EA041251B1 - APPLICATION OF A PHARMACEUTICAL COMPOSITION FOR THE TREATMENT OR PREVENTION OF INFECTIONS IN THE INCISED AREA DURING SURGICAL INTERVENTION - Google Patents

Description

This application claims priority under U.S. Provisional Application No. 62/058809, filed Oct. 2, 2014, entitled Compositions and Methods for Treating and Preventing Surgical Site Infections, the contents of which are incorporated herein by reference in their entirety.

Technical field

The present invention relates generally to the use of sustained release pharmaceutical compositions for the treatment or prevention of infections in the surgical incision site.

State of the art

Surgical site infection (SSI), i.e. infection within or near surgical incisions that develops within 30 days of surgery is a common hospital-acquired infection that accounts for 15% of all hospital-acquired infections and is the most common hospital-acquired infection in patients after surgery. SSI is associated with increased morbidity and mortality, ranging from wound discharge associated with infection of the superficial layers of the skin to life-threatening conditions such as severe sepsis. SSI is causing an increased economic burden on health care systems, including additional increases in postoperative hospital stays and costs.

The onset of surgical site infection is caused by contaminants that exist in operating areas and are resistant to administered antimicrobial agents. In most cases of SSI, the source of the pathogen is the natural flora of the skin, mucous membranes, or hollow internal organs of the patient. After the skin is incised, the underlying tissue is exposed to excess endogenous flora. Staphylococcus aureus is the most frequently shed organism in SSI, causing 15-20% of SSI cases occurring in the hospital. Other organisms routinely isolated from SSI include gram-negative bacilli, coagulase-negative staphylococci, Enterococcus spp. and Escherichia coli. An increasingly important pathogen is methicillin-resistant S. aureus (MRSA), which causes more than 50% of hospital-acquired S. aureus infections in the US and Europe and complicates treatment due to multiple antibiotic resistance. Some yeast species and viral pathogens are also risk factors.

Surgical site infections are a significant clinical problem in orthopedic, spinal, digestive, cardiovascular, breast and many other clinical procedures involving skin incision. For example, a serious complication after cardiac surgery, which is accompanied by high morbidity and mortality, reaching 40%, is wound infection of the area after sternotomy (mediastinitis). Patients with wound site infection after sternotomy require longer hospital stays, repeated surgical interventions, prolonged antibiotic therapy, and experience a significant deterioration in quality of life and well-being. The cost of treating these patients and the financial burden on health care systems is estimated to be 3 times higher than that of patients undergoing open cardiac surgery without any infection.

Typically, biofilm colonization of the surgical site renders pathogens resistant to antimicrobial agents as well as to other interventions such as debridement to treat wound infection. Indeed, in recent years, despite the development of new surgical technologies, new antibiotics, new technologies for diagnosing postoperative infections and wound care technologies, the incidence of surgical site infections has not decreased.

International publication WO 2010/007623, co-authored by the author of the present invention and other authors, the contents of which are incorporated into this application by reference, discloses compositions for drug delivery for controlled release of the active ingredient, containing a lipid matrix containing a biodegradable polymer. Said drug delivery compositions are capable of incorporating a broad range of one or more biologically active molecules and releasing them at a predetermined rate over periods ranging from several days to several months.

In the international publication WO 201420610, one of the co-authors of which is the author of the present invention, the contents of which are incorporated into this application by reference, compositions, methods and medical devices for the treatment of bone cavities and bone defects are disclosed, including the step of applying the composition to a bone cavity or bone defect site containing a matrix, which provides a local prolonged release of at least one antibiotic agent in the area of the bone cavity. Surgical site infections continue to be a major public health problem. There is a need in the art for treatments to prevent and treat surgical site infections locally in the surgical site.

Brief description of the invention

The present invention provides the use of a pharmaceutical composition containing a biodegradable mineral substrate, wherein said mineral substrate is impregnated or its surface is coated in whole or in part with a matrix composition containing (a) a biodegradable polyester selected from the group consisting of PLA (polylactic acid), PGA (polyglycolic acid), PLGA (copolymer of lactic acid and glycolic acid);

(b) a first lipid component containing cholesterol;

(c) a second lipid component containing phosphatidylcholine containing fragments of fatty acids containing at least 14 carbon atoms; and (d) a pharmaceutically active agent containing an antibiotic agent selected from the group consisting of doxycycline or doxycycline hyclate, for the treatment or prevention of an infection of the soft tissue incision area associated with a surgical operation, where the specified mineral substrate consists of particles of calcium β-orthophosphate ( β-TCP) and where the specified pharmaceutical composition is intended for local administration in the area of soft tissue incision.

A substrate impregnated and/or surface coated, in whole or in part, with the matrix composition described herein is intended for topical application in soft tissues and hard organs during surgical procedures and provides local prevention and treatment of infection by reducing the overall infection rate after surgery. and by reducing or eradicating soft tissue infections that may exist prior to surgery. In some embodiments, the drug-eluting substrate described herein prevents or inhibits the formation of a biofilm that may form in and around the surgical site, thereby preventing or inhibiting surgical site infections. Inhibition of biofilm formation at the surgical site refers to the inhibition of biofilm formation on surfaces such as biological tissues and/or materials or devices that may be used during surgery (e.g., wound tissue, necrotic cells, biomaterials, and surgical implants (e.g., suture materials and solid stainless steel wire products)). In accordance with some embodiments of the present invention, the drug-eluting substrate described herein is capable of eradicating an existing biofilm.

Upon application to the surgical site of a substrate impregnated and/or surface coated, in whole or in part, with the matrix composition described herein, said substrate provides locally controlled drug release at and near the surgical site for a predetermined extended period of time. , preferably from a few days to a few weeks, thereby preventing or eradicating tissue infection.

A substrate impregnated or coated, in whole or in part, with a matrix composition in accordance with some embodiments of the present invention is preferably administered to a subject who has or is at risk of developing an infection, prior to or during treatment of the subject using a method that may cause infection and/or the formation of a biofilm in the subject. According to some embodiments of the present invention, said method may be any surgical procedure, such as orthopedic surgery (e.g., hip arthroplasty, knee arthroplasty, total joint replacement, trauma), spine surgery, digestive organ surgery ( esophagus, stomach, small intestine, large intestine, rectum, colon, appendix, liver, pancreas, gallbladder, gastric ulcer, gastric cancer, open gastric anastomosis, appendectomy, colectomy, cholecystectomy, vagotomy, open surgery biliary tract surgery, small bowel surgery, colorectal surgery), cardiac surgery (eg, coronary artery bypass surgery, cardiothoracic transplant surgery, heart apparatus insertion surgery), hernia repair, vascular surgery, cesarean transection, prostatectomy, obstetrical and gynecological surgeries (eg, hysterectomy), head and neck cancer surgeries, transplant surgeries (eg, lung, liver, pancreas, kidney), neurosurgical interventions (eg, insertion of a deep brain stimulation implant), and plastic surgery (eg, breast reconstruction, mastectomy).

According to some embodiments of the present invention, the infection includes at least one of a superficial incisional infection, a deep incisional infection, and an organ/space infection.

According to some embodiments of the present invention, said use is suitable for at least one of reducing the overall incidence of infections after surgery, eradicating soft tissue infections that may have existed prior to surgery, inhibiting biofilm formation at the incision site, and eradicating existing biofilm at the incision site. soft tissues.

In some embodiments, the infection includes methicillin-resistant S. aureus (MRSA).

According to some embodiments of the present invention, said surgery is selected from orthopedic surgery, spinal surgery, gastrointestinal surgery, cardiac surgery, hernia repair, angioplasty, caesarean section, prostatectomy, obstetrical and gynecological surgery, head cancer surgery and neck, transplant surgery, neurosurgery and plastic surgery.

According to some embodiments of the present invention, said surgical operations on the organ of the digestive system are selected from the operation on the esophagus, stomach, small intestine, large intestine, rectum, colon, appendix, liver, pancreas, gallbladder, gastric ulcer, gastric cancer, creation open gastric anastomosis, appendectomy, colectomy, cholecystectomy, vagotomy, open biliary tract surgery, small intestine surgery and colorectal surgery.

In some embodiments of the present invention, said surgery is a cardiac surgery.

In some embodiments of the present invention, said mineral substrate is comprised of particles having an average particle size of less than about 200 microns (μm), preferably less than about 150 microns (μm), more preferably, said biodegradable substrate is composed of particles having an average particle size which is in the range of 50-150 microns (μm), even more preferably said majority of the particles are spherical and/or spherical.

According to some variants of implementation of the present invention, the specified phosphatidylcholine contains fragments of fatty acids containing 14-18 carbon atoms.

According to some embodiments of the present invention, said pharmaceutically active agent further comprises an active agent selected from the group consisting of an antibiotic agent, an antiseptic agent, an anti-inflammatory agent, an antifungal agent, and any combination thereof; or said pharmaceutically active agent further comprises at least one anti-inflammatory agent.

According to some variants of implementation of the present invention, the mass ratio of total lipids to the specified biodegradable complex polyester is from 1:1 to 9:1, inclusive.

According to some variants of implementation of the present invention, the specified coated biodegradable mineral substrate contains approximately 60-90% wt./wt. biodegradable mineral substrate and 10-40% wt./wt. matrix composition, while preferably specified coated biodegradable mineral substrate contains approximately 70-90% wt./wt. biodegradable mineral substrate and 10-30% wt./wt. matrix composition.

According to some variants of implementation of the present invention, the matrix composition contains (a) 15 - 25% wt./wt. copolymer of lactic acid and glycolic acid (PLGA);

(b) 5-15% w/w cholesterol;

(c) 50-70% w/w mixtures of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-distearoylsn-glycero-3-phosphocholine (DSPC), wherein the ratio of DPPC to DSPC is from 5:1 to 2:1; and (d) 7-12% w/w. doxycycline or doxycycline hyclate.

According to some embodiments of the present invention, the specified mineral substrate is approximately 80-90% wt./wt. and the matrix composition is approximately 10-20% wt./wt. from the total weight of the pharmaceutical composition.

According to some variants of implementation of the present invention, the pharmaceutical composition is prepared in the form of a paste.

Other implementation options and the full scope of the scope of the present invention will be clear from the detailed description below. However, it should be understood that the detailed description and specific examples, while describing the preferred embodiments of the present invention, are provided by way of illustration only, as various changes and modifications within the spirit and scope of the present invention will be apparent to those skilled in the art at based on this detailed description.

Brief description of the drawings

In FIG. 1 shows the accumulation profile of released doxycycline hyclate from hydrated (5% serum at 37° C.) calcium orthophosphate (TCP) particles (~100 μm) impregnated with a matrix composition consisting of PLGA, cholesterol, DPPC, DSPC and doxycycline hyclate.

- 3 041251

In FIG. Figure 2 shows the accumulation profile of released doxycycline hyclate from hydrated (5% serum at 37°C) polyvinyl alcohol (PVA) particles impregnated with a matrix composition consisting of PLGA, cholesterol, DPPC, DSPC and doxycycline hyclate after hydration in 5% serum at 37 °C.

In FIG. 3A, 3B, 3C and 3D are SEM images of uncoated PVA globular particles with a porous rough surface (FIGS. 3A and 3B, magnifications of 500 and 25,000 times, respectively) and similar PVA particles impregnated with a matrix composition consisting of PLGA. , cholesterol, DPPC, DSPC, and doxycycline hyclate (FIGS. 3C and 3D, 500- and 25,000-fold magnifications, respectively). In FIG. 3C and 3D show that the outer and inner surfaces of the particles are coated.

In FIG. 4 shows the results of evaluating the effectiveness of calcium orthophosphate beads (~100 µm) coated with a matrix composition containing doxycycline, in accordance with some embodiments of the present invention (test article), in reducing bacterial proliferation after infection induction in a surgical site infection model (SSI) obtained by intramuscular implantation of the investigational device in combination with Staphylococcus aureus in Sprague-Dawley (SD) rats. Implantation of uncoated TCP particles together with Staphylococcus aureus was used as a control.

Detailed description of the invention

The present invention provides a pharmaceutical composition for treating or preventing a surgical site infection associated with a surgical operation, comprising the step of introducing into the surgical site a biocompatible, biodegradable substrate impregnated and/or whose surface is coated, in whole or in part, with a matrix composition that provides locally controlled and prolonged release of at least one pharmaceutically active agent in the area of surgical intervention in a subject in need thereof. In particular, the matrix composition contains (a) a biocompatible polymer;

(b) a first lipid component containing a sterol;

(c) a second lipid component containing at least one phospholipid containing fragments of fatty acids containing at least 12 carbon atoms; and (d) a pharmaceutically active agent selected from the group consisting of an antibiotic agent, an antiseptic agent, an anti-inflammatory agent, an antifungal agent, and any combination thereof.

In this application, the term area of surgical intervention refers to the area created by any incision of the skin or internal organs, made according to a specific medical purpose. An area of surgery that is open refers to areas of surgery in which medical personnel have direct physical access to the area of interest. The surgical site may include, but is not limited to, organs, muscles, tendons, ligaments, connective tissue, and the like.

The methods of the present invention can also be used to treat open wounds. In this application, open wounds refer generally to an injury in which the normal integrity of tissue structures is disrupted, and more specifically to a type of injury in which the skin is torn, cut, or pierced. Open wounds include, but are not limited to: incisions or incised wounds, cuts, penetrating wounds, festering wounds, burns, etc.

Methods for preventing or inhibiting surgical site infections can be used to prevent or inhibit postoperative infection in a contaminated or potentially contaminated operation, wherein the postoperative infection may be any of a superficial incisional infection, a deep incisional infection, and an organ/space infection. The present invention also discloses methods for treating surgical site infections, including preoperative, intraoperative and/or postoperative infections. These preoperative and/or postoperative infections may be associated with biofilm formation. A preoperative infection may include a biofilm formed due to a disease or condition in an organ, tissue, or body system (eg, bone, skin, abdomen, urinary tract, etc.). Said disease or condition may be selected from, for example, infections associated with a medical device, infections associated with an orthopedic implant, infections associated with biliary stents and catheters.

The method for suppressing, preventing, and treating surgical site infection according to embodiments of the present invention may be in addition to standard methods for reducing bacterial inoculum, such as proper surgical site preparation, systemic prophylactic antibiotics, cell-based therapy, and host stimulation by perioperative supplemental oxygenation, maintaining normothermia and glycemic control.

