DESCRIPTION Technical Part This invention is related to gel systems containing vancomycin microspheres for controlled drug release and serratiopeptidase in order to inject to the area of medical device-related infection.

In biofilm-based medical device-related osteomyelitis, biofilm causes a high resistance to used antibiotics and slow or incomplete penetration of antibiotic to infected area. Therefore, it is needed that the drug delivery systems which do not require surgical procedure and provide biofilm eradication and local, sustained and desired amount of drug in the site of infection for the treatment of biofilm-based medical device-related osteomyelitis. The prior art, vancomycin loaded microspheres and pluronic thermosensitive gel formulations are known. But, subject of this invention which is a gel system containing vancomycin HCI, vancomycin HCI loaded microspeheres and serratiopeptidase in the same formulation, which can be administered directly to the infection area is not available. Designed thermosensitive gel system characterized in that it comprises antibiotic loaded microspheres, free antibiotic and proteolytic enzyme, wherein the proteolytic enzyme is serratiopeptidase.

A biodegradable polymer was selected to prepare of the microspheres. Various biodegradable natural polymers such as polysaccharides (dextran or cellulose), chitin, chitosan, proteins (collagen.fibrin, gelatin, albumin) and synthetic polymers such as aliphatic polyesters [ poly(glycolide) (PGA), poly(lactide) (PLA), poly(lactide-co-glycolide) (PLGA), poly(e-caprolactone) (PCL), poly(3-hydroxybutyrate(PHB), poly (3-hydroxybutyrate-co-3- hydroxyvalerate) (P(HBco-HV)), Poly (anhydrides) can be used. In order to prepare microsphere formulation poly(E-caprolactone) (PCL) (Sigma Aldrich) which is biodegradable, biocompatible and FDA approved polymer was selected. This synthetic and non-toxic polymer is a member of aliphatic polyesters and it has semicrystalline structure.

l The commercially available PCL types are mentined below:

• Mw 14.000 Mn 10.000

· Mn 60.000

• Mn 70.000-90.000

Mw: weight average molecular weight

Mn: number average molecular weight

In order to provide in situ gel formation, using a thermosensitive gel which utilize temperature change as the trigger that determines their gelling behavior without any additional external factor such as organic solvent, copolymerization or gelling agent is foreseed. Poloxamer is chosen to obtain thermosensitive gel. Poloxamers are non ionic copolymers of polyoxyethylene -polyoxypropylene. Poloxamer 407 has thermosensitive gelling properties in water and it is also known by the BASF trade name Pluronic F127 (Sigma Aldrich). Poloxamer 407 was used in sample formulations.

Serratiopeptidase (Speciality Enzymes and Biochemicals Co.) is a proteolitic enzyme which is isolated from non-patgogenic Serratia spp. It is used in dose of 5-10 mg (10 000-20 000 units) three times a day to reduce inflammation and edema which is caused by trauma, infection, airway obstruction or chronic venous insufficiency. Optimal pH values are between pH 8,5-9,5 and optimal temperature is 40 °C for its activity. Serratiopeptidase is also stable between pH 5.5-9.5 at 40 °C, however it looses its activity at 60 °C in 10 minutes.

Vancomycin is a glycopeptide antibiotic. It is preferred first-line therapy for the treatment of methicillin-resistant Staphylococcus aureus (MRSA) infection. Hydrochloride salt of vancomycin (vancomycin HCI) (Sandoz) was used in this invention. Preparation of Microspheres

Polymer (600 mg) was dissolved in 3 mL of dichlorometane to obtain organic phase for acquiring inner phase of multiple emulsion. This organic phase was added to 1 mL of aqueous phase including 0.05 or 0.1 % (w/v) polyvinyl alcohol) (PVA) and drug ( 0 or 20 % (w/w) of polymer amount) and was wortexed for 2 minutes. This dispersion was injected to 150 mL of distilled water including 0.1 % (w/v) PVA in two minutes and was mixed (Silverson L4RT) at 1000 rpm for 1 hour in order to obtain multiple emulsion. With the purpose of evaporation of dichlorometane, this emulsion was diluted with distilled water including 0.05 % (w/v) PVA and was mixed (Silverson L4RT) at 800 rpm for 1 hour at room temperature. The microsphere suspension was then vacuum filtered and washed with distilled water two times to remove PVA residue. The microspheres were frozen at -20°C followed by 48 hours lyophilization.

Blank and drug loaded PCL microspheres were sterilized at a dose of 25kGy (fix dose rate 3.62 kGy/h ) using a ⁶⁰ Co source at room temperature. Preparation of Blank Thermosensitive Pluronic Gels

Blank thermosensitive Pluronic F127 gels were prepared using cold method. Pluronic F127 (20 % w/v) was dissolved in phosphate buffered saline (PBS) and was stirred with magnetic stirrer at +4°C for 12 hours. Blank thermosensitive Pluronic F127 gel formulations were sterilized in autoclave at 121 °C for 15 minutes.

Preparation of Thermosensitive Pluronic Hydrogels Including Serratiopeptidase, Vancomycin Hydrochloride and Vancomycin Hydrochloride Loaded Microspheres

Serratiopeptidase, vancomycin hydrochloride and vancomycin hydrochloride loaded microspheres (in powder form) were added to the sterile thermosensitive Pluronic gel and mixed in sterile conditions for 30 minutes. In Vitro Drug Release Studies

In vitro release experiments were carried out by dialysis bag method. PBS (pH 7.4) was used as release medium. 0.5 mL of thermosensitive gel formulations were placed inside the dialysis membrane (MWCO 300,000 Da).The bags were fitted into the tube containing 2 mL of PBS and the tubes were fully immersed in a thermostated shaker (50 rpm) bath system at 37°C. At 1., 3., 6., 12., 24., 48. hours, the whole release medium was withdrawn and vancomycin hydrochloride content was determined by HPLC.

Evaluation of In Vitro Antibiofilm Activity

100 ί of thermosensitive pluronic gels including different amount of vancomycin HCI, serratiopeptidase and vancomycin HCI loaded PCL microspheres were added to 96 well plates containing biofilm. Then the plates were incubated at 37 °C on a horizontal shaker at 60 rpm for 24 hours. The wells without gels were used as a control group. After incubation, the wells were washed with PBS twice to remove planktonic cells and then the low-energy sonication was applied to the plates to take up the biofilm.

The survived cells were serially diluted and were seeded into the plates including TSA (Tryptic Soy Agar). After 24 hours of incubation, colonies formed were counted.

in vivo test The formation of biofilm-based infection was limited to a very small area in the group of treated experimental animals.