Biodegradable systems for the intra-articular delivery of “Lornoxicam”

Abstract

Knee osteoarthritis, is one of the rheumatic diseases for which an effective cure does not currently exist. Current treatment measures are directed towards controlling inflammation, pain relief, minimizing disability and improving joint function. This goal is achieved by different classes of drugs among which nonsteroidal anti-inflammatory drugs (NSAIDs), are the most commonly used. However, oral administration of such drugs is accompanied by numerous side effects, which would be avoided by their local injection in the intra-articular cavity. The main challenge of local intra-articular injection is the rapid clearance of drugs from the synovial cavity. Thus the aim of this thesis was to formulate lornoxicam (NSAID) delivery systems for treatment of osteoarthritis with sustained release properties to be administered via the intra-articular route. Thus the work in this thesis was divided into three chapters: - Chapter 1: preparation and characterization of lornoxicam loaded chitosan/tripolyphosphate microspheres - Chapter 2: Preparation and characterization of lornoxicam liposomes loaded in depot-forming polymeric implants - Chapter 3: In-vivo Evaluation of Intra-articular lornoxicam chitosan microspheres and liposomes loaded in depot forming polymeric implants

Chapter 1: preparation and characterization of lornoxicam loaded chitosan/tripolyphosphate microspheres In this chapter, lornoxicam loaded chitosan/TPP microspheres were prepared by ionotropic gelation technique. A preliminary screening experiment was conducted to study proper conditions for chitosan/TPP microspheres preparation and results revealed thatincreasing chitosan concentration from 0.03% to 0.05%, 0.1% and 0.15% significantly increased the particle size from 3.15 ± 0.03 to 3.82 ± 0.02, 4.13 ± 0.04 and 6.43 ± 0.04 µm, respectively. Moreover, a significant increase in particle size was observed by increasing chitosan molecular weight from low, medium to high with respective values of 4.13 ± 0.04, 4.88 ± 0.06 and 7.14 ± 0.04 µm at a fixed chitosan concentration of 0.1%w/v. Decreasing chitosan: TPP mass ratio from 4:1 to 1:2 led to a significant increase in the microspheres size from 183 ± 6.3 nm to 9.7±0.6 µm, respectively for 0.1%w/v chitosan and aggregation of the formulae took place at 1:4 chitosan: TPP mass ratio. Upon increasing pHs of both chitosan and TPP solutions the chitosan/TPP microspheres experienced an increase in their particle sizes with values ranging from 3.25±0.04 to 7.12±0.06 µm. Again, increasing cross linking time from 15 min to 30 min led to an increase in the particle size of the formed microspheres while further increase to 60 min did not significantly change the particle size. From the preliminary screening study, the optimum fabrication conditions for chitosan/TPP microspheres intended for the intra-articular delivery were found to be: medium molecular weight chitosan with 0.1% w/v, 1:1 chitosan: TPP mass ratio with 4.5 chitosan solution pH and 9 TPP solution pH and 30 mins crosslinking time. This formula had particle size of 4.88 ± 0.06 µm and was designated as (F1). A factorial design experiment for the optimization of lornoxicam EE% in the plain (F1) chitosan/TPP microspheres was conducted. The studied factors were chitosan solution pH, TPP solution pH and drug concentration. Results revealed that increasing chitosan solution pH from 4 to 4.5 led to a significant increase in lornoxicam EE%with values ranging from 13.5±0.3% to 59.5±2.2%. Decreasing TPP solution pH from 9 to 8 led to a significant increase in the lornoxicam EE%. Increasing drug concentration from 1% to 2% led to a significant increase in the lornoxicam EE%. Lornoxicam loaded chitosan/TPP microspheres showed particle size ranging from 3.57 ± 0.02 to 6.12 ± 0.00 µm, which is acceptable for the intra-articular drug delivery. The in-vitro release studies showed a biphasic pattern of lornoxicam release from chitosan microspheres with an initial burst release followed by sustainment of drug release over approximately one week.Scanning electron micrographs revealed the formation of spherical shape microspheres with indentations appearing on the surface of microspheres.DSC and FTIR studies showed the disappearance of lornoxicam characteristic peaks in the drug loaded chitosan microspheres suggesting presence of lornoxicam as molecular dispersion within the polymer matrix.