According to some embodiments of the present invention, the substrate used in

- 4 041251 compositions described herein, is a bioabsorbable hydrophilic material that is biocompatible (i.e., has low toxicity, causes only a weak reaction to a foreign body in a living body, and may have good affinity for body tissues) , bioabsorbability (i.e., biodegradable) and hydrophilicity, but which has low water solubility or is insoluble in water and, in addition, has a solid form at ambient temperature and is moldable. Any materials with the listed properties can be used without restrictions. Bioabsorbable hydrophilic materials, in accordance with some embodiments of the present invention, include mineral substrates, natural polymeric substrates, and synthetic derivatives thereof. Non-limiting examples of mineral substrates include hydroxyapatite, fluorapatite, hydroxyapatite, wollastonite, apatite/wollastonite glassceramics, anorthite, calcium fluoride, calcium sulfate, calcium carbonate, tetracalcium phosphate, α-calcium orthophosphate (α-TCP), β-calcium orthophosphate (β- TCP), amorphous calcium phosphate, calcium hydrogen orthophosphate, agrelite, devitrite, canasite, phlogopite, monetite, brushite, octacalcium phosphate, whitlockite, cordierite, berlinite, combite, phosphoric acid crystals, sodium hydrogen orthophosphate and other types of phosphate-based bioceramics. Non-limiting examples of natural polymeric substrates include gelatin, hyaluronic acid, hyaluronic acid derivatives such as hyaluronic acid polyionic complex, triethanolamine alginate, casein, keratin, myosin and/or fibroin, collagen, collagen derivatives such as succinylated collagen or methylated collagen, chondrotin sulfate, chitosan, chitosan derivatives such as methylpyrrolidone-chitosan, polyaminogalactosamine. In some embodiments, the substrate is a water-soluble synthetic polymer such as, for example, polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), polyacrylic acid (PAA), K-(2-hydroxypropyl)methacrylamide (HPMA), poly-( 2-alkyl-2-oxazolines), polyphosphoesters (PPE), polyphosphates and polyphosphonates. According to some embodiments of the present invention, the substrate is polyvinyl alcohol (PVA). According to some embodiments of the present invention, the substrate is a bioabsorbable hydrophobic material, such as, for example, a biodegradable polyester selected from the group consisting of PLA (polylactic acid), PGA (polyglycolic acid), PLGA (copolymer of lactic acid and glycolic acid) and their combinations.

In some embodiments of the present invention, the substrate is dense. In some embodiments of the present invention, the substrate is porous. According to some variants of implementation of the present invention, the substrate is formed in the form of particles (or granules). The substrate particles are typically spherical or spheroidal. According to some embodiments of the present invention, the substrate particles, which do not have to be spherical and/or spheroidal, but preferably have a spherical and/or spheroidal shape, may have an average diameter, for example, at least about 30 microns, at least about 40 microns, at least about 50 microns, at least about 60 microns, at least about 70 microns, at least about 80 microns, at least about 90 microns, at least about 100 microns, in the range from 50 to 200 microns, in the range from 50 to 180 µm, in the range from 70 µm to 150 µm, in the range from 80 to 120 µm, in the range from 50 to 100 µm and in the range from 70 to 100 µm, not more than about 500 µm, not more than about 400 µm, max. approx. 350 µm, max. approx.

300 µm, max. approx. 250 µm, max. approx. 200 µm, max. approx.

180 µm, max. approx. 150 µm, max. approx. 140 µm, max. approx.

130 µm, max. approx. 120 µm, max. approx. 110 µm, max. approx.

100 µm. According to some embodiments of the present invention, the substrate particles are in the form of a powder. In accordance with some embodiments of the present invention, the substrate may be in any form (eg, sponges, nets, sheets, or fibers). One skilled in the art will appreciate that the shape and/or size of the substrate can be adjusted, before or after coating or impregnation with the matrix composition, as needed (eg, type, size, and location of the incision). Each possibility represents a separate embodiment of the present invention.

According to some embodiments of the present invention, the biocompatible polymer in the coating matrix composition comprises a polyester selected from the group consisting of PLA (polylactic acid), PGA (polyglycolic acid), PLGA (copolymer of lactic acid and glycolic acid)) and combinations thereof. In accordance with some variants of implementation of the present invention, the biocompatible polymer is 5-30% of the matrix. In some embodiments of the present invention, the biocompatible polymer is polyethylene glycol (PEG), preferably PEG, having a molecular weight up to and including 10,000 Da.

According to specific embodiments of the present invention, the first lipid contains at least one sterol. According to some embodiments of the present invention, the sterol

- 5 041251 is a phytosterol. In some embodiments of the present invention, the sterol is a zoosterol. In accordance with specific embodiments of the present invention, the sterol is cholesterol. According to some variants of implementation of the present invention, the first lipid component contains a mixture of sterols. In some embodiments of the present invention, the first lipid component is substantially free of non-sterol lipids. According to some variants of implementation of the present invention, the first lipid component is 5-40% wt./wt. matrix. According to some preferred embodiments of the present invention, the sterol is cholesterol and is up to 50% wt./wt. from the total lipid content in the specified matrix composition. The total lipid content refers to the weight of all lipids in the matrix composition, for example, the first lipid component, the second lipid component and any additional lipid additive included in the matrix. In accordance with specific embodiments of the present invention, the first lipid and polymer are non-covalently linked.

In some embodiments of the present invention, each phospholipid fatty acid chain contains at least 12 carbon atoms. In some embodiments of the present invention, each phospholipid fatty acid chain contains no more than 18 carbon atoms. In some embodiments of the present invention, the fatty acid chains of the phospholipid are fully saturated. In some embodiments of the present invention, at least one of the fatty acid chains of the phospholipid is unsaturated (eg, contains at least one double bond). In some embodiments, both fatty acid chains of the phospholipid are unsaturated. In some embodiments, the second lipid comprises a phospholipid selected from the group consisting of phosphatidylcholine, a mixture of phosphatidylcholines, phosphatidylethanolamine, and combinations thereof. In some embodiments, the second lipid comprises a mixture of phosphatidylcholines. According to some embodiments of the present invention, the second lipid component further comprises an additional phospholipid selected from the group consisting of phosphatidylserine, phosphatidylglycerol, and phosphatidylinositol. According to some variants of implementation of the present invention, the second lipid component is 30-80% wt./wt. matrix composition.

According to some embodiments of the present invention, the pharmaceutically active agent is included in the matrix composition. According to some embodiments of the present invention, the pharmaceutically active agent is an antibiotic agent. According to some embodiments of the present invention, the pharmaceutically active agent is an antiseptic agent. According to some embodiments of the present invention, the pharmaceutically active agent is an anti-inflammatory agent. In accordance with some embodiments of the present invention, the pharmaceutically active agent is a steroid or non-steroidal anti-inflammatory drug.

In accordance with some embodiments of the present invention, the pharmaceutically active agent is 1-20% wt./wt. matrix composition. In accordance with some embodiments of the present invention, the pharmaceutically active agent is approximately 5-15% wt./wt. matrix composition. In accordance with some typical embodiments of the present invention, the pharmaceutically active agent is approximately 8-12% wt./wt. matrix composition.

In accordance with some variants of implementation of the coated substrate used to prevent and/or treat infections in the area of surgical intervention, is approximately 60-90% wt./wt. substrate and 10-40% wt./wt. matrix composition described in this document. According to some variants of implementation of the present invention, the coated substrate is approximately 70-90% wt./wt. substrate and 10-30% wt./wt. matrix composition. In accordance with some variants of the invention, the coated substrate is approximately 80-90% wt./wt. substrate and 10-20% wt./wt. matrix composition. In accordance with some variants of implementation of the present invention, the coated substrate is approximately 85-90% wt./wt. substrate and 10-15% wt./wt. matrix composition.

According to some embodiments of the present invention, the coating matrix composition has a highly organized multilayer structure, in which the polymer and lipids are organized in a plurality of alternating layers. According to some variants of implementation of the present invention, the matrix composition contains a continuous structure, devoid of internal gaps and/or free volume. According to some embodiments of the present invention, the matrix composition is saturated with lipids, which indicates that the space between the polymer layers or the polymer backbone is filled with lipid molecules in combination with a pharmaceutical agent to the extent that additional lipid fragments can no longer be included in the matrix to a noticeable extent. .

According to some embodiments of the present invention, the matrix composition can

- 6 041251 able to release at least 30% of the pharmaceutical agent by zero order kinetics. Without being limited by a particular theory or mechanism of action, the authors of the present invention believe that the specified organized structure or substructure of the matrix composition according to the present invention is one of the main reasons for the release rate of the zero order of the drug or drugs from the matrix composition after its hydration. Therefore, the zero order release rate can be explained by the slow and continuous exfoliation of the drug along with the formulation components from the hydrated surface layer(s) of the highly organized lipid and polymer layers. According to some variants of implementation of the present invention, the matrix according to the present invention is water resistant. In this regard, water cannot readily, or not at all, diffuse into the matrix, and the pharmaceutically active agent trapped between the layers cannot readily, or not at all, diffuse out of the matrix. In accordance with some embodiments of the present invention, the drug is released from the matrix compositions disclosed herein with the gradual destruction of the surface of the matrix, which allows for sustained release over several days to several weeks. The biocompatible substrate retains its three-dimensional structure during the release of the pharmaceutical agent due to the hydrophobic matrix composition that coats or impregnates the substrate. The gradual destruction of the matrix composition will eventually lead to the exposure of the surface of the substrate. Exposure of the biodegradable substrate to biological body fluids initiates its destruction and removal without trace amounts in the treated surgical area. In a specific embodiment, the present invention provides methods for suppressing, preventing or treating a surgical site infection associated with a surgical operation, comprising the step of introducing into the surgical site a biodegradable substrate impregnated and/or the surface of which is covered, in whole or in part, with a matrix composition containing (a) biodegradable polyester;

(b) a sterol;

(c) phosphatidylcholine containing fragments of fatty acids containing at least 14 carbon atoms; and (d) an antibiotic agent.

According to another implementation variant of the matrix composition contains at least 50% lipids by weight of the matrix. In another embodiment, the matrix composition contains at least 40% phospholipids by weight of the matrix. In some embodiments of the present invention, the biodegradable, slow release coating composition contains at least 10% polymer by matrix weight. According to some embodiments of the present invention, the biodegradable slow release coating composition (matrix) contains at least 5% of the antibiotic by weight of the matrix. In another embodiment, the matrix composition is homogeneous. In some embodiments, the polymer, the sterol non-covalently bound to it, and the phospholipid form a structurally ordered lipid-rich matrix composition that is substantially free of water. In some embodiments, the substrate coated/impregnated with the matrix composition is selected from calcium orthophosphate particles or polyvinyl alcohol particles.

In some embodiments of the present invention, the matrix composition comprises (a) a biodegradable polyester selected from PLA, PGA, and PLGA;

(b) cholesterol that is non-covalently linked to a biodegradable polyester;

(c) at least one phospholipid containing fragments of fatty acids containing 16-18 carbon atoms; and (d) an antibiotic agent.

According to another implementation variant of the matrix composition contains at least 50% lipids by weight of the matrix. In another embodiment, the matrix composition contains at least 40% phospholipids by weight of the matrix. In some embodiments of the present invention, the biodegradable, slow release coating composition contains at least 10% polymer by matrix weight. According to some embodiments of the present invention, the biodegradable slow release coating composition (matrix) contains at least 5% of the antibiotic by weight of the matrix. In some embodiments of the present invention, the phospholipid is phosphatidylcholine. In some embodiments of the present invention, the phosphatidylcholine is a mixture of phosphatidylcholines. In some embodiments of the present invention, the phosphatidylcholine(s) contains saturated fatty acid moieties, i. e. fatty acid chains do not contain carbon-carbon double bonds. In some embodiments of the present invention, the phospholipid is selected from the group consisting of DMPC, DPPC, DSPC, DOPC, and any

- 7 041251 battle of their combination. In some embodiments of the present invention, the phospholipid is selected from DPPC, DSPC, and any combination thereof. In some embodiments of the present invention, the phospholipid is selected from DMPC, DPPC, and any combination thereof. In some embodiments of the present invention, the phospholipid is selected from DMPC, DPPC, DOPC, and any combination thereof. In some embodiments, the polymer, its associated cholesterol, and the phospholipid form a structured, lipid-rich matrix composition that is substantially free of water. In some embodiments, the substrate coated/impregnated with the matrix composition is selected from calcium orthophosphate particles or polyvinyl alcohol particles.

In some embodiments, the matrix composition contains (a) polyethylene glycol (PEG);

(b) cholesterol that is non-covalently linked to the polymer;

(c) at least one phospholipid containing fragments of fatty acids containing 14-18 carbon atoms; and (d) an antibiotic agent.

According to another implementation variant of the matrix composition contains at least 50% lipid by weight of the matrix. In another embodiment, the matrix composition contains at least 40% phospholipids by weight of the matrix. In some embodiments of the present invention, the biodegradable sustained release coating composition contains at least 10% polymer by matrix weight. According to some embodiments of the present invention, the biodegradable sustained release coating composition (matrix) contains at least 5% of the antibiotic by weight of the matrix. In some embodiments of the present invention, the phospholipid is phosphatidylcholine. In some embodiments of the present invention, the phosphatidylcholine is a mixture of phosphatidylcholines. According to some embodiments of the present invention, the phosphatidylcholine(s) contains(at) saturated fatty acid moieties, i. e. fatty acid chains do not contain carbon-carbon double bonds. In some embodiments of the present invention, the phospholipid is selected from the group consisting of DMPC, DPPC, DSPC, DOPC, and any combination thereof. In some embodiments of the present invention, the phospholipid is selected from DPPC, DSPC, and any combination thereof. In some embodiments of the present invention, the phospholipid is selected from DMPC, DPPC, and any combination thereof. In some embodiments of the present invention, the phospholipid is selected from DMPC, DPPC, DOPC, and any combination thereof. In some embodiments, the polymer, its associated cholesterol, and the phospholipid form a structured, lipid-rich matrix composition that is substantially free of water. In some embodiments, the substrate coated/impregnated with the matrix composition is selected from calcium orthophosphate particles or polyvinyl alcohol particles.

In this application, the terms preventing or preventing infection of the surgical site refer to inhibiting or eradicating the replication of bacteria in and around the surgical site, inhibiting the transmission of bacteria or preventing the settlement of bacteria in and around the surgical site, or alleviating the symptoms of a disease that may be caused by infection. Treatment will be considered therapeutic if the bacterial load is reduced.

Methods according to some embodiments of the present invention are useful for preventing or inhibiting biofilm formation in and around a surgical site in a subject in need thereof. Inhibition of biofilm formation in a surgical site refers to the inhibition of biofilm formation on surfaces such as biological tissues and/or materials or devices that may be used or implanted during surgery. In some embodiments of the present invention, the drug-eluting substrate described herein is also capable of causing eradication of an existing biofilm formed prior to surgery.

In this application, the term biofilm is defined according to its usual meaning in the art as a structured community of microorganisms that grow while attached to a surface and develop a mucilaginous layer of extracellular polymers, with the microbial communities embedded in a protective environment. The surfaces to which the biofilm is attached may be inert or living surfaces (eg, wound tissue, dead cells, biomaterials, and surgical implants (eg, sutures and solid stainless steel wire products)). The biofilm community may include bacteria, fungi, yeasts, protozoa, and other microorganisms. Biofilms that are commonly found associated with human tissue and organ surfaces are often bacterial biofilms.

