Formulation of liposome-encapsulated piroxicam for improved therapeutic efficacies and reduced systemic toxicities

Piroxicam, a frequently prescribed nonsteroidal anti-inflammatory drug for treating various types of musculoskeletal disorders, has been associated with low oral bioavailability and delayed onset of its therapeutic activities. A prolonged usage of piroxicam also leads to broad spectrum of untoward reactions such as gastrointestinal, haematological, hepatic and renal complications. In present work, liposomal drug delivery system, a promising lipid-based nanoparticle technology, was exploited with the aim to improve therapeutic index of piroxicam. Different in vitro and in vivo experimental models were employed to reach a proof of concept for present liposomal piroxicam formulation, as well as to elucidate the underlying mechanisms of actions. A simple and reproducible proliposomes method was successfully being developed to produce liposome-encapsulated piroxicam. The most optimum liposomal formulation (ProlipoTM Duo; 12 mg piroxicam per g ProlipoTM; 10 hours hydration time; no size reduction treatment) produced highest amount of actual drug been entrapped in liposomes (800.4 mg/g ProlipoTM) and exhibited a satisfactory entrapment efficiency (15.36%). This formulation also yielded a homogenous liposomes population (polydispersity index = 0.45) with small particle size (359.95 nm). The prepared liposome-encapsulated piroxicam was stable at both room and refrigerated (2-8 ºC) temperature for at least 4 weeks. In lipopolysaccharide-stimulated RAW 264.7 macrophages model, the potential of this optimized liposomal piroxicam formulation in reducing both cytotoxicity and in vitro inflammatory responses were demonstrated. Liposome-encapsulated piroxicam exhibited a significantly (P<0.05) stronger inhibition of proinflammatory mediators (nitric oxide, tumour necrosis factor-α, interleukin-1β and prostaglandin E2) than piroxicam of equivalent dosages. Liposome-encapsulated piroxicam also resulted in significant (P<0.05) production of an anti-inflammatory cytokine (interleukin-10). Present research work also showed that the intragastrically administered liposomal piroxicam formulation was able to improve drug’s therapeutic effects in various animal models. The acetic acid-induced abdominal writhing test, formalin-induced paw-licking test as well as carrageenan-induced mechanical and thermal hyperalgesia test evidenced an increased and longer lasting peripherally-mediated antinociceptive activities by present liposomal piroxicam formulation. A lower dosage of liposomal piroxicam formulation was also shown to inhibit the Brewer’s yeast-induced hyperthermia significantly (P<0.05). In addition, carrageenan-induced paw oedema test and cotton pellet-induced granuloma test showed that liposome-encapsulated piroxicam possessed significantly (P<0.05) stronger acute and chronic anti-inflammatory effects than piroxicam, even if lower drug dosages were used to treat animals. The enhanced in vivo therapeutic activities were attributed to a better modulation in production of different inflammatory mediators such as nitric oxide, tumour necrosis factor-α, interleukin-1β and interleukin-10. Liposomal formulation also significantly (P<0.05) enhanced the inhibition of cyclooxygenase-2, but not cyclooxygenase-1 enzyme. In addition, sub-acute toxicity study revealed that rats treated with liposomal formulation exhibited less signs of toxicity. In contrast to an equivalent dosage of liposome-encapsulated piroxicam, the piroxicam caused significantly (P<0.005) lower concentration of red blood cell, haemoglobin, haematocrit, lymphocytes and platelet in complete blood count. Biochemical analysis (liver and kidney function tests) as well as histopathology assessment indicated that present liposomal formulation was able to reduce piroxicam-induced hepatotoxicity significantly (P<0.05), but not the renal dysfunction. Moreover, liposomal formulation also significantly (P<0.05) reduced macroscopic and microscopic gastric lesion in repeatedly treated rats. Further toxicity study showed that liposomal formulation resulted in a significantly (P<0.05) higher gastric mucus and pH level than equivalent dosage of piroxicam. A lower drug dosage of liposome-encapsulated piroxicam also significantly (P<0.05) increased the level of serum gastrin. In contrast to piroxicam, the liposome-ncapsulated piroxicam did not cause significant (P<0.05) reduction of prostaglandin E2 in gastric mucosa. Hence, liposomal formulation was believed to exert its gastroprotective effects through the augment of gastric mucosa defence. As a conclusion, present works successfully developed a practical liposomal piroxicam formulation with improved therapeutic efficacies and reduced systemic toxicities.

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