Chapter 2: Preparation and characterization of lornoxicam liposomes loaded in depot-forming polymeric implants The aim of this chapter was to prepare and characterize a thermosenstive in-situ forming intra-articular depot formulation of lornoxicam liposomes, this system should aggregate into a gel form at body temperature forming depot for sustained drug release, while being solution at room temperature thereby sustaining lornoxicam release in the intra-articular cavity. Lornoxicam loaded multilamellar liposomes were prepared using thin film hydration technique, and several experiments were conducted to optimize the EE% of lornoxicam in the prepared vesicles and the results revealed that increasing cholesterol content from (7:4) to (7:6) increased EE% from 73.6±0.1% to 80.4±1.6% till a certain PC:CH molar ratio i.e. (7:7) beyond which the EE% decreased to 69.7±3.9%. Incorporation of the positive charge inducer, stearylamine resulted in an increase in the EE% of lornoxicam relative to the neutral liposomes and the negatively charged liposomes. Increasing stearylamine concentration from 2 to 4%w/w led to an increase in the EE% from 81.6±1.9 to 82.3±0.07% till reaching an optimum value of 86.6±0.5% at 7% w/w beyond which EE% decreased to 67.3±0.9% at 10%w/w stearylamine. Optimum lipid concentration was found to be 20mg/ml that achieved the highest EE%.Increasing the drug concentration from 1 to 2 mg/10ml led to increased amount of drug loaded in liposomes, while drug precipitation took place at 3mg/10ml. Therefore, optimum drug concentration was found to be 2 mg/10ml. The particle size of the prepared lornoxicam liposomes ranged from 4.6±0.01 to 8.70±0.15µm, which is regarded suitable for the intra-articular delivery.Differential scanning calorimetry (DSC) study showed complete molecular dispersion of lornoxicam indicating its entrapment inside the liposomal formula.In-vitro release study proved the ability of liposomal formulae to sustain lornoxicam release for a period up to 2 days, where, increasing cholesterol concentration decreased lornoxicam release rate. The positively charged liposomes showed more sustained drug release followed by neutral then negatively charged liposomes. Increasing stearylamine concentration led to a decrease in the percent release. The increase in drug concentration increased the percent drug released. Release from all liposomal formulae followed Higuchi diffusion release mechanism. Stability studies of liposomes proved that liposomal formula L15 was stable for a period of up to 2 months showing no significant change in the particle size nor the EE%. Lornoxicam loaded multilamellar liposomes were able to sustain lornoxicam release for only 2 days which was not sufficient for the intra-articular delivery goal, so for further prolongation of the drug residence time in the joint cavity a new in-situ forming depot polymeric implant was designed in order to incorporate the prepared lornoxicam liposomes thus extending its release time.The results of preparation and characterization of this formula which is based on poloxamer 407, poloxamer 188 and sodium hyaluronate revealed that increasing P407 concentration from 16 to 18%w/w decreased the Tsol-gel from 28.0±0.0 to 25.0±0.0 ˚C, while addition of P188 at 5 and 10%w/w increased the Tsol-gel with values reaching 42.5±0.0 ˚C for 10%w/w P188.Sodium hyaluronate added at two concentrations 0.4 and 0.6%w/w to P407/P188 gels decreased their Tsol-gel, where increasing the sodium hyaluronate concentration decreased the Tsol-gel at all the studied P407 and P188 concentrations. By addition of liposomal formula L15 to different P407/P188/HA a decrease in the Tsol-geltook place with values ranging from 27.0 ±0.6 to 35.0±1.1 ˚C. The optimum depot forming polymeric system L15G incorporating L15 liposomal formula, composed of P407/P188/HA, 16/10/0.4 %w/w,havingTsol-gel of 35.0±1.1˚C was suitable for intra-articular delivery route.Viscosity measurements showed that, addition of lornoxicam liposomes L15 to plain in-situ forming gels significantly increased its viscosity with value 0f 1.90±0.01 Pa.s at 100 r.p.m. compared to the plain gel formula at the same r.p.m. showing 0.68±0.04 Pa.s at 37 ˚C.Increasing temperature from 25 to 37˚C increased the measured viscosity with value of 0.27±0.0 to 1.9±0.01 Pa.s at 100 r.p.m., respectively, which ensured depot formation at body temperature.Pseudoplastic shear thinning behavior of the prepared thermosenstive gels was observed due to HA addition.pH of L15G was 7.37±0.0 which is considered suitable for the intra-articular delivery route.The particle size of L15 liposomal formula decreased from 7.15±0.20 to 5.45±0.02 µm upon incorporation in the in-situ gelling formulae P407/P188/HA, 16/10/0.4 %w/w.Syringeability force measurement of L15G was found to be 4.55±0.5N which is considered suitable for intra-articular delivery.TEM of the prepared L15G revealed that the incorporation of liposomes in polymeric based in-situ gels did not affect the liposomal shape or stability.Release study of the L15G showed marked reduction in the drug release rate relative to L15, showing almost half the amount of lornoxicam released after 4 hrs. L15G was able to sustain lornoxicam release for a period of 8 days relative to liposomal formula which only sustained drug release for 2 days.