In this application, the term subject refers to a human, a patient who has an infection, develops an infection (biofilm formation is clinically evident or detectable by one of skill in the art, but the biofilm is not yet fully formed), or who is at risk of developing an infection (biofilm formation is still clinics are not detected

- 8 041251 stomy or a person skilled in the art, however, it is known that the patient is at risk of developing a biofilm due to a disease or is awaiting a surgical operation, such as, for example, cardiac surgery or graft implantation). The term subject refers to a mammal, preferably a human, who is to be treated or is being treated by a clinician (physician, nurse, or other medical practitioner) for the treatment of a disease, condition, operation, or routine examination.

The term controlled release refers to controlling the rate and/or amount of the pharmaceutically active agent(s) delivered by the matrix compositions of the present invention. Controlled release may be continuous or intermittent and/or linear or non-linear.

The term sustained release means that the pharmaceutically active agent is released over an extended period of time.

General characteristics of the matrix composition used to coat the substrate.

The matrix composition used to impregnate or coat a biodegradable substrate in accordance with some embodiments of the present invention contains (a) a biocompatible polymer;

(b) a first lipid component containing at least one sterol that is non-covalently linked to a biocompatible polymer;

(c) a second lipid component containing at least one phospholipid containing fragments of fatty acids containing at least 12 carbon atoms; and (d) a pharmaceutically active agent.

The matrix compositions provide sustained release of the pharmaceutically active agent at the site of the surgical intervention in the body of the subject in need thereof.

According to specific embodiments of the present invention, the polymer and lipids form a structured, lipid-rich matrix composition that is substantially free of water. According to some variants of implementation of the present invention, the matrix composition has a highly organized multilayer structure, in which the polymer and lipids are organized in several alternating layers. In some embodiments, the biocompatible coating matrix contains at least about 50% total lipids by weight.

According to some variants of implementation of the present invention, the matrix composition contains at least 10% biocompatible polymer by weight. According to some variants of implementation of the present invention, the matrix composition contains approximately 10-30% polymer by weight. According to some variants of implementation of the present invention, the matrix composition contains approximately 15-25% polymer by weight. According to some variants of implementation of the present invention, the matrix composition contains approximately 20% polymer by weight. According to some embodiments of the present invention, the biocompatible polymer is at least 10% wt./wt., at least 11% wt./wt., at least 12% wt./wt., at least 13% wt./ wt., at least 14% wt./wt., at least 15% wt./wt., at least 16% wt./wt., at least 17% wt./wt., at least 18% w/w, at least

19% w/w, at least 20% w/w, at least 21% w/w, at least

22% w/w, at least 23% w/w, at least 24% w/w, at least

25% w/w, at least 26% w/w, at least 27% w/w, at least

28% wt./wt., at least 29% wt./wt., at least 30% wt./wt. matrix.

In accordance with some embodiments of the present invention, the polymer is a biodegradable polyester. In accordance with some embodiments of the present invention, the polyester is selected from the group consisting of PLA (polylactic acid). The term PLA refers to poly-L-lactide, poly-O-lactide and poly-DL-lactide. In another embodiment, the polymer is PGA (polyglycolic acid). In another embodiment, the polymer is PLGA (lactic acid-glycolic acid copolymer). The PLA contained in PLGA may be any PLA known in the art, such as a mixture of enantiomers or a racemate. PLGA according to the methods and compositions of the present invention in another embodiment has a lactic acid/glycolic acid ratio of 50:50. In another embodiment, the ratio is 60:40. In another embodiment, the ratio is 75:25. In another embodiment, the ratio is 85:15. In another embodiment, the ratio is 90:10. In another embodiment, the ratio is 95:5. In another embodiment, the ratio is another ratio that is suitable for obtaining a sustained or sustained release profile under in vivo conditions. PLGA may be a random or block copolymer. Each possibility represents a separate embodiment of the present invention. It should be emphasized that the polymer can be of any size or length (ie any molecular weight).

- 9 041251

According to another embodiment of the present invention, the biodegradable polyester may be selected from the group consisting of polycaprolactone, polyhydroxyalkanoate, polypropylene fumarate, polyorthoether, polyanhydride, and polyalkyl cyanoacrylate, provided that the polyester contains a hydrogen bond acceptor moiety. According to another embodiment of the present invention, the biodegradable polyester is a block copolymer containing a combination of any two monomers selected from the group consisting of PLA, PGA, PLGA, polycaprolactone, polyhydroxyalkanoate, polypropylene fumarate, polyorthoether, polyanhydride, and polyalkyl cyanoacrylate. In another embodiment, the biodegradable polyester is a random copolymer containing a combination of any two of the monomers listed above. Each possibility represents a separate embodiment of the present invention.

The term biodegradable refers to a substance that will degrade over time through hydrolytic action, enzyme action and/or other similar mechanisms in the human body. The term biodegradable further implies that the substance can be broken down or degraded in the body to non-toxic components after or during the period when the therapeutic agent has been or is being released.

The term biocompatible refers to a substance that will not cause significant tissue irritation or necrosis in the target tissue.

According to some embodiments of the present invention, the matrix composition contains up to 40% wt./wt. a first lipid component containing a sterol that is non-covalently linked to a biocompatible polymer. In accordance with some variants of implementation of the present invention, the sterol is approximately 30% wt./wt. by weight of the matrix composition. According to some variants of implementation of the present invention, the matrix composition contains approximately 5-40% wt./wt. the first lipid component containing sterol. According to some variants of implementation of the present invention, the matrix composition contains approximately 5-30% wt./wt. sterol. According to some variants of implementation of the present invention, the matrix composition contains approximately 5-20% wt./wt. sterol. According to some variants of implementation of the present invention, the matrix composition contains approximately 5-15% wt./wt. sterol. According to some variants of implementation of the present invention, the matrix composition contains approximately 7-13% wt./wt. sterol. According to some variants of implementation of the present invention, the matrix composition contains approximately 9-11% wt./wt. sterol. In accordance with some typical implementation options matrix composition contains approximately 10% wt./wt. sterol. According to some embodiments of the present invention, the sterol is at least 5% wt./wt., at least 6% wt./wt., at least 7% wt./wt., at least 8% wt./ wt., at least 9% wt./wt., at least 10% wt./wt., at least 11% wt./wt., at least 12% wt./wt., at least 13% w/w, at least 14% w/w, at least 15% w/w, at least 16% w/w, at least 17% w/w ., at least 18% wt./wt., or at least 19% wt./wt. matrix. According to some embodiments of the present invention, the sterol is at most 20% w/w, at most 19% w/w, at most 18% w/w, at most 17% w/w, at most 16% w/w, max 15% w/w, max 14% w/w, max 13% w/w, max 12% w/w, max 11% w/w, max 10% w/w, max 9% w/w, max 8% w/w, max 7% w/w, max 6% w/w or not more than 5% wt./wt. matrix. In some preferred embodiments, the sterol is cholesterol.

According to some variants of implementation of the present invention, the matrix composition contains at least about 30% wt./wt. the second lipid component containing at least one phospholipid containing fragments of fatty acids containing at least 12 carbon atoms. According to some variants of implementation of the present invention, the matrix composition contains at least about 40% wt./wt. the second lipid component containing at least one phospholipid containing fragments of fatty acids containing at least 12 carbon atoms. According to some variants of implementation of the present invention, the matrix composition contains approximately 40-75% wt./wt. the second lipid component containing at least one phospholipid containing fragments of fatty acids containing at least 12 carbon atoms. According to some variants of implementation of the present invention, the matrix composition contains approximately 50-70% wt./wt. the second lipid component containing at least one phospholipid containing fragments of fatty acids containing at least 12 carbon atoms. In accordance with some typical implementation options matrix composition contains approximately 60% wt./wt. the second lipid component containing at least one phospholipid containing fragments of fatty acids containing at least 12 carbon atoms. According to some embodiments of the present invention, the second lipid component containing at least one phospholipid containing fatty acid fragments containing at least 12 carbon atoms is at least

- 10 041251

40% w/w, at least 45% w/w, at least 50% w/w, at least 55% w/w, at least 60% w/w ., at least 65% wt./wt. or at least 70% wt./wt. matrix. According to some embodiments of the present invention, the second lipid component containing at least one phospholipid containing fragments of fatty acids containing at least 12 carbon atoms is not more than 75% wt./wt., not more than 70% wt./wt. , not more than 65% wt./wt. matrix. According to some variants of implementation of the present invention, the second lipid component contains at least one molecule of a phospholipid containing fragments of fatty acids containing at least 14 carbon atoms. According to some variants of implementation of the present invention, the second lipid component contains at least one molecule of phosphatidylcholine containing fragments of fatty acids containing at least 14 carbon atoms. In accordance with some embodiments of the present invention, the phosphatidylcholine molecules of the composition comprise DMPC. In accordance with some embodiments of the present invention, the phosphatidylcholine molecules of the composition comprise DPPC. In accordance with some embodiments of the present invention, the phosphatidylcholine molecules of the composition comprise DSPC. According to some embodiments of the present invention, the matrix composition contains DOPC. According to some variants of implementation of the present invention, the matrix composition contains a mixture of DOPC with a second phospholipid containing fragments of fatty acids containing at least 14 carbon atoms. According to some variants of implementation of the present invention, the matrix composition contains a mixture of DMPC and DPPC. Typically, the ratio of DMPC to DPPC in the composition is from about 10:1 to 1:10. In accordance with some variants of implementation of the present invention, the matrix composition contains a mixture of DPPC and DSPC. Typically, the ratio of DPPC to DSPC in a formulation is about 10:1 to 1:1; preferably 5:1 to 2:1; more preferably, the ratio between DPPC and DSPC in the formulation is about 3:1. In accordance with some embodiments of the present invention, the matrix composition contains approximately 50-70% wt./wt. mixtures of DMPC and DPPC. According to some variants of implementation of the present invention, the matrix composition contains approximately 50-70% wt./wt. mixtures of DPPC and DSPC.

In some embodiments of the present invention, the lipid:polymer weight ratio of the composition of the present invention is between 1:1 and 9:1, inclusive. According to another implementation variant, the ratio is from 2:1 to 9:1 inclusive. According to another implementation variant, the ratio is from 3:1 to 9:1 inclusive. According to another implementation variant, the ratio is from 4:1 to 9:1 inclusive. According to another implementation variant, the ratio is from 5:1 to 9:1 inclusive. According to another implementation variant, the ratio is from 6:1 to 9:1 inclusive. According to another implementation variant, the ratio is from 7:1 to 9:1 inclusive. According to another implementation variant, the ratio is from 8:1 to 9:1 inclusive. According to another implementation variant, the ratio is from 1.5:1 to 9:1 inclusive. Each possibility represents a separate embodiment of the present invention.

It should be emphasized that the period of sustained release using the compositions according to the present invention can be programmed taking into account the biochemical and/or biophysical properties of the biopolymer and lipid. In particular, the rate of polymer degradation and the fluidity of lipids should be considered. For example, a PLGA (85:15) polymer will degrade more slowly than a PLGA (50:50) polymer. Phosphatidylcholine (12:0) is more fluid (less rigid and less ordered) at body temperature than phosphatidylcholine (18:0). Therefore, for example, the release rate of a drug included in a matrix composition containing PLGA (85:15) and phosphatidylcholine (18:0) will be lower than that of a drug included in a matrix consisting of PLGA (50:50) and phosphatidylcholine (14:0). Another aspect that will determine the rate of release includes the physical characteristics of the entrapped or embedded drug. In addition, the release rate of drugs can be further controlled by adding other lipids to the matrix composition, some of which are described below.

According to some variants of implementation of the present invention, the matrix composition contains approximately 1-20% wt./wt. pharmaceutically active agent. According to some variants of implementation of the present invention, the matrix composition contains approximately 5-15% wt./wt. pharmaceutically active agent. According to some variants of implementation of the present invention, the matrix composition contains approximately 8-12% wt./wt. pharmaceutically active agent. According to some variants of implementation of the present invention, the matrix composition contains approximately 10% wt./wt. pharmaceutically active agent. According to some embodiments of the present invention, the pharmaceutically active agent is at least 1% wt./wt., at least 2% wt./wt., at least 3% wt./wt., at least 4% wt. /wt., at least 5% wt./wt., at least 6% wt./wt., at least 7% wt./wt., at least 8% wt./wt., at least at least 9% w/w, at least

- 11 041251

10% w/w, at least 11% w/w, at least 12% w/w, at least 13% w/w, at least 14% w/w ., at least 15% wt./wt., at least 16% wt./wt., at least 17% wt./wt., at least 18% wt./wt. or at least 19% wt./wt. matrix. According to some embodiments of the present invention, the pharmaceutically active agent is at most 20% w/w, at most 19% w/w, at most 18% w/w, at most 17% w/w, no more than 16% wt./wt., no more than 15% wt./wt., no more than 14% wt./wt., no more than 13% wt./wt., no more than 12% wt./wt., not more than 11% wt./wt., not more than 10% wt./wt., not more than 9% wt./wt., not more than 8% wt./wt., not more than 7% wt./wt., not more than 6% wt./wt., not more than 5% wt./wt. matrix.

According to some embodiments of the present invention, the pharmaceutically active agent is an antibiotic agent. According to some embodiments of the present invention, the pharmaceutically active agent is an antifungal agent. According to some embodiments of the present invention, the pharmaceutically active agent is an antiseptic agent. According to some embodiments of the present invention, the pharmaceutically active agent is an anti-inflammatory agent. In accordance with some embodiments of the present invention, the pharmaceutically active agent is a steroidal or non-steroidal anti-inflammatory drug. According to some embodiments of the present invention, a plurality of pharmaceutically active agents are included in the matrix composition, for example, a combination of two or more antibiotic agents, a combination of one or more antibiotic agents and one or more antifungal agents, a combination of one or more antibiotic agents and one or more non-steroidal anti-inflammatory drugs (NSAIDs). According to some embodiments of the present invention, the pharmaceutically active agent is included in the matrix composition. Each possibility represents a separate embodiment of the present invention. According to some embodiments of the present invention, the pharmaceutically active agent has low water solubility. In another embodiment, the pharmaceutically active agent is hydrophobic. In another embodiment, the pharmaceutically active agent is amphipathic.

The term hydrophobic refers to a material that has a solubility in distilled water at ambient temperature of less than about 1 g per 100 ml, or less than about 0.5 g per 100 ml, or less than about 0.1 g per 100 ml.

As used herein, a pharmaceutically active agent having low water solubility refers to a material whose solubility in distilled water at ambient temperature is less than about 3 g per 100 ml, or less than about 2 g per 100 ml, or 1-2 g per 100 ml.

