistant to antibiotics. Moreover, there is a growing number of people rejecting the use of synthetic drugs in favor of natural remedies. 25

It should be emphasized that essential oils, collagen and zeolite are natural compounds, non-toxic and environmentally friendly materials for humans and the environment. 27

US Patent No. 0077281 discloses medical patches used to treat colds, by releasing essential oil, by evaporation. This patch comprises a support layer (permeable to gas and water vapor) and a hydrophilic polymeric matrix connected to the support layer (with pressure-sensitive adhesion properties). The patches can cure colds through aromatherapy, but not skin infections. WO 02062361 relates to a composition containing propolis and essential oils, with antiviral capacity. WO 9817262 discloses a 33 essential oil carrier in the form of patches for the skin. This invention involves the use of topical application aromatherapeutic oils to reduce the symptoms of jet-lag 35 during long-term travel, or to relieve anxiety, insomnia, heartburn, detoxification and energizing the body. The patents described above have the advantage of 37 developing products that cure by aromatherapy, have antiviral properties and reduce the symptoms of "jet lag", but in comparison with our invention, they do not have the ability to treat the infection and to regenerate the damaged tissues, in the same time. Patent FR 2743722 describes a cosmetic mask of creamy consistency, based on clay and essential oil, for the treatment of acne. AU 259508 relates to packages of active materials, which are made of polyethylene, polypropylene, polyester, polyamide and other substrates based on 43 hydrocarbons incorporated in essential oil / zeolite mixtures prepared by the gas-solid reaction.

These materials are effective in extending freshness and improving the quality of fresh fruits, vegetables and flowers. US Patent No. 034149 discloses an essential oil material incorporated in plastic foil for the protection or preservation of horticultural products and 47 foods. Combinations of essential oil and zeolite are also used in cosmetics and the packaging industry, with good results, but they cannot be used in medicine as medical devices for the treatment of skin infections.

RO 128361 Β1

The preparation of collagen biomaterials in different forms (sponges, membranes) has been presented in many previous patents. For example, US Patent No. 6969523 discloses collagen and glycosaminoglycan-based biomaterials for skin regeneration, US Patent No. 4412947 relates to a material in the form of porous collagen foil, used as a dressing for wounds, burns, and hemostatic, US Patent No. 0168400. collagen-based dressings / synthetic resins used in the treatment of several types of wounds, such as accidental wounds or stents. Another wound dressing for chronic wounds is based on oxidized cellulose and collagen, as described in EP 1325754. Although many collagen-based medical devices have been developed for use as bandages, they do not contain essential oils, zeolite or their combination, which has the role of treating the infection. The advantage of our invention is that the developed biomaterial not only repairs damaged tissue, but also treats the infection caused by S. aureus and P. aeruginosa.

The technical problem solved by the invention consists in selecting polymeric support (collagen), zeolite (clinoptilolite, tribomechanically activated) and essential oils (Thymbra spicata L. and Origanum syriacum L. var. Bevanii), and combining them in such proportions that to produce biomaterials with antibacterial action and absorbent properties.

The collagen biomaterials, according to the invention, remove the disadvantages mentioned by the fact that they consist of the following components, expressed in gravimetric percentages related to 100% dry collagen:

a) a natural polymer, fibrillar collagen type I, obtained from calf skin dermis, in gel form, with native triple helical structure, with a content of 2 ... 2,5% dry collagen,

b) 0.5-1% natural zeolite, activated tribomechanical clinoptilolite which, in suspension form 0.5-1% in distilled water, is mixed in collagen gel,

c) 2 ... 4% essential oil of O. syriacum or 4 ... 8% oil of 7. spicata which, in the form of a 40 ... 50% solution in ethyl alcohol, is added over component b), and

d) 0.002 ... 0.003% cross-linking agent, selected from glutaraldehyde, formaldehyde and tannic acid, which is mixed in the final composition as a solution of 0.2 ... 0.3% in distilled water.

The process for obtaining spongy collagen biomaterials consists in that, prior to that, the zeolite is treated with essential oil by mixing the suspension of zeolite in distilled water with the solution of essential oil in ethyl alcohol, so that the oil is adsorbed in the zeolite cavities, and be released under control. The resulting slurry is added over collagen gel, homogenized, adjusted to pH 7.4 with 1M sodium hydroxide, and distilled water is added until the final composition contains 1.2% dry collagen, then a blending agent is added. crosslinking. The obtained composition, in suspension form, is immediately frozen at -2O ...- 6O ° C, after which it is dried by lyophilization, 24 ... 48 h, at a pressure of 0.1 ... 0.001 mbar and the final temperature from 3O ... 35 ° C.