In accordance with some embodiments of the present invention, the pharmaceutically active agent used in the methods of some embodiments of the present invention is an antibiotic agent selected from the group consisting of penicillin antibiotics, cephalosporin antibiotics, macrolide antibiotics, tetracycline antibiotics, glycylcycline antibiotics, fosfomycin antibiotics , aminoglycoside antibiotics and new quinolone antibiotics. Non-limiting examples of antibiotics include amoxicillin, amoxicillin/clavulanic acid, ampicillin/sulbactam, penicillin, metronidazole, clindamycin, chlortetracycline, demeclocycline, oxytetracycline, amikacin, gentamicin, kanamycin, neomycin, netilmicin, streptomycin, tobramycin, cefadroxil, cephalopyrin, cefazolin, cefazolin, Cefradin, Cephacon, Cepamanol, Cepametazole, Cephonicide, Cephotan, Cephoxin, Coffpopodsim, Cefprosil, Cefeporsim, Cefdinir, Cefephoperzon, Cefotrazim, Ceftazim, Ceftibuten, Ceftizim, Ceiffrixon, Cephysin, Clamicin, Claramperis, Clamicaran, Clamitirus, Mistemicin, Clamage, Mistyriper, Mistyripin. , тролеандомицин, бакампициллин, карбенициллин, клоксациллин, диклоксациллин, метициллин, мезлоциллин, нафциллин, оксациллин, пиперациллин, тикарциллин, циноксацин, ципрофлоксацин, эноксацин, грепафлоксацин, левофлоксацин, ломефлоксацин, налидиксовую кислоту, норфлоксацин, офлоксацин, спарфлоксацин, сулфизоксазол, сульфацитин, сульфадиазин, sulfamethoxazole, dapsone, aztreonam, bacitracin, capreomycin, chloramphenicol, clofazimine, colistimethate, colistin, cycloserine, fosfomycin, furazolidone, urotropine, nitrofurantoin, pentamidine, rifabutin, rifampin, spectinomycin, tigecycline, trimethoprim, antibiotic trimetrexate glucuronate, vancomycin, chlorhexidine and ertapenem. In accordance with some embodiments of the present invention, the antibiotic agent is a peptide antibiotic. Each antibiotic is a separate embodiment of the present invention.

In some preferred embodiments, the antibiotic agent according to the methods and compositions of the present invention is a tetracycline. In one embodiment, the tetracycline is doxycycline. In another embodiment, the antibiotic is a hydrophobic tetracycline. Non-limiting examples

- 12 041251 hydrophobic tetracyclines include 6-dimethyl-6-deoxytetracycline, 6-methylenetetracycline, minocycline (also known as 7-dimethylamino-6-dimethyl-6-deoxytetracycline) and 13-phenylmercapto-a6-deoxytetracycline. In another embodiment, the antibiotic is selected from the group consisting of doxycycline, tetracycline, and minocycline.

In another embodiment, the antibiotic is doxycycline or doxycycline hyclate.

Doxycycline can be effectively used to treat surgical site infections caused by many types of Gram-negative and Gram-positive bacteria and to treat a number of conditions. It is important to note that doxycycline is very effective against Staphylococcus aureus (S. aureus), the most common bacteria causing surgical site infections. In addition, bacteriological testing shows significant doxycycline susceptibility in methicillin-resistant Staphylococcus aureus (MRS A). The minimum inhibitory concentrations (MICs) of doxycycline against common bacteria as well as Staphylococcus aureus are relatively low and can be as low as 0.1 μg/mL (against Staphylococcus aureus), providing high in vivo activity against surgical site infections .

In accordance with some embodiments of the present invention, the pharmaceutically active agent used in the methods in accordance with some embodiments of the present invention is an antifungal agent selected from the group consisting of amphotericin B complex and cholesterol sulfate, natamycin, amphotericin, clotrimazole, nystatin, lipid complex amphotericin B, fluconazole, flucytosine, griseofulvin, itraconazole, ketoconazole, benzoic acid and salicylic acid, betamethasone and clotrimazole, butenafine, carbola-fuchsin, ciclopirox, clioquinol, clioquinol and hydrocortisone, clotrimazole, econazole and hydrocortisone , ketoconazole, miconazole, naftifine, nystatin, nystatin and triamcinolone, oxyconazole, sodium thiosulfate, sulconazole, terbinafine, tolnaftate, triacetin, undecylic acid and its derivatives, butoconazole, clotrimazole, sulfonamides, terconazole and thioconazole.

According to some embodiments of the present invention, the matrix composition of the present invention may contain, in addition to the antibiotic agent and/or antifungal agent, another pharmaceutically active agent selected from steroids and/or non-steroidal anti-inflammatory drugs (NSAIDs).

Any suitable NSAID may be included in the matrix composition for delayed and/or controlled release. Non-limiting examples of NSAIDs include ibuprofen, flurbiprofen, sodium aminosalicylate, magnesium choline trisalicylate, choline salicylate, diclofenac, diflunisal, etodolac, fenoprofen, indomethacin, ketoprofen, ketolac tromethamine, magnesium salicylate, meclofenamate, mefenamic acid, nabumetone, pyrobutazine, fenoxycamsen, oxaproxen , salsalat, sulindak and tolmetin. Each of the listed NSAIDs is a separate embodiment of the present invention.

Any suitable non-steroidal anti-inflammatory drug may be included in the matrix composition. Non-limiting examples of steroidal anti-inflammatory drugs (SAIDs) for use in the compositions of the present invention include, but are not limited to, corticosteroids such as betamethasone, betamethasone valerate, cortisone, dexamethasone, dexamethasone 21-phosphate, fludrocortisone, flumethasone, fluocinonide, fluocinonide desonide, fluocinolone , fluocinolone acetonide, fluocortolone, halcinonide, halopredone, hydrocortisone, hydrocortisone 17-valerate, hydrocortisone 17-butyrate, hydrocortisone 21-acetate, methylprednisolone, prednisolone, prednisolone 21-phosphate, prednisone, triamcinolone, triamcinolone acetonide, cortodoxone, fluoroacetonide, flufluoroacetonide diacetate, flurandrenolone acetonide, medrisone, amcinafel, amcinafide, betamethasone and its other esters, chlorprednisone, clorcortelone, descinolone, desonide, dichlorizone, difluprednate, flucloronide, flumethasone, flunisolide, flucortolone, fluoromethalone, fluperolone, fluprednisolone, fluprednisone, paramethazine, para-meprednisone cortiz она ацетат, гидрокортизона циклопентилпропионат, кортодоксон, флуцетонид, флудрокортизона ацетат, флурандренолона ацетонид, медризон, амцинафал, амцинафид, бетаметазон, бетаметазона бензоат, хлоропреднизона ацетат, клокортолона ацетат, дезцинолона ацетонид, дезоксиметазон, дихлоризона ацетат, дифлупреднат, флуклоронид, флуметазона пивалат, флунизолида ацетат , fluperolone acetate, fluprednisolone valerate, paramethasone acetate, prednisolamate, prednival, triamcinolone hexacetonide, cortivazole, formocortal and nivazol.

According to specific embodiments of the present invention, the matrix composition is substantially free of water. As used herein, the term substantially free of water refers, in one embodiment, to a composition containing less than 5% water by weight. In another embodiment, the term refers to a composition containing less than 4.5% water by weight. In another embodiment, the term refers to a composition containing less than 4.0% water by weight. In another embodiment, the term refers to a composition containing less than 3.5% water.

- 13 041251 by weight. In another embodiment, the term refers to a composition containing less than 3.0% water by weight. In another embodiment, the term refers to a composition containing less than 2.5% water by weight. In another embodiment, the term refers to a composition containing less than 2.0% water by weight. In another embodiment, the term refers to a composition containing less than 1.5% water by weight. In another embodiment, the term refers to a composition containing less than 1.0% water by weight. According to another implementation variant, the term refers to the absence of such amounts of water that affect the water-resistant properties of the composition. In another embodiment, the term refers to a composition made without the use of any aqueous solvents. In another embodiment, preparing a composition using the substantially water-free process described herein provides lipid saturation. Saturation with lipids provides the ability of the matrix composition to resist general degradation under in vivo conditions; therefore, the matrix composition is capable of mediating sustained release on a scale of days, weeks, or months.

In another embodiment, the matrix composition is substantially free of free water. In another embodiment, the term refers to a composition that does not contain detectable amounts of unbound water. The term free water refers to free water that is not part of the thin film of water (usually a few molecules thick) that forms on the surface of macromolecules (eg, phospholipids and polymers). The total amount of water in the composition can be determined by any method known in the art, such as Karl Fischer titration and loss on drying. The ratio of the amounts of bound and unbound water can be determined, for example, using differential scanning calorimetry (DSC).

Technological platform of the substrate, impregnated or completely or partially coated with a matrix composition used in the methods according to the present invention.

According to some embodiments of the present invention, the coating matrix composition has a highly organized multilayer structure, in which the polymer and associated cholesterol form one type of layer, the phospholipids form a second type of layer, and these two types of layers are organized as a plurality of alternating or quasi-alternating layers.

According to some variants of implementation of the present invention, the coating matrix composition according to the present invention contains a continuous structure, devoid of internal gaps and/or free volume. According to some embodiments of the present invention, the coating matrix composition is saturated with lipids, this indicates that the space between the polymer layers or the polymer backbone is filled with lipid molecules in combination with a pharmaceutically active agent (for example, an antibiotic agent and/or an antifungal agent), to the extent that additional lipid fragments cannot be incorporated into the matrix to a noticeable extent.

Coating matrix compositions disclosed in this description are saturated with lipids. As used herein, the term lipid-rich refers to saturation of the matrix composition polymer with a first lipid component (eg, cholesterol) and a second lipid component (eg, phospholipids) in combination with any pharmaceutical agent present in the matrix and any other lipids that may be present. The matrix composition is saturated with any lipids present. In another embodiment, lipid saturation refers to the filling of internal gaps (void volume) within the lipid matrix defined by the outer boundary of the polymer backbone. The gaps are filled with phosphatidylcholines in combination with cholesterol and possibly another type of lipid and antibiotic agent present in the matrix, to the extent that the additional lipid moieties can no longer be incorporated into the matrix to any appreciable extent. The lipid-rich matrices of the present invention have the additional advantage that they do not require a synthetic emulsifier or surfactant such as polyvinyl alcohol; therefore, matrix compositions according to the present invention, as a rule, essentially do not contain polyvinyl alcohol.

According to some embodiments of the present invention, the coating matrix composition is capable of releasing at least 30% of the active agent in zero order kinetics when maintained in an aqueous environment (when hydrated). In some embodiments of the present invention, at least 40% of the pharmaceutically active agent is released from the matrix composition in zero order kinetics when maintained in an aqueous environment. According to some embodiments of the present invention, at least 50% of the pharmaceutically active agent is released from the matrix composition in zero order kinetics when maintained in an aqueous environment. Without being limited by a particular theory or mechanism of action, the authors of the present invention believe that the organized structure or substructure of the matrix composition according to the present invention is one of the main reasons for the release rate according to the zero-order kinetics of the drug or drugs

- 14 041251 from the matrix composition after its hydration. Therefore, the zero order release rate can be attributed to the slow and continuous cleavage of the hydrated surface layer(s) from the highly organized lipid and polymer layers with concomitant drug release as the surface layer components are removed from the matrix. It has been suggested that the process slowly repeats itself, releasing the drug(s) at a constant rate over days, weeks or even months until the matrix is completely destroyed. Without wishing to be bound by any theory, the present inventors believe that the polymer forms a first type of layer, and that the phospholipid(s) form a second type of layer, and that these layers alternate, i.e. (polymer)-(phospholipid)-(polymer)-(phospholipid); in this application, the term quasi-alternation is used to refer to a situation in which there is an alternation of more than one copy of a certain type of layer, for example (polymer)(phospholipid)-(phospholipid)-(polymer)-(phospholipid)-(phospholipid)-( polymer). It is assumed that the cholesterol molecules are located between the two layers, the polar head group is directed towards the polymer and the hydrophobic part is located between the phospholipid molecules.

In some embodiments of the present invention, the matrix composition comprises a plurality of mixed layers of polymer and phospholipid, as described above, that do not form microspheres, micelles, reverse micelles, or liposomes. According to some variants of implementation of the present invention, the matrix composition does not contain micelles, reverse micelles or liposomes.

In accordance with some variants of implementation of the present invention, the matrix according to the present invention is water resistant. Thus, water cannot easily or not at all diffuse into the inner layers of the matrix, and the pharmaceutically active agent trapped between the inner layers can hardly or not at all diffuse out of the matrix. More specifically, a composition is provided wherein the base (e.g., the portion of the composition that is surrounded by an outer surface, said outer surface being exposed to the environment) is not exposed to water, or is exposed to such an extent that the amount of penetrating water is small and insufficient to cause degradation. or destruction of the matrix base. Without wishing to be bound by any theory or mechanism of action, the inventors of the present invention believe that the water-resistant properties of the matrix composition, along with its unique multilayer structure, allow the matrix to provide a sustained release, for example, give the matrix the ability to release at least 30% of the pharmaceutically active agent (eg, antibiotic agent) from the composition in zero order kinetics for several days, weeks, and even months when the composition is maintained in an aqueous environment at physiological temperature.

The effectiveness of the drug is usually determined by its local concentration. The local concentration, in turn, is determined by the ratio of the rate of accumulation of the drug that is released from the product, and the rate of its excretion through physical distribution in the surrounding tissues, as well as through neutralization and / or degradation. An optimal drug delivery system should release the drug in accordance with biological demand so as to create an effective concentration in close proximity to the target and for a sufficient period of time necessary to produce the desired biological effect. This can be achieved by releasing the active form of the drug in the vicinity of the target at a rate that will result in an effective concentration that is above the minimum effective concentration but below the toxic level, and for the required period of time necessary to produce an effective therapeutic effect.

One way to more effectively control the local impact of a drug is to control the rate at which it is delivered. The rate of receipt is due

1) drug release profile;

2) release rate; And

3) duration of release.

The listed parameters are closely related; while the release rate is highly dependent on the specific formulation, the duration of release depends on two factors: the release rate and the size of the drug reservoir.

The matrix composition of the present invention, containing a combination of specific lipids and polymers loaded with a drug, preferably an antibiotic agent, not only determines the drug release rate profile, but also provides control of the release rate over an extended stage by zero order kinetics. Without wishing to be bound by any theory or mechanism of action, the present inventors believe that the most effective profile will combine an initial release that will provide an effective local drug concentration, followed by a sustained zero-order kinetic release over a sufficiently long period of time, for example, up to 2 months, up to 7 weeks, up to 6 weeks, up to 5 weeks, up to 4 weeks, up to 3 weeks, up to 2 weeks, preferably

- 15 041251 at least 3-4 weeks. The initial release must be limited so as to leave a sufficient reservoir to support the subsequent sustained release.

According to some embodiments of the present invention, if the composition is maintained in an aqueous environment at physiological temperatures, from 1 to 50% of the specified pharmaceutically active agent is released from the matrix composition by the end of the first day, from 10 to 100% of the specified pharmaceutically active agent is released from the matrix composition by the end the first week, 20 to 100% of said pharmaceutically active agent is released from the matrix composition by the end of the first two weeks and 30 to 100% of said pharmaceutically active agent is released at the end of the first three weeks. According to some embodiments of the present invention, if the composition is maintained in an aqueous environment at physiological temperatures, at least 10%, but not more than 60% of the pharmaceutically active agent is released at the end of the first week, at least 20%, but not more than 80% of the pharmaceutically active agent is released at the end of the second week, at least 30% of the pharmaceutically active agent is released at the end of the third week. At least 40% of the pharmaceutically active agent is released at the end of the third week. At least 50% of the pharmaceutically active agent is released at the end of the third week. At least 60% of the pharmaceutically active agent is released at the end of the third week. In preferred embodiments, the pharmaceutically active agent is an antibiotic agent.