The resulting biomaterials have a spongy structure (fig. 1), with pore size

10 ... 100 pm (fig. 2B). The biomaterials have at least 96% triple helical structure (fig. 3), and gradually release the essential oil adsorbed into the zeolite structure (fig. 4A). The density of the biomaterials is about 0.055 g / cm ³ , and they absorb more than 3400% water, about 34 g / g, compared to the dry matter collagen (fig. 5). The microbiological results (Fig. 6) show that, compared to the antibiotic, the biomaterials according to the invention show a much better antibacterial activity for both bacteria studied, P. aeruginosa and S. aureus. Furthermore, biomaterials have been shown to be effective in treating decubitus scales (Fig. 7).

RO 128361 Β1

Brief description of the drawings 1

Fig. 1 shows the spongy biomaterial obtained by lyophilizing a suspension containing collagen, zeolite and O. syriacum oil. 3

Fig. 2 shows electron microscopy images of the spongy biomaterial obtained by lyophilizing a suspension containing (A) collagen, (B) collagen, zeolite and O. 5 syriacum oil.

Fig. 3 shows the FT-IR spectrum of the spongy biomaterial obtained by lyophilizing a suspension containing 7 collagen, zeolite and O. syriacum oil. The ratio of the absorbents between the amide III and the corresponding amide at 1450 cm ^{-1 has} a triple helical structure with an integrity of about 96%.

Fig. 4 shows the release capacity of the oils of O. Syriacum (left) and 7. 11 spicata L. (right), previously absorbed on zeolite, from collagen (A), compared to the oils of Origanum syriacum L. var. bevanii (left) and T. spicata L. (right), mixed 13 directly with collagen (B), determined by TGA under isothermal conditions (2 h at 30 ° C).

Fig. 5 presents the water absorption of biomaterials containing: 15

a) only collagen (Coli);

b) collagen, zeolite and oil of T. Spicata L. (Coll-Z-T), and 17

c) collagen, zeolite and O. Syriacum oil (Coll-Z-O).

Fig. 6 shows the influence of the essential oils on the microbial activity, where: 19 (A) shows that the biomaterial containing only collagen and O. syriacum oil has no effect on P. aeruginosa; 21 (B) shows that biomaterial containing only collagen and oil of O. Syriacum has no effect on S. aureus; 23 (C) shows the antimicrobial activity of the biomaterial containing collagen, zeolite and O. syriacum oil on P. aeruginosa; 25 (D) shows the antimicrobial activity of the biomaterial containing collagen, zeolite and O. syriacum oil on S. aureus; 27 (E) shows the antimicrobial activity of the biomaterial containing collagen, zeolite and T. spicata L. oil on P. aeruginosa; 29 (F) shows the antimicrobial activity of the biomaterial containing collagen, zeolite and T. spicata L. oil on S. aureus. 31

Fig. 7 presents the evolution of a decubitus ladder: a) as such and treated with biomaterial from collagen, zeolite and T. spicata L. oil at b) 2 weeks and c) 4 weeks. 33

The advantages of the invention in relation to the prior art

Application of the invention leads to the following advantages: 35

- reducing the toxic effect of synthetic drugs by using natural components that are friendly to the human body and the environment (collagen, natural zeolite and 37 essential oils extracted from plants);

- obtaining natural systems with intelligent properties, manifested by the controlled release 39 of the essential oils and controlled antibacterial action, with applications in medicine, surgery, cosmetics; 41

- tissue regeneration, due to collagen, balancing the pH of the affected skin, due to the ion exchange action of zeolite, and treatment of infections caused by P. 43 aeruginosa and S. aureus bacteria, due to the antibacterial action of the essential oils;

- The process according to the invention is simple, applicable at room temperature 45 (23 ... 28 ° C), for a maximum of 48 hours, with low energy consumption and with simple equipment, specific for obtaining and characterizing biomaterials. 47

RO 128361 Β1

Detailed description of the invention

Fibrillar collagen type I, in the form of gel obtained from bovine skin, having a collagen concentration of 0.8 ... 2.5% (w / w), is preferred to be used in this invention. Fat and ash should be lacking. The pH of the gel is 1.5 ... 3.8.

The zeolite used in this invention is a natural zeolite, clinoptilolite, which should have a Mohs hardness of 3.5 ... 4, a specific density of 2.1 ... 2.6 g / cm ³ and the following composition: Na ₂ O 2.1 ... 3.4%; K ₂ O 4.1 ... 5.2%; AI ₂ O ₃ 11.3 ... 12.1%; SiO ₂ 66.7 ... 67.1%; H ₂ O

6.5 .. .13.3%, MgO 0 ... 1.1%; CaO 0 ... 4.9% and FeO 0 ... 2.7%.