In accordance with some embodiments of the present invention, as shown (see examples 1 and 2), substrate particles (eg, calcium orthophosphate or polyvinyl alcohol) impregnated/coated with a matrix composition containing approximately 15-25% wt./wt. PLGA, approximately 5-15% wt./wt. cholesterol, approximately 50-70% wt./wt. a mixture of DPPC and DSPC, in which the ratio of DPPC and DSPC is from about 5:1 to 2:1, and about 7-12% wt./wt. doxycycline show an initial release of up to about 35% of the included antibiotic, and preferably up to 30% of the included antibiotic. The amount of drug that is released immediately after hydration is clinically safe and leaves most of the drug (at least 65%) for sustained delivery for at least 30 days, and can increase local doxycycline levels to 10-50 MICs or more.

A substrate impregnated or coated, in whole or in part, with a matrix composition used in the methods of the present invention gradually releases a pharmaceutically active agent (e.g., an antibiotic agent) at a constant release rate (about 1.5-5% (weight fraction of the pharmaceutical agent released per day/total weight of the pharmaceutically active agent initially encapsulated in the matrix composition)), which provides a local drug concentration that is at least 10 times higher than the minimum inhibitory concentration (MIC) of the antibiotic against pathogens that are most common in infections of the area surgical intervention (for example, Staphylococcus aureus bacteria), for a period of up to 5 weeks.

A substrate impregnated or coated, in whole or in part, with a matrix composition used in the methods of the present invention allows a wide variety of one or more biologically active molecules to be included and released at a predetermined rate over a period of several days to several weeks.

A substrate impregnated or coated, in whole or in part, with a matrix composition used in the methods of the present invention releases the pharmaceutically active agent locally at a predictable delayed rate. Therefore, therapeutic drug levels can be maintained locally at the surgical site (eg, incision) while maintaining low or absent systemic levels. Due to the prolonged local release of the pharmaceutical agent, a small and safe dose of the local pharmaceutical agent, which, in some cases, does not exceed a single dose usually administered intravenously, is very effective in eradicating local bacterial infections of the surgical site. For example, the amount of antibiotic (e.g., doxycycline) in 5 g of substrate impregnated or coated, in whole or in part, with the matrix composition used in the methods of the present invention is approximately equal to the amount of antibiotic in a single dose, which is usually administered intravenously, or in one pill ( or tablet) for oral administration.

In addition, the coating matrix composition acts as a reservoir in which the incorporated pharmaceutical agent is protected. Unlike standard polymer-based delivery systems, this property can protect the sensitive drug reservoir not only from biodegradants such as enzymes, but also from chemical degradation due to in vivo solubility of materials and hydration. If a prolonged action is required, then this property becomes extremely important.

The term zero order release rate or zero order release kinetics means a constant, linear, continuous, delayed and controlled release rate of a pharmaceutically active agent from a polymeric matrix, i. the dependence of the amount of released pharmaceutically active agent on time is linear. According to some embodiments of the present invention, at least 30% of the pharmaceutically active agent is released from the matrix composition in zero order kinetics at a rate of approximately 1-7, 1.5-6, 1.5-5, 2-4, 1.5-3 % (mass fraction of the pharmaceutically active agent released per day/total weight of the pharmaceutically active agent initially encapsulated in the composition).

Each possibility is a separate embodiment of the present invention.

Lipids.

Phospholipids are phosphoglycerides containing one phosphatidyl bond on the backbone of glycerol and fatty acids in the other two positions. However, it should be clearly understood that phosphoglycerides containing hydrocarbon chains other than fatty acid moieties, including alkyl chains, alkenyl chains, or any other hydrocarbon chain containing at least 12 carbon atoms, or alternatively at least 14 carbon atoms are included in the scope of the present invention. The bond may be an ether bond, instead of an acyl bond that occurs in phospholipids.

Phosphatidylcholine refers to a phosphoglyceride containing a phosphorylcholine head group. Said phospholipid consists of a choline head group and glycerophosphoric acid with various fatty acid moieties. Fragments of fatty acids, as a rule, are natural. In some embodiments of the present invention, the fatty acid moieties are saturated. In some embodiments of the present invention, the fatty acid moieties are unsaturated. Saturated refers to the absence of a double bond in the hydrocarbon chain. In another embodiment, the fatty acid moieties contain at least 12 carbon atoms. According to another implementation variant of the fragments of fatty acids contain 14 carbon atoms. According to another implementation variant of the fragments of fatty acids contain 16 carbon atoms. According to another implementation variant of the fragments of fatty acids contain 18 carbon atoms. According to another implementation variant of the fragments of fatty acids contain 14-18 carbon atoms. According to another implementation variant of the fragments of fatty acids contain 14-16 carbon atoms. According to another implementation variant of the fragments of fatty acids contain 16-18 carbon atoms. In another embodiment, the fatty acid moieties are selected such that the gel-liquid-crystal transition temperature of the resulting matrix is at least 40°C. In another embodiment, both fatty acid moieties are arachidonyl. Each possibility represents a separate embodiment of the present invention.

According to another embodiment of the present invention, the phosphatidylcholine is a natural or synthetic phosphatidylcholine. According to one embodiment, the phosphatidylcholine is a symmetrical phosphatidylcholine (i.e., a phosphatidylcholine in which the two fatty acid moieties are identical (e.g.) dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), 1,2-distearoyl-sn-glycero- 3-phosphocholine (DSPC), dioleoylphosphatidylcholine (DOPC) In another embodiment, the phosphatidylcholine is an asymmetric phosphatidylcholine (e.g., 1-palmitoyl-2-stearoylphosphatidylcholine (PSPC); 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), 1-stearoyl- 2-arachidonylphosphatidylcholine (SAPC), 2-arachidonyl-1-palmitoyl-sn-glycero-3-phosphocholine (APPC) In another embodiment of the present invention, the phosphatidylcholine is any other phosphatidylcholine known in the art.Each phosphatidylcholine is separate embodiment of the present invention.

In accordance with some embodiments of the present invention, at least one phosphatidylcholine in a matrix composition useful for preventing and/or treating surgical site infections is selected from the group consisting of DMPC, DPPC, DSPC, DOPC, and any combination thereof. In another embodiment, at least one phosphatidylcholine is selected from DMPC, DPPC, or combinations thereof. In another embodiment, at least one phosphatidylcholine is selected from DPPC, DSPC, or combinations thereof. In another embodiment, at least one phosphatidylcholine is selected from DMPC, DPPC, or combinations thereof. In another embodiment, at least one phosphatidylcholine is selected from DMPC, DOPC, or combinations thereof.

Phosphatidylethanolamine consists of a combination of glycerol esterified with two fatty acids and phosphoric acid, while the phosphate group is combined with ethanolamine. In one embodiment, the fatty acid moieties may be saturated or unsaturated. In another embodiment, the fatty acid moieties contain at least 14 carbon atoms. In another embodiment, the fatty acid moieties contain at least 16 carbon atoms. According to another implementation variant of the fragments of fatty acids contain 14 carbon atoms. According to another implementation variant of the fragments of fatty acids contain 16 carbon atoms. According to another implementation variant of the fragments of fatty acids contain 18 carbon atoms. According to another implementation variant of the fatty acid fragments contain 14-18 carbon atoms

- 17 041251 genera. According to another implementation variant of the fragments of fatty acids contain 14-16 carbon atoms. According to another implementation variant of the fragments of fatty acids contain 16-18 carbon atoms. In another embodiment, the fatty acid moieties are selected such that the gel-liquid-crystal transition temperature of the resulting matrix is at least 40°C. The two fatty acids may be the same or different, and are typically attached to positions 1 and 2 of the glycerol moiety. Non-limiting examples of suitable phosphatidylethanolamines include dimethyldimyristoylphosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE), diuroylphosphatidylethanolamine (DLPE), distearoylphosphatidylethanolamine (DSPE), dioleoylphosphatidylethanolamine (DOPE), 1-palmitoyl-2-oleylphosphatidylethanolamine (POPE), 1-oleyl-2palmitoylphosphatidylethanolamine (POPE), and dielaidoylphosphatidylethanolamine (DEPE). In another embodiment, the phosphatidylethanolamine is any other phosphatidylethanolamine known in the art. Each phosphatidylethanolamine is a separate embodiment of the present invention.

Sterol, in one embodiment, refers to a steroid containing a hydroxyl group at position 3 of the A-ring. In accordance with some variants of implementation of the present invention, the sterol is up to about 40% wt./wt. by weight of the matrix composition. According to another implementation variant of the sterol in accordance with the methods and compositions according to the present invention is a zoosterol. In another embodiment, the sterol is cholesterol.

In another embodiment, the composition of the present invention further comprises a lipid other than phosphatidylcholine, phosphatidylethanolamine, or sterol. In another embodiment, the additional lipid is a phosphoglyceride. In another embodiment, the additional lipid is selected from the group consisting of phosphatidylserine, phosphatidylglycerol, and phosphatidylinositol. In another embodiment, the additional lipid is selected from the group consisting of phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, and sphingomyelin. In another embodiment, the additional lipid is selected from the group consisting of phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, sphingomyelin, and ceramides. According to another implementation variant of the specified composition contains a combination of any 2 or more of the above additional lipids. In another embodiment, the polymer, phosphatidylcholine, phosphatidylethanolamine, sterol, and additional lipid(s) are included in the matrix composition. Each possibility represents a separate embodiment of the present invention.

Additional components.

According to another implementation variant of the matrix composition in accordance with the methods and compositions according to the present invention additionally contains free fatty acids. Non-limiting examples of free fatty acids that may be included in the coating matrix composition according to the present invention are selected from omega-6 fatty acids, omega-9 fatty acids, a free fatty acid containing 14 or more carbon atoms, a free fatty acid containing 16 or more more than carbon atoms, a free fatty acid containing 16 carbon atoms, a free fatty acid containing 18 carbon atoms, a free fatty acid containing 16-22 carbon atoms, a free fatty acid containing 16-20 carbon atoms, a free fatty acid containing 16 -18 carbon atoms, free fatty acid containing 18-22 carbon atoms, free fatty acid containing 18-20 carbon atoms, linoleic acid, linolenic acid and oleic acid. In another embodiment, the free fatty acid is another suitable free fatty acid known in the art. According to another implementation variant of the free fatty acid increases the flexibility of the matrix composition. In another embodiment, the free fatty acid retards the release rate under in vivo conditions. In another embodiment, the free fatty acid improves the stability of controlled release under in vivo conditions. The fatty acid may be saturated or unsaturated. In another embodiment, the inclusion of a saturated fatty acid containing at least 14 carbon atoms increases the gel-liquid transition temperature of the resulting matrix composition. Each type of fatty acid is a separate embodiment of the present invention.

In another embodiment, the matrix composition according to the methods and compositions of the present invention further comprises tocopherol (e.g., E307 (α-tocopherol), β-tocopherol, E308 (γ-tocopherol), E309 (δ-tocopherol). According to certain embodiments of the present invention, tocopherol may be included in the matrix, instead of or in addition to the first lipid containing a polar group (eg, sterol, cholesterol).Each embodiment is a separate embodiment of the present invention.

According to another implementation variant of the matrix composition in accordance with the methods and compositions according to the present invention additionally contains physiologically acceptable buffer salts, which are well known in the art. Non-limiting examples

- 18 041251 physiologically acceptable buffer salts include phosphate buffers. A typical phosphate buffer contains 40 parts NaCl, 1 part KCl, 7 parts Na ₂ HPO ₄ -2H ₂ O and 1 part KH ₂ PO ₄ . In another embodiment, the buffer salt is any other physiologically acceptable buffer salt known in the art. Each possibility represents a separate embodiment of the present invention.

Therapeutic ways.

Methods for implementing the present invention, aimed at preventing and treating surgical site infections, meet medical needs that currently do not have effective solutions and are of great interest to the medical community. The methods of the present invention provide for the treatment and prevention of local infection required during and/or after surgery. The methods of the present invention reduce the overall incidence of infections and overcome or reduce existing infections, including infections with nosocomial resistant bacteria. The methods of the present invention can be used to treat and prevent postoperative infections in various tissues and solid organs.

In some embodiments of the present invention, methods of preventing, inhibiting, or treating a surgical site infection in a subject are useful for suppressing surgical site infections in general, and in particular, surgical site infections associated with orthopedic surgery (e.g., hip, knee, joint, total joint replacement, trauma), spine surgery, digestive system surgery (eg, esophagus, stomach, small intestine, colon, rectum, colon, appendix, liver, pancreas, gallbladder, ulcer stomach, gastric cancer, open gastric bypass, appendicitis, colectomy, cholecystectomy, vagotomy, open biliary tract surgery, small bowel surgery, colonic and rectal surgery), cardiac surgery (eg, aorto bypass, cardiothoracic transplantation, implantation of cardiac devices), hernia repair, angioplasty, caesarean section, prostatectomy, obstetric and gynecological surgery (eg, hysterectomy), head and neck cancer surgery, transplantation (eg, lung, liver, pancreas) , kidney), neurosurgery (eg, deep brain stimulation implant), and plastic surgery (eg, breast reconstruction, mastectomy).

In accordance with specific embodiments, the present invention provides methods for preventing, inhibiting or treating a wound infection in the sternum region associated with cardiac surgery, including the step of applying to the surface of the halves of the sternum and / or surrounding soft tissues of a biodegradable substrate impregnated and / or the surface of which coated, partially or completely, with a matrix composition containing (a) a biocompatible polymer;

(b) a first lipid component containing a sterol;

(c) a second lipid component containing at least one phospholipid containing fragments of fatty acids containing at least 12 carbon atoms; and (d) a pharmaceutically active agent selected from the group consisting of an antibiotic agent, an antiseptic agent, an anti-inflammatory agent, an antifungal agent, and any combination thereof.

According to some embodiments of the present invention, the pharmaceutically active agent is an antibiotic agent. In some embodiments of the present invention, the antibiotic agent is doxycycline or doxycycline hyclate. Methods for preventing, inhibiting, or treating a sternum wound infection associated with cardiac surgery further include preventing or suppressing biofilm formation on a sternum wound following cardiac surgery. In this application, the term wound infection of the sternum includes complications of superficial and deep wounds of the sternum.

In accordance with specific embodiments, the present invention provides methods for preventing, inhibiting, or treating wound infection of the sternum associated with cardiac surgery, comprising the step of applying to the surface of the halves of the sternum and/or surrounding soft tissues calcium orthophosphate particles or polyvinyl alcohol particles impregnated/coated with a matrix composition containing approximately 15-25% wt./wt. PLGA, approximately 5-15% wt./wt. cholesterol, approximately 50-70% wt./wt. a mixture of DPPC and DSPC, in which the ratio of DPPC and DSPC is from about 5:1 to 2:1 and about 7-12% wt./wt. doxycycline.