A variety of essential oils can be used with favorable effects in this invention. Preferred essential oils are obtained from T. spicata and O. syriacum, mainly due to the bacterial effect against P. aeruginosa and S. aureus, and their antioxidant activity. The oil composition of T. spicata L. should have 70 ... 72% carvacrol, 8 ... 10% o-cymene,

3 .. .7% y-terpinene, 0.3 ... 1.4% β-myrcene, 0.2 ... 0.6% thymol, and O. syriacum oil should have 41 ... 45% carvacrol, 10 ... 16% cymene, 4.2 ... 4.8% γ-terpinene, 12 ... 14% thymol and 2 ... 3% caryophyllene.

The invention will be explained in more detail by the following examples.

Example 1 In this invention a type I fibrillar collagen gel with a gravimetric concentration of 2%, 98% water and a pH of about 2 ... 3 was used. A suspension of natural zeolite, thermomechanically activated clinoptilolite (Megamin type) was previously prepared by mixing 0.5% zeolite (relative to collagen in dry matter) with distilled water, 2% oil in O. syriacum (relative to collagen in substance dried) was dissolved in 40% ethanol, and 6.7% of this solution was mixed with 6.7% suspension of zeolite in water. After homogenization, the obtained suspension was incorporated into the gel, the pH adjusted to 7.4 with IM sodium hydroxide, and water was added until the final composition had 1.2% collagen, then cross-linked with 0 , 25% glutaraldehyde. The suspension was immediately frozen at about -40 ° C. Lyophilization took place at 0.001 mbar and the final temperature of 35 ° C. The resulting biomaterial was a sponge foam with absorbent properties and antibacterial effect on S. aureus and P. aeruginosa bacteria.

Example 2

The suspension was obtained by the process described in Example 1, except for the amount of O. syriacum oil used, which was 4% (relative to dry collagen). The freezing and freeze-drying process were similar to those described in Example 1. The resulting biomaterial was a spongy form with absorbent properties and antibacterial effect on S. aureus and P. aeruginosa bacteria.

Example 3

The suspension was obtained as in the procedure described in Example 2, except that the oil used was T. spicata. The freezing and freeze-drying processes were similar to those described in Example 1. The resulting biomaterial was a spongy form, with absorbent properties and antibacterial effects on S. aureus.

Example 4

The slurry was prepared by the process described in Example 3, except for the amount of T. spicata oil used, which was 8% (relative to dry collagen). The freezing and freeze-drying processes were similar to those described in Example 1. The resulting biomaterial was spongy, with absorbent and antibacterial properties on the S. aureus and P. aeruginosa bacteria.

The biomaterial obtained in this invention is a dressing with antimicrobial properties for wounds that absorb the exudate, helping to heal the wound.

Claims (2)

Claims 1 1. Spongy collagen biomass materials, based on natural zeolite and essential oils, 3 characterized in that they have the following composition, expressed in gravimetric percentages related to 100% dry collagen: 5 a) fibrillar collagen type I, a natural polymer, obtained from the calf skin dermis, in gel form, with native triple helical structure, with a content of 2 ... 2,5% collagen 7 dry substance, b) 0.5 ... 1% natural zeolite, tribomechanically activated clinoptilolite, 9 c) 2 ... 4% essential oil of Origanum syriacum L. var. beverages or 4 ... 8% oil Thymbra spicata L., and 11 d) 0.002 ... 0.003% cross-linking agent, selected from glutaraldehyde, formaldehyde and tannic acid. 13 2. Process for obtaining collagen biomaterials with zeolite and essential oils, having the composition defined in claim 1, characterized in that, prior to this, 15 zeolites, in the form of suspension 0.5 ... 1% in distilled water, are treated. with essential oil in the form of a 40 ... 50% solution in ethyl alcohol, for the oil to be absorbed into the zeolite cavities, 17 and to be released controlled, the resulting suspension is added over collagen gel, homogenized, pH adjusted to 7, 4 with 1M sodium hydroxide, and add distilled water until 19 the final composition contains 1.2% dry collagen, then crosslink with crosslinking agent, which is mixed in the final composition as a 0.2 solution. .0.3% in distilled water 21, the obtained suspension is immediately frozen at -2O ...- 6O ° C, then dried by lyophilization, 24 ... 48 h, at a pressure of 0.1 ... 0.001 mbar and final temperature of 3O ... 35 ° C. 2. 3