In accordance with some embodiments of the present invention, a substrate impregnated or coated, in whole or in part, with a composition in accordance with the methods of the present invention, can be introduced into the surgical site (for example, in the incision)

- 19 041251 directly or proximal to the incision site. According to some embodiments of the present invention, the impregnated/coated substrate may be administered to the surgical site (eg, incision site) by spraying the impregnated/coated substrate particles onto and near the surgical site. According to some variants of implementation of the present invention, the coated substrate is made in the form of a powder. According to some embodiments of the present invention, the coated substrate particles can be sprayed over and near the surgical site using a saline shaker container or dispenser. As used herein, a salt shaker container or dispenser refers to a container defining a cavity (eg, tubular cavity) for storing coated substrate powder, which has at least one opening through which coated substrate particles can be dispensed. In another embodiment, a substrate impregnated or coated, in whole or in part, with a composition in accordance with some embodiments of the present invention may be injected into and near the surgical site (eg, incision area). In another embodiment, substrates in the form of a sponge, foam, or sheet impregnated or coated, in whole or in part, with a composition in accordance with some embodiments of the present invention, can be introduced into the surgical site, by placing them above or near the surgical site, for example, by covering the area of surgical intervention with at least one part of a gelatin or collagen sponge, foam or sheet impregnated or coated with a matrix composition. In another embodiment, biocompatible substrates impregnated or coated, in whole or in part, with a composition in accordance with some embodiments of the present invention can be made into a paste and dispensed into and around the surgical site. Typically, the paste-like structure is obtained by hydrating the drug-coated substrate disclosed herein with an aqueous solution prior to use. In accordance with some embodiments of the present invention, hydration should be performed no more than 2 hours before applying the resulting paste to the surgical site, preferably 1 hour before applying the resulting paste to the surgical site, more preferably no more than 30 minutes before applying to the surgical area. In some embodiments of the present invention, a paste texture will be achieved if the amount of aqueous solution (eg, saline) mixed with the drug coated substrates is between 0.1:1 and 1:1 w/w. respectively; preferably from 0.3:1 to 0.6:1 wt./wt. respectively.

The present invention provides methods for preventing or treating a surgical site infection associated with a surgical operation, comprising the step of introducing into the surgical site a biodegradable substrate impregnated and/or the surface of which is coated, in whole or in part, with a matrix composition that provides localized controlled and prolonged releasing at least one pharmaceutically active agent at the surgical site. According to some embodiments of the present invention, the matrix composition contains a plurality of pharmaceutically active agents. According to some embodiments of the present invention, the substrate coated with the matrix composition of the present invention can be administered essentially as a single ingredient (the substrate is not used as a component of a mixture with other ingredients). Alternatively, the substrate coated with the matrix composition may be applied to the surgical site as a combination of two or more populations of substrates having different coatings. For example, the methods may include the step of administering to the surgical site a combination of a first population of coated substrates containing one antibiotic agent mixed with a second population of coated substrates containing a different antibiotic agent.

As described above, the amounts, ratios, and types of ingredients forming the matrix composition of the present invention can be varied so as to adjust the polymer-lipid matrix according to the biophysical/biochemical properties of the drug, the therapeutically effective dose of the drug, and the desired release rate and/or or duration of drug release. The methods of the present invention therefore include the step of introducing into the surgical site a combination of two or more populations of coated substrates, each of which is capable of releasing a drug at a different rate and/or duration, said drug in different populations of coated substrates may be identical or various. Without wishing to be bound by any theory or mechanism of action, the present inventors believe that the introduction into the surgical site of a combination of coated substrate populations, each containing a different drug formulation, is formulated to provide release at a predetermined rate and/ or duration, provides the clinician or specialist with greater flexibility in adjusting the treatment protocol according to medical need. A non-limiting example includes a combination of two populations of drug-eluting substrates, one of which contains

- 20 041251 the first antibiotic agent released within 3-4 weeks and the second population of drug coated substrates contains the second antibiotic agent released within 12 weeks. It should be emphasized that substrates coated/impregnated with a matrix composition in accordance with embodiments of the present invention may be provided to the clinician or specialist as a premixed combination of two or more populations of coated substrates, or preferably as a single ingredient (which is not included in the mixture with other ingredients) to be mixed by a qualified professional prior to application to the surgical site.

Methods for obtaining matrix compositions.

To obtain compositions according to the present invention can be used in any suitable way, which will allow to obtain a homogeneous dispersion of the polymer and lipids in a water-resistant matrix. Preferably, according to some embodiments, the methods of the present invention do not use water at any stage of the manufacturing process.

Preferably, the matrix compositions of the present invention are prepared by processes that do not involve the formation of emulsions and in which aqueous media are generally not used. The preparation of emulsions, which are then subjected to drying, necessarily leads to the formation of vesicles or microspheres. To obtain coated articles, a mixture of polymer, lipids and antibiotics with appropriate selected volatile organic solvents will be used to coat the desired surface.

In some embodiments, the polymer and sterol are mixed with the appropriate volatile organic solvent(s) of choice, and the phospholipids, together with the active pharmaceutical agent, are mixed with their respective solvent or solvents of choice prior to mixing with the polymer/sterol mixture.

In some embodiments, the present invention provides a method for preparing a matrix composition, said method comprising the following steps:

(a) mixing in a first volatile organic solvent (i) a biodegradable polyester and (ii) sterols; and (b) mixing separately in a second volatile organic solvent (i) the active agent, (ii) phosphatidylcholine or a mixture of phosphatidylcholines, and optionally (iii) an additional lipid component such as, for example, phosphatidylethanolamine;

(c) mixing and homogenizing the products obtained in steps (a) and (b); and (d) bringing the substrate into contact with the homogeneous mixture obtained in step (c).

In another embodiment, the phosphatidylethanolamine may be included in the volatile organic solvent of step (a) instead of or in addition to the phosphatidylethanolamine added to the volatile organic solvent of step (b). In another embodiment, the biodegradable polyester is selected from the group consisting of PLA, PGA and PLGA. In another embodiment, the biodegradable polyester is any other suitable biodegradable polyester known in the art. In some embodiments of the present invention, the first volatile organic solvent is a non-polar solvent. In some embodiments of the present invention, the second volatile organic solvent is a water-miscible solvent. Where the active agent is a protein or peptide, it is important to select solvents that do not denature or reduce the activity of the protein.

According to another embodiment of the present invention, the mixture of step (a) containing the volatile organic solvent is homogenized before mixing with the solution of step (b). In another embodiment, the volatile organic solvent or volatile organic solvent mixture used in step (a) may be identical to or different from the volatile organic solvent or organic solvent mixture used in step (b). According to another embodiment of the present invention, the mixture of step (b) is homogenized before mixing with the mixture of step (a). In another embodiment, the polymer in the mixture of step (a) is saturated with lipids. In another embodiment, the matrix composition is saturated with lipids. In a preferred embodiment, the polymer and phosphatidylcholine are included in the matrix composition. In another embodiment, the active agent is also included in the matrix composition.

According to another implementation variant, each step of the production method essentially does not include the use of an aqueous solution. According to another implementation variant, each step essentially does not include the use of water or any aqueous solution.

When mixed, a homogeneous mixture is formed. The substrate to be coated or impregnated with the matrix composition is combined with said homogeneous mixture.

The production method further includes the step of evaporating the solvent present in the product of step (d). Evaporation of the solvent is usually carried out by heating the product obtained in step (d). Heating is continued until the solvent is completely eliminated and at normal temperature in the range from room temperature to 60°C, preferably at

- 21 041251 temperature below 50°C, more preferably at a temperature of 45°C or below, more preferably at a temperature of 30°C or below. In some embodiments of the present invention, low vacuum (eg, 300-600 psi) is used in the solvent evaporation steps. In another embodiment, the vacuum drying step is carried out after the solvent evaporation step. Each possibility represents a separate embodiment of the present invention.

Methods for determining lipid saturation.

The following method can be used to determine the degree of lipid saturation.

(i) After manufacture, the matrix composition is hydrated and isolated by centrifugation or filtration. Lipids that have not been included in the matrix form free micelles or liposomes and are present in the supernatant. The total lipid content of the supernatant and matrix is quantified. Therefore, the content of included and free lipids is determined for different formulations having different ratios of lipid:polymer at the very beginning. Therefore, the actual, experimental, maximum lipid/polymer ratio is determined.

(ii) After manufacture, the matrix composition is hydrated with a solution containing tritium-labeled water, washed with a solution containing no tritium, and recovered by centrifugation or filtration, and the amount of water included in the polymer mass is quantified. These steps are repeated with various lipid:polymer ratios to determine the amount of lipid required to saturate the free volume in the matrix composition.

Experimental data section

Abbreviations used.

Phosphoethanolamine - PE;

phosphatidylcholine - PC;

1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine - DMPE (14:0);

1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine - DPPE (16:0);

1,2-distearoyl-sn-glycero-3-phosphocholine - DSPC (18:0);

1,2-dipalmitoyl-sn-glycero-3-phosphocholine - DPPC (16:0);

calcium orthophosphate (TCP);

polyvinyl alcohol (PVA).

Example 1 A method for preparing a substrate (eg, calcium orthophosphate particles, polyvinyl alcohol particles) coated/impregnated with a matrix composition containing doxycycline, in accordance with some embodiments of the present invention (for the treatment and prevention of surgical site infections).

Review: two mixtures were created to obtain lipid-rich polymer matrices.

1. The biodegradable polymer and the first lipid component (eg, sterol) are mixed with a volatile organic solvent, which is mixed to form a lipid rich polymer matrix solution or suspension as measured by differential scanning calorimetry (DSC).

2. The active agent and the second lipid component (eg, at least one phospholipid) are mixed with a second volatile organic solvent to form a second solution or suspension.

3. Both solutions or suspension were combined and mixed until equilibrium was reached.

4. The substrate (e.g. gelatin sponge, collagen foam, mineral substrate) is then mixed with the solution obtained in step 3.

5. The organic solvents were then evaporated to provide a substrate coated and/or impregnated with a lipid-rich, drug-containing polymer matrix.

typical protocol.

Particles of calcium orthophosphate with an average diameter of 100 μm were coated with a matrix composition suitable for delayed release of doxycycline using the following method.

1. Preparation of stock solutions.

1.1. Stock solution PLGA 75:25 (300 mg/ml in ethyl acetate) - PLGA 75/25 was weighed into a volumetric flask. Ethyl acetate was added to the required volume. The solution was stirred until complete dissolution of the PLGA particles.

1.2. The original solution of cholesterol (30 mg/ml in ethyl acetate) - cholesterol was weighed into a volumetric flask. Ethyl acetate was added to the required volume. The solution was stirred until complete dissolution of cholesterol.

1.3. The original solution of doxycycline (210 mg/ml in methanol) - doxycycline was weighed into a volumetric flask. Methanol was added to the required volume. The solution was stirred until complete dissolution of doxycycline.

1.4. A stock solution of DPPC (206 mg/mL and DSPC 69 mg/mL in methanol/ethyl acetate (9/14)) - DPPC and DSPC was weighed into a volumetric flask. Methanol/ethyl acetate (9/14) was added to the required volume. Solution

- 22 041251 were incubated at 45°C for 5 minutes and stirred until complete dissolution of the phospholipids.

2. Preparation of coating solution.

Solution A: 5 volumes of cholesterol stock solution were mixed with 1 volume of PLGA stock solution. The mixture contained 50 mg/ml PLGA and 25 mg/ml cholesterol. Solution B: 18 volumes of doxycycline solution were successfully mixed with 82 volumes of phospholipid solution (see section 1.4). The mixture contained 225 mg/ml phospholipids (56 mg/ml DSPC and 169 mg/ml DPPC) and 37.5 mg/ml doxycycline.

Solution AB: 2 volumes of solution B was mixed with 3 volumes of solution A, the resulting solution contained 30 mg/ml PLGA 75/25, 15 mg/ml cholesterol, 90 mg/ml phospholipids and 15 mg/ml doxycycline.

3. Substrate coating.

1.5 g of Substrate (eg, calcium orthophosphate powder (particle diameter 100 μm), polyvinyl alcohol (PVA) powder, polylactic acid (PLA) powder) was weighed into a 30 mm glass Petri dish. 1.5 ml of AB solution was added to the cup.

The Petri dish was placed in a vacuum oven at a set temperature of 45°C and a partial vacuum (~610 mmHg) was applied until the solvents completely evaporated (the presence of solvents was not detected), the oven was turned off and a full vacuum was applied to remove any residual solvents (overnight) .

The dried coated calcium orthophosphate powder was transferred to a light protected vial and stored at 4°C.

Drug release profile: The coated substrate was hydrated (5% serum at 37° C.) and the release of doxycycline from the calcium orthophosphate particles impregnated/coated with the matrix formulation was monitored and quantified by HPLC. The release profile is shown in Fig. 1.

Example 2 Drug Release from PVA Particles Impregnated/Coated with a Sustained Release Compound of the Invention.

Coated/impregnated PVA particles were prepared as described in Example 1 above.

After hydration (5% serum at 37° C.), the release of doxycycline from the PVA particles impregnated/coated with the matrix formulation was detected and quantified by HPLC. The release profile is shown in Fig. 2.

As follows from FIG. 2, the release during the first hour was approximately 45% doxycycline in the coating matrix. Further investigation of the release during the first hour revealed that if the collected sample was centrifuged (settled) and further sieved (45 µm filter) or vice versa (sieved and centrifuged again), the amount of doxycycline released during the first hour and detected with using HPLC was at least 50% lower (eg ~20%). Without wishing to be bound by any particular theory or mechanism of action, it is believed by the present inventors that the collected sample (prior to centrifugation and/or filtering of the collected sample) also contains, in addition to the doxycycline molecules that have been released from the formulation, small fragments of coated PVA particles. Coated and uncoated PVA particles were further examined by FT-IR and SEM.

Using FT-IR, no data was obtained indicating an interaction between the PVA particles and the coating matrix.

SEM images of uncoated PVA particles show globular particles having a porous, rough surface (FIGS. 3A and 3B). Images of coated PVA particles indicate that the outer and inner surfaces of the particles are coated (FIGS. 3C and 3D).

While not wishing to be bound by any theory or mechanism of action, the present inventors believe that the use of PVA may be advantageous due to the fact that it dissolves shortly after degradation of the drug-containing coating matrix. Exposure of the PVA to body fluids will initiate its destruction and removal without leaving any marks on the treated surgical area.

Example 3 Drug release from an absorbable gelatin sponge containing a sustained release formulation of the present invention.

A 1x1 cm piece of collagen sponge was placed in a 20 ml vial. 0.4 ml of AB solution (as described in Example 1 above) was added to the vial and the vial was left closed at room temperature for 10 minutes. A piece of collagen sponge impregnated with AB solution was transferred to a 4 ml vessel to evaporate the solvents, followed by vacuum drying overnight. The obtained piece of collagen sponge was used for experiments to study the release.

After hydration (5% serum at 37° C.), the release of doxycycline from the piece of gelatin sponge impregnated with the matrix composition into the environment was detected and quantified by HPLC.

Example 4 Preclinical Study of TCP Beads Coated/Impregnated with a Matrix Composition According to Embodiments of the Present Invention for the Treatment of Intramuscular Surgical Site Infection.

- 23 041251

The purpose of this study is to evaluate the effectiveness of calcium orthophosphate beads (~100 µm) coated with a matrix composition containing doxycycline according to some embodiments of the present invention (test article) in reducing bacterial proliferation after infection induction in a surgical site infection model (SSI) , which is carried out by intramuscular implantation of the study device in combination with Staphylococcus aureus (ATCC 25923) in Sprague-Dawley rats. The preparation of the test article is described in Example 1.

All preclinical studies were performed in accordance with the guidelines for conducting animal experiments in the State of Israel and in accordance with the requirements of the Research Institute's Ethics Committee.

Animal model: male Sprague-Dawley™ SD™ rats at 11-12 weeks of age.

Bacterial inoculum: Staphylococcus aureus from source ATCC 25923 was obtained in a ready-to-use form and divided into two appropriate concentrations of 1x10 ⁷ and 1x10 8 cfu/ml.

Test material: synthetic calcium orthophosphate (TCP) - CamBioceramic granules with a diameter of 50-100 μm, coated with a matrix composition containing doxycycline, as described in example 1, which were obtained in the form of a ready-to-use powder. Before the start of the surgical operation, the carrier material was aseptically divided into aliquots of 50±2 mg and placed in glass vials (one aliquot per implantation site).

The composition of the study groups and doses.

In table. 1 below shows the experimental groups in the study. _______________________________________________________________Table 1

No. groups Number of animals Bacterial inoculum Treatment Suggested observation period (days after implantation) Conc. suspension and S. aureus (ATCC 25923) (cfu/ml) Implanted S. aureus volume (ATCC 25923) (µl) Implanted material Implanted volume (mg) Method of implantation 1 n=2 No processing 14 2 n=6 1x10 ⁷ 50 TCP 50 Implantation in the intramuscular pocket formed in the right gluteal muscle 3 n=6 Test item 4 n=6 1x10 ⁷ 50 TCP 50 5 n=6 Test item

Research methods.

Preoperative drugs: Animals were administered an opioid analgesic (buprenorphine at a dose of ~0.075 mg/kg) by subcutaneous injection approximately 1-2 hours before surgery. In all cases, the animals were subjected to general anesthesia by isoflurane inhalation (2-4% in oxygen at a flow rate of 0.8-1.2 l/min).

Implantation of test materials into the intramuscular pocket was performed in each animal (excluding untreated animals) under aseptic conditions as follows: the animal was placed on a heated surface in the prone position. A longitudinal skin incision was made in the lumbar region of the back, approximately 2 cm to the right of the spine, and extended to visualize the gluteal muscles. A pocket measuring approximately 2 cm in length and 1 cm in depth was formed in the gluteal muscles using a blunt incision technique or with a surgical scalpel.

The studied material was brought into the pocket directly from the paper for weighing, pulling the edges of the pocket with tweezers. Then, keeping the edges of the pocket pulled back, 50 µl of the bacterial suspension (at the appropriate concentration) or saline (in the control sample) was pipetted into the formed pocket. The muscles and skin were closed with simple interrupted surgical sutures using appropriate sutures and surgical clamps, respectively.

The closed incision was cleared of blood debris by rinsing with saline, followed by application of Polydine solution.

Postoperative care: Animals were injected subcutaneously with buprenorphine at a dose of ~0.075 mg/kg twice a day for 3 days after surgery.

Observations and studies: duration - ~14 days. Controlled clinical signs and body weight. The incision areas were checked and evaluated once a day. In addition, to assess local response in deeper tissues, surgical sites were mechanically palpated and scored on a 5-point scale as follows: 0=no palpable nodule, 1=slightly palpable nodule, 2=0.25-0.5 cm nodule in diameter, 3=nodule >0.5 cm in diameter, 4=pronounced abscess.

The entire area of implantation in all animals was collected, weighed and individually placed in labeled vials filled with 10 ml of saline. The vials were placed on ice until microbiological assay was performed.

Quantitative determination of bacteria in tissue: each area of interest was aseptically cut into small pieces using sterile scissors, thereby exposing the contents of the pocket.

A suspension of ground tissue in saline was shaken for 1 min.

Prepared 10-fold dilution of the suspension in saline (up to 10 ^-5 for the highest concentration of inoculum).

Samples were then plated on selective agar and blood agar. After incubation at 37°C for 24-48 hours, the number of isolated bacteria was quantified in each medium and expressed as the number of colony forming units per sample.

The results of the evaluation of the effectiveness are presented in table. 2 below and shown in FIG. 4.

table 2

Treatment Method of implantation Quantitative determination of bacteria in the body animal number implanted myatepial S. aureus Conc. suspensions (ATCC 25923) (cfu/ml) cfu/sample cfu/g sample 1 No processing 150 44 2 150 38 1 TCP (Group 1) Without induction (control) Implantation in the intramuscular pocket formed in the right gluteal muscle 650 89 2 1500 313 3 TCP (Group 2) 1x10 ⁷ 316000 43288 4 310000000 62000000 5 280000000 42424242 6 1830000000 338888889 8 660000000 146666667 9 Test item (Group 3) 1x10 ⁷ 800000 150943 10 79000 17174 12 1150000 396552 13 1300000 265306 14 142000 33023 15 TCP (Group 4) 1x10 ⁸ 8950000 2355263 16 243000 65676 17 24500 3356 20 2470000000 475000000 21 Test item (Group 5) 1x10 ⁸ 77000 17907 22 182000 49189 24 2000000 263158 25 35500 8452 26 306000 62449

Conclusion: The results obtained in this study, including macroscopic observations, results of microbiological assay and histological evaluation, clearly indicate that the substrate (eg, synthetic calcium orthophosphate) coated with a formulation containing doxycycline significantly reduced bacterial proliferation. in an intramuscular model of surgical site infection (SSI) in SD rats.

Histopathology - 14 days after surgery, the animals were sacrificed and the entire implantation area was macroscopically assessed for the degree of inflammatory response. Animals #7, 11, 18 and 23 from groups #2, 3, 4 and 5, respectively, were then collected, weighed and sent for microbiological evaluation to quantify the bacteria in the samples.

Section preparation: Histological processing was performed as follows, each sample was decalcified, cut, embedded in paraffin and cut into 3 sections, each approximately 5 μm thick, at intervals of approximately 500 μm from each other. Each of the 3 sections was stained with hematoxylin and eosin (H&E).

Histological evaluation: The amount of residual implant material and the presence of bacterial colonies were assessed and scored according to the following scoring scale: 0=no implant remnants/bacterial colonies observed, 1=one or two small foci of residual materials/bacterial colonies, 2=multiple small foci, with or no large lesion, residual material/bacterial colonies, 3=several large foci of residual material/bacterial colonies, 4=abundant residual materials/bacterial colonies filling the surgical site.

Results: macroscopic assessment of the studied areas at autopsy.

In general, virtually all animals assigned to the TCP treatment groups (i.e. groups 2 and 4) had a 0.5-2 cm bulge in the center of the surgical area. adhesion of the skin to the inner tissue.

In contrast, only slight tissue elevation was detected in the study areas in 2 out of 6 and 1 out of 6 animals assigned to groups 3 and 5, respectively.

Histopathological observations: animals No. 7 and No. 18 in groups 2 and 4, respectively - TCP in combination with 1x10 ⁷ and 1x10 ⁸ CFU / ml of bacteria: an intramuscular pocket in the gluteal muscle contained groups of polymorphonuclear cells (abscess formation), necrotic tissues and granules of the carrier of the test products (TCP) surrounded by granulomatous (ie macrophages, giant cells) reaction. The entire active site is surrounded by an early maturing capsular reaction. Numerous colonies of bacteria have also been identified near and within abscesses. The formation of an abscess was rated at 3 points.

Animals #11 and #23 from groups 3 and 5 respectively - study device in combination with 1 x 10 ⁷ and 1 x 10 ⁸ cfu/ml of bacteria: intramuscular pocket in the gluteal muscle contained granules of calcium orthophosphate (TCP) (which is one of the components of the implanted test device) surrounded by a granulomatous (ie, macrophages, giant cells) reaction and sporadic minimal (grade 1) mononuclear cell infiltrates, indicating superior ability to reduce bacterial proliferation.

Conclusion: Groups 2 and 4 specimens (TCP only implantation): The intramuscular pocket in the gluteal muscle contained a vehicle-associated abscess (TCP) for pellets, and numerous bacterial species were also present. There were no obvious differences between any of the components and/or scores when comparing the reactions that were observed in the samples from groups 2 and 4.

Groups 3 and 5 Samples (Study Device): The intramuscular pocket in the gluteal muscle contained clusters of calcium orthophosphate (TCP) granules surrounded by a granulomatous (i.e., macrophages, giant cells) reaction and sporadic minimal mononuclear cell infiltrates, indicative of superior ability reduce bacterial proliferation. There were no obvious differences between any of the components and/or scores when comparing the reactions that were observed in the samples from groups 3 and 5. It can be concluded that the test compound is very effective in reducing the proliferation of bacteria in the surgical site.

EXAMPLE 5 Eradication of Formed Biofilm in the Presence of TCP Particles Coated with a Matrix Composition in accordance with some embodiments of the present invention.

The efficacy of calcium orthophosphate beads coated with the matrix composition according to embodiments of the present invention in eradicating formed biofilm was measured using the MBEC™ (Minimum Biofilm Eradication Concentration Concentration) Physiological and Genetic Quantification Method.

Overview of the MBEC™ Assay Method: The MBEC™ assay method allows you to determine the operating parameters needed to propagate and process various bacterial biofilms in a high throughput screening quantitative assay. The assay device consists of a plastic cap with ninety-six (96) pegs and a matching receiving plate with ninety-six (96) individual wells, working volume

- 26 041251 which is not more than 200 µl. Biofilm forms on stakes in a batch culture model (i.e. no nutrients are added or removed to a single well) with gentle agitation.

The formed biofilm is transferred to a new receiving plate to study the effectiveness of the disinfectant agent.

Description of the sample.

Each studied set of samples included the following groups, indicated in table. 3 below.

Table 3

Code Sample Description contact time Concentrations A β-TCP β-calcium orthophosphate (β-TCP) 24±2 hours 0.3, 1, 3, 10 and 30% w/v (mg/µl), therefore, 200 µl contains 0.6, 2, 6, 20 and 60 mg of sample, respectively. IN Researched product Test product formulation: β-TCP beads coated/impregnated with matrix composition containing doxycycline 24±2 hours 0.3, 1, 3, 10 and 30% w/v (mg/µl), therefore, 200 µl contains 0.6, 2, 6, 20 and 60 mg of sample, respectively. WITH β-TCP +DOxycycline 5 Doxycycline hyclate, non-formulated β-calcium orthophosphate (β-TCP) and free (non-formulated) doxycycline hycate (10 mg/mL and 5 mg/mL solutions in distilled water 24±2 hours 0.3, 1, 3, 10 and 30% w/v (mg/µl), so 200 µl contains 0.006 at 3%, 0.06 at 3%, 0.6, 2, 6, 20, and 60 mg TCP, respectively, to be soaked 6.72, 224 and 672 mg doxycycline, respectively.

Organisms tested: Staphylococcus aureus (strains associated with osteomyelitis); source: ATCC 29213; dilution/challenging medium: 1000xTSB+10% human serum, 24h.

Culture medium/agar: trypsin soy broth/trypsin soy agar for aerobic conditions for 24 h.

Test Method Overview: High Throughput Antimicrobial Susceptibility Pilot Process with P&G MBEC™ Quantitative Assay Using Hydroxyapatite Coating. This standard protocol was divided into several small steps, each of which is described in detail in the following sections.

1. Obtaining a culture/seed.

Using a frozen stock suspension (at -70° C.), the first subculture of Staphylococcus aureus was streaked onto OCA (organism-specific agar). The plates were incubated under appropriate propagation conditions for 20±2 hours and then stored at 4°C.

The second subculture, selected from the first subculture, was streaked on the OCA. The plates were incubated under the appropriate propagation conditions for 20 ± 2 h. A single colony from the second subculture was aseptically selected from the OCA plate and inoculated in 50 ml of sterile liquid bacterial propagation broth, followed by incubation under the appropriate propagation conditions for 20 ± 2 h ( at 150 shakes per minute).

The inoculum was adjusted to an approximate cell density of 10 ⁶ cfu/ml.

Samples (100 µl) of the diluted organism were used to test the inoculum by serial dilution and spotting for OCA in triplicate.

Preparing a tablet for provocation.

150 µl of the remaining diluted body suspension was placed in each of the appropriate wells of the P&G MBEC™ device, except for the wells for sterility control (Table 5). The device was placed on an orbital shaker (PO shakes per minute) in a humidified incubator at a temperature of 37±1°C.

Sample sterility control: Pegs were broken off from the BGCH wells using burnt pliers. Each peg was placed in 200 µl of neutralizer. The pegs were sonicated for 30 min. The isolation suspension was then serially diluted and spotted on the OAS. The resulting cultures were used to test for biofilm expansion.

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200 μl of sterile TSB was added to the GC and SC-M wells of the challenge plate, respectively.

These wells were used as a sterility control (SC) and growth control (GC) in each study for each organism. BGCh was used to test for biofilm expansion. Wells N - control of the toxicity of the neutralizer and wells N: 50 - control of the efficiency of the neutralizer.

Table 4

Tablet for provocation

β-TCP Researched product β-TCP + free doxycycline Gentamycin A A A IN V V WITH WITH WITH A thirty% thirty% thirty% thirty% thirty% thirty% thirty% thirty% thirty% 32 32 32 A A A V V V With With With IN 10% 10% 10% 10% 10% 10% 10% 10% 10% 16 16 16 A A A V V V With With With WITH 3.0% 3.0% 3.0% 3.0% 3.0% 3.0% 3.0% 3.0% 3.0% 8.0 8.0 8.0 A A A V V V With With With D 1.0% 1.0% 1.0% 1.0% 1.0% 1.0% 1.0% 1.0% 1.0% 4.0 4.0 4.0 A A A V V V With With With E 0.3% 0.3% 0.3% 0.3% 0.3% 0.3% 0.3% 0.3% 0.3% 2.0 2.0 2.0 A A A 0.3% 0.3% 0.3% F SC-A SC-A SC-A SC-B SC-B SC-B (0.06 (0.06 (0.06 1.0 ι, ο ι, ο mcg mcg mcg dox) dox) dox) A A A 0.3% 0.3% 0.3% G N:50 N:50 N:50 N N N SC-C SC-C SC-C (0.006 (0.006 (0.006 mcg mcg mcg dox) dox) dox) H BGCH BGCH BGCH SC SC SC GC GC GC GC GC GC

Using a sterile 96-well microtiter plate, the following steps were performed under aseptic conditions to prepare the challenge plates listed in Table 1. 4.

Neutralization control: 200 µl of neutralizer was added to 300 µg of doxycycline in N:50 wells (final concentration of doxycycline in D/E (neutralizer) is 1.5 mg/ml).

Neutralizer toxicity control: 200 µl of neutralizer was added to N wells.

Sterility control of the biocidal agent: 60 mg of β-TCP, test device and β-TCP+doxycycline were added to SC wells A-C.

Biofilm Formed Antimicrobial Challenge: The biofilm formed on the lid of the MBEC device was washed by immersing the lid in saline (˜30 s) to remove planktonic cells. The lid was then placed on top of the challenge plate and incubated on a rotary shaker at 110 rpm at 35±2°C for 24±2 hours.

Biofilm isolation: After incubation (above), planktonic cells were washed off the biofilm by immersing the lid in saline (~20-30 s). The lid was then transferred to neutralizer/isolation plates and placed in an ultrasonic bath (˜30 min) to separate the surviving biofilm.

Determination of planktonic MBCs: 20 µl from each well of the challenge plate was withdrawn and placed in the appropriate wells of a fresh 96-well plate containing 180 µl of DE neutralizer. The tablet was incubated at 35±2°C for 24±2 hours. The results of the determination of MBC were visually assessed after incubation.

Bacterial cell reduction, log ₁₀ : After sonication, 100 µl from each well of an MBEC™ plate was transferred to the first 12 empty wells of the first row of a 96-well plate

- 28 041251 for microtiter and further diluted 10 times and made in the next 8 rows of holes (dilution 100-10 ⁷ ). 5 µl from each well was then used for spot seeding on OCA plates. Agar plates were incubated at 37±1° C. and colonies were counted after approximately 24-48 hours of incubation. The arithmetic mean of the number of colonies counted on the plates was calculated. 100 µl of sterile neutralizer was added to each well of the isolation plate to bring the volume back up to 200 µl. The newly filled plate was incubated at 35±2°C for 24±2 hours.

Comparative MBEC results were determined after 24±2 hours of incubation using a tablet reader.

Density in log10 for one peg was calculated as follows:

log 10 (cfu/peg)=log 10 [(X/B)(D)], where X is the average number of cfu;

B - planted volume (0.02 ml); And

D - breeding.

The total biofilm accumulation was determined by calculating the average of the calculated log10 density values.

The reduction in bacterial cell count (log10) for each dilution was calculated as follows: log10 reduction=mean log10 for growth control -mean log10 for test sample

Results.

The average values of log10 cfu/peg are presented in table. 5.

Table 5

Mean log10 cfu/peg values for isolated bacteria

A 1 2 3 Average Standard deviation 30.0% 3.90 3.60 3.60 3.70 0.17 10.0% 3.60 3.90 3.60 3.70 0.17 3.0% 3.60 3.78 3.60 3.66 0.17 1.0% 3.60 3.60 3.60 3.70 0.17 V 1 2 3 Average Standard deviation 30.0% 0.00 0.00 0.00 0.00 0.00 10.0% 0.00 0.00 0.00 0.00 0.00 3.0% 0.00 0.00 0.00 0.00 0.00 1.0% 1.91 2.45 0.00 1.45 1.29 0.3% 2.08 3.30 3.08 2.82 0.65 With 1 2 3 Average Standard deviation 30.0% 0.00 0.00 0.00 0.00 0.00 10.0% 0.00 0.00 0.00 0.00 0.00 3.0% 2.90 3.20 3.08 3.06 0.15 1.0% 3.60 3.60 3.90 3.70 0.17 0.3% 2.90 3.60 3.56 3.35 0.39 D (ng) 1 2 3 Average Standard deviation 32 0.00 0.00 0.00 0.00 0.00 16 0.00 0.00 0.00 0.00 0.00 8.0 0.00 0.00 0.00 0.00 0.00 4.0 1.61 2.30 0.00 1.31 1.18 2.0 2.30 0.00 2.78 1.69 1.49 1.0 4.72 2.60 2.30 3.21 1.32 0.5 3.90 2.90 3.64 3.48 0.52

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Data on the logarithmic decrease in the number of bacterial cells (log10) are presented in Table. 6.

Table 6

Logarithmic reduction in the number of bacterial cells (log10)

Decrease in bacterial cells (log 10) Comparison (%) LogR T-test S/NS 30.0% 3.70 0.00 S 10.0% 3.70 0.00 S A compared to B 3.0% 3.66 0.00 S 1.0% 2.25 0.02 S 0.3% 0.98 0.03 S Decrease in bacterial cells (log 10) Comparison (%) LogR T-test S/NS And compared to C 30.0% 3.70 0.00 S 10.0% 3.70 0.00 S 3.0% 0.60 0.00 S 1.0% 0.00 0.50 NS 0.3% 0.45 0.07 NS Decrease in bacterial cells (log 10) Comparison (%) LogR T-test S/NS GC versus D 32 4.59 0.00 S 16 4.59 0.00 S 8.0 4.59 0.00 S 4.0 3.29 0.00 S 2.0 2.90 0.00 S 1.0 1.38 0.02 S 0.5 1.11 0.00 S

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The results of the visual determination of MVS and MVES are presented in table. 7.

Table 7

Results of visual determination of MVS and MVES

MWEC 1 2 3 4 5 6 7 8 9 10 eleven 12 A + + + - IN + + + - WITH + + + - - - + + + - + - D + + + - - - + + + + + + E + + + + + + + + + + + + F - - - - - - + + + + + + G + + + + + + - - - + + + H - - - - - - + + + + + + MVS 1 2 3 4 5 6 7 8 9 10 eleven 12 A + + + - IN + + + - - - - - - - - WITH + + + - D + + + - - - - - - - + + E + + + - - - - - - - + + F - - - - - - - - - + + + G + + + + + + - - + + + H - - + - - - + + + + + +

Conclusion: logarithmic bacterial cell reduction data (log10) indicated that the test product (TCP beads coated with a matrix formulation according to embodiments of the present invention) was capable of causing preformed biofilm death at a minimum concentration of 3.0% and was effective even at 1.0% (death >99%). In contrast, doxycycline together with P-TCP when used not in the form of a composition was effective at concentrations of 10% or higher.

Example 6 Inhibition of Biofilm Formation in the Presence of TCP Particles Coated with a Matrix Composition in accordance with some embodiments of the present invention.

The efficacy of calcium orthophosphate beads coated with a matrix composition according to embodiments of the present invention in inhibiting biofilm formation was assessed by calculating logarithmic bacterial reduction values using the MBEC™ (Minimum Biofilm Eradication Concentration Concentration) Physiological and Genetic Quantification Assay (system described above in Example 5).

Culture/inoculum preparation was carried out in accordance with the method described above in example 5.

Obtaining a tablet for provocation.

Table 8

Staphylococcus aureus A SC-A SC-A SC-A Bl Bl Bl Al Al Al IN SC-B SC-B SC-B B2 B2 B2 A2 A2 A2 WITH N:50 N:50 N:50 B3 B3 B3 A3 A3 A3 D N N N B4 B4 B4 A4 A4 A4 E B5 B5 B5 A5 A5 A5 F G H BGCH BGCH BGCh

Provocation plate preparation design: SC wells were used for sterility control for each experiment; GC was used as a propagation control; BGCh was used to control biofilm formation; wells N were used to control the toxicity of the neutralizer; wells N:50 were used for performance control.

Using a sterile 96-well microtiter plate, the following steps were performed under aseptic conditions to prepare the above challenge plates.

- 31 041251

Performance Control: 150 µl Neutralizer was added to 672 µg doxycycline per well

N:50 (final concentration of doxycycline in D/E was 4.48 mg/ml).

Neutralizer toxicity control: 150 µl of neutralizer was added to N wells.

Sterility control of the biocidal agent: 60 mg of test article was added to SC wells.

60 mg of TCP and the test product were added in accordance with the scheme presented in table. 8 in columns 1-9 (n=3). 150 μl of inoculated media was added to each well of a 96-well biofilm/challenging plate, except for sterility control wells.

Antimicrobial challenge to inhibit biofilm formation: The lid was transferred to the challenge plate and incubated on a rotary shaker at 110 rpm at 35±2°C for 24±2 h. dip the cap into the rinse plate (200 µl of saline per well) for 30 s. After the specified contact time, the MBEC™ cap was transferred to a neutralizer plate (200 µl of neutralizer per well).

The plate was placed in a sonicator and sonicated for 30 min to separate the surviving biofilm.

Determination of planktonic MBC and the value of the logarithmic reduction was determined as described above in example 5. The average value of log10 recovery of bacterial cells are presented in table. 9 below.

Table 9 ______Mean log10 bacterial cell isolation______

A 1 2 3 Average Standard deviation 30.0% 4.60 5.38 4.90 4.96 0.39 10.0% 5.30 5.56 5.45 5.43 0.13 3.0% 4.90 5.30 5.08 5.09 0.20 1.0% 5.38 5.51 5.60 5.50 oh and 0.3% 5.60 5.20 5.60 5.47 0.23 V 1 2 3 Average Standard deviation 30.0% 0.00 0.00 0.00 0.00 0.00 10.0% 0.00 0.00 0.00 0.00 0.00 3.0% 0.00 0.00 0.00 0.00 0.00 1.0% 0.00 0.00 0.00 0.00 0.00 0.3% 0.00 0.00 0.00 0.00 0.00

Table 10

Logarithmic decrease in the number of bacterial cells

Decreased number of bacterial cells (log 10) Comparison (%) LogR T-test S/NS 30.0% 4.96 0.00 S 10.0% 5.43 0.00 S A compared to B 3.0% 5.09 0.00 S 1.0% 5.50 0.00 S 0.3% 5.47 0.00 S

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The results of the visual determination of MVS and MVES are presented in table. eleven.

bacteria throughout the challenge period and at all TCP concentrations tested.

Test compound B caused complete death of the bacteria inoculated into the test wells at each test concentration. The MBC data indicate that all cells died and were not suppressed at the concentrations tested. Example 7: Preclinical Study of TCP Particles Coated/Impregnated with a Matrix Composition According to Embodiments of the Present Invention for the Treatment of Sternal Breast Infections.

The purpose of this study was to evaluate the effectiveness of calcium orthophosphate beads (-100 µm) coated with a matrix composition containing doxycycline in accordance with some embodiments of the present invention (test article) in reducing bacterial proliferation after infection induction in a surgical site infection model (SSI). ), which was achieved by implantation into the sternum of the study device in combination with Staphylococcus aureus (ATCC 25923) in New Zealand white rabbits. The preparation of the test article is described in Example 1.

Description.

Sternum injury at half the depth of the sternum was performed in New Zealand white rabbits. 12 female New Zealand white rabbits were randomly divided into two equal groups of 6 animals. Animals were subjected to median sternotomy using a control device (uncoated TCP) or test device (TCP particles coated/impregnated with the matrix composition prepared as described in Example 1) mixed with a specific calibrated dose of bacterial inoculum, which was introduced into the gap formed (e.g. , chest injury).

Rabbits were anesthetized with ketamine (30 mg/kg), xylazine (5 mg/kg) and atropine (1 to 3 mg/kg) intramuscularly and maintained on isoflurane after intubation.

Sternotomy was performed using standard aseptic techniques. Carried out damage to the sternum in half the depth of the sternum. Equal amounts of the control article or test articles mixed separately with the bacterial inoculum were applied to cover the cut surface of the bone and the time to hemostasis was recorded. The sternum was closed surgically (sternum halves were fastened with a monofilament suture and the incision was closed in layers). General health was monitored daily. Animals were sacrificed after 6 weeks. Each sternum was collected for radiological, histological, hematological and mechanical quantitative studies to assess the healing of the sternum.

The foregoing description of specific embodiments fully discloses the general nature of the invention, which allows, using current knowledge, to easily modify and/or adapt for various applications, these specific implementations without

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Claims (11)

undue experimentation without departing from the general concept of the present invention, and therefore it is intended that such adaptations and modifications be understood within the meaning and range of equivalents of the disclosed embodiments. It should be understood that the phraseology or terminology used herein is to describe and not limit the present invention. The means, materials, and steps for implementing the various disclosed functions may take various alternative forms without departing from the scope of the present invention. CLAIM 1. The use of a pharmaceutical composition containing a biodegradable mineral substrate, where the specified mineral substrate is impregnated or its surface is covered in whole or in part with a matrix composition containing (a) a biodegradable polyester selected from the group consisting of PLA (polylactic acid), PGA ( polyglycolic acid), PLGA (copolymer of lactic acid and glycolic acid); (b) a first lipid component containing cholesterol; (c) a second lipid component containing phosphatidylcholine containing fragments of fatty acids containing at least 14 carbon atoms; and (d) a pharmaceutically active agent containing an antibiotic agent selected from the group consisting of doxycycline or doxycycline hyclate, for the treatment or prevention of an infection of the soft tissue incision area associated with a surgical operation, where the specified mineral substrate consists of particles of β-calcium orthophosphate ( β-TCP) and where the specified pharmaceutical composition is intended for local administration in the area of soft tissue incision. 2. Use according to claim 1, characterized in that the infection comprises at least one of a superficial incisional infection, a deep incisional infection, and an organ/space infection. 3. Use according to claim 1 or 2, suitable for at least one of reducing the overall incidence of infections after surgery, eradicating soft tissue infections that may have existed prior to surgery, inhibiting biofilm formation at the incision site, and eradicating existing biofilm at the soft tissue incision site . 4. Use according to any one of claims 1 to 3, characterized in that the infection includes methicillin-resistant S. aureus (MRSA). 5. Use according to any one of claims 1 to 4, characterized in that said surgical operation is selected from orthopedic surgeries, spinal surgeries, surgeries on the organ of the digestive system, cardiac surgeries, hernia excision, angioplasty, caesarean section, prostatectomy, obstetric and gynecological surgery, head and neck cancer surgery, transplant surgery, neurosurgery and plastic surgery. 6. The use according to claim 5, characterized in that said surgical operations on the organ of the digestive system are selected from operations on the esophagus, stomach, small intestine, large intestine, rectum, colon, appendix, liver, pancreas, gallbladder, ulcer stomach, gastric cancer, open gastric anastomosis, appendectomy, colectomy, cholecystectomy, vagotomy, open biliary tract surgery, small intestine surgery, and colorectal surgery. 7. Use according to claim 5, characterized in that said surgical operation is a cardiac operation. 8. Use according to any one of claims 1 to 7, characterized in that said mineral substrate consists of particles having an average particle size of less than about 200 microns (μm), preferably less than about 150 microns (μm), more preferably said biologically the degradable substrate consists of particles having an average size in the range of 50-150 microns (µm), even more preferably said majority of the particles are spherical and/or spherical in shape. 9. Use according to any one of claims 1 to 8, characterized in that said phosphatidylcholine contains fatty acid fragments containing 14 to 18 carbon atoms. 10. Use according to any one of claims 1 to 9, characterized in that said pharmaceutically active agent further comprises an active agent selected from the group consisting of an antibiotic agent, an antiseptic agent, an anti-inflammatory agent, an antifungal agent, and any combination thereof; or said pharmaceutically active agent further comprises at least one anti-inflammatory agent. 11. Use according to any one of claims 1 to 10, characterized in that the weight ratio of total lipids to said biodegradable polyester is between 1:1 and 9:1 inclusive. -