Vertebral fractures (VF) are common in patients suffering from osteoporosis and osteomyelitis and lead to severe pain at the fracture site. VF is a condition associated with acute back pain and may lead to chronic pain and loss of height. Upon stress induced by VF, the human body transmits alarming signals mediated by inflammation. Inflammation, in turn, is mediated through a number of chemical factors like bradykinin, histamine, prostaglandin and interleukin-1. First-line treatment of VF is associated with the administration of non-steroidal anti-inflammatory drugs (NSAIDs) that reduce levels of the aforementioned chemical mediators, thereby relieving pain and inflammation [1-3]. However, complete VF treatment demands prolonged drug release in order to provide round-the-clock management of pain and inflammation.

As an example, ketorolac tromethamine (KT) is a widely used NSAID for the management of moderate to severe pain associated with trauma, post-operative pain, breakthrough pain of cancer, VF as well as spinal injuries and is usually administered through oral, intramuscular or intravenous routes. KT acts by inhibiting cyclooxygenase-1 (COX-1) and COX-2 and has been reported to be 800 times more potent than aspirin [4-5]. Moreover, unlike narcotic analgesics, it is non-addictive and free of any respiratory side effects. However, since KT has a biological half-life (t½) of 4 to 6 h, its frequent dosing is required in order to manage the chronic pain for a prolonged period.

Therefore, biodegradable, implantable drug delivery systems have been designed to release the drug of choice at the target site (for e.g., fractured vertebra) for an extended period of time. In this regard, various dosage forms or delivery systems including surgical implants and transdermal drug delivery systems have been investigated for prolonged drug release [6-8]. However, implantation of the aforementioned devices to a target site demands surgical intervention [9,10], thereby reducing patient compliance. Recently, in situ gel systems have received considerable attention since they are in liquid state (before administration) and can transform into a stiff gel (viscous) format upon injection to the body cavity. In situ gel formation allows slow drug release, primarily by diffusion across the gel matrix. Additionally, in situ gels are advantageous in comparison to implantable drug delivery systems with respect to ease of their administration thereby leading to improved patient compliance [11].

Although a variety of biodegradable polymers have been employed for in situ gel formation, this study deals with the fabrication of alginate-based in situ gels for intrathecal delivery of KT. Alginates are naturally occurring anionic polysaccharides obtained from marine brown algae and consist of two monomeric units; β-D-mannuronic acid and α-L-guluronic acid. Sodium salt of alginic acid (sodium alginate) shows gelation properties as a result of crosslinking of branched chain structure containing acidic contents with multivalent cations such as Ca2+, Ba2+, and Al3+ [12-15]. Therefore, dilute aqueous solutions of alginate form firm gels upon addition of di- and trivalent ions (commonly present in simulated biological fluid (SBF)).

The present work deals with the development of alginate-based in situ gels for the delivery of KT. Intrathecal administration of these in situ gels could lead to reduced dosage frequency, prolonged drug release and ultimately improved patient compliance for the potential treatment of VF.

In the first set of experiments, ion activated in situ gels were prepared using the cold method. Only those formulations that gelled instantaneously (< 1 min) following their contact with SBF were selected for further study ().

Physicochemical characterization of the gels

Visual inspection revealed the formation of a translucent matrix following incubation of the formulations with SBF. The prepared gels were transparent at all pH values. Further, pH of all formulations was in acceptable range () and the clarity of all formulations was satisfactory. Further, the formulations were slight yellow in colour.

The drug content was found to be in the acceptable range for all formulations. Percentage drug content in all formulations was in the range of 98.3 - 103.3 % indicating uniform drug distribution in the studied formulations.

Viscosity and rheological properties of KT loaded gels

Three best formulations (G2, G4 and G7) were evaluated for viscosity using Brookfield viscometer DV II+Pro (). The formulations exhibited pseudo-plastic rheology as evidenced by shear thinning and increase in shear rate as a function of angular velocity. The viscosity was however found to be dependent on polymeric content of the formulations and followed the order, G7>G2>G4.

In-vitro drug release

In vitro drug release studies were carried out for all formulations using simulated biological fluid (SBF) as the dissolution medium. It was found that the percentage drug release was 88.67, 86.59, 89.43, 88.28, 97.12, 96.98, 84.43 and 91.38 % for the formulation G1, G2, G3, G4, G5, G6, G7 and G8, respectively at the end of 12 h. In order to gain insight into drug release mechanism from ion activated in situ gels, the release data was subjected to different modes of data treatment. The results indicated a zero order drug release profile from alginate based in situ gels, potentially as a result of diffusion from the gel matrix.

In vivo analgesic and anti-inflammatory activity of KT loaded gels

On the basis of physiochemical properties, rheological properties and in vitro release profile, formulation G7 was selected for determining the in vivo analgesic and anti-inflammatory potential of KT-loaded biodegradable implants on Wistar rats ().

Analgesic and anti-inflammatory activities of KT loaded G7 gels were found to be 54.28 % and 51.6 % respectively, and the effect was comparable to that of the free drug. The anti-inflammatory activity was presented in terms of inhibition (%) of paw edema using the standard carrageenan induced hind rat paw edema model.

Taken together, the results of this study demonstrated the potential of KT loaded in situ gels (G7) in VF treatment due to high drug content, good rheological properties as well as analgesic and anti-inflammatory activity on Wistar rats.

Biodegradable polymers have shown potential as drug delivery systems in the form of implants and devices for bone repair. Apart from being non-mutagenic and non-cytotoxic, biodegradable polymers are metabolized in the body and eliminated by normal physiological pathways [17,18]. Of the various fabrication systems available, in situ gels show potential for local as well as systemic controlled drug delivery systems [19-21]. Further, since intrathecal KT (free drug) pre-treatments have been reported to reduce the spinal cord ischemic and VF injuries [22], this study employed in situ gels for intrathecal KT administration due to the disadvantages associated with the administration of free KT (e.g., poor drug stability, reduced half-life, frequent administration and hence reduced patient compliance).

Sodium alginate was used for the fabrication of ion activated in situ gels due to the property of its aqueous solutions to transform into stiff gels through ionic gelation in presence of divalent cations of SBF (Ca²⁺). Further, alginate-based in situ gels have been reported to preserve their integrity without undergoing dissolution or erosion for prolonged period of time [23]. Moreover, the method used for fabrication of alginate-based in situ gels in the present study was simple and reproducible. Only those formulations were selected that demonstrated satisfactory attributes in terms of gelling capacity in the pre-formulation studies.

The initial drug release pattern from alginate based in situ gels was characteristic of hydrophilic matrices. This initial fast release of KT could be attributed to the fact that alginate gels were formulated in water and hence the polymer was completely hydrated. Incubation in SBF resulted in gelation and formation of a pre-hydrated matrix wherein hydration and water penetration no longer limited the drug release thereby leading to an apparent diffusion based controlled release. In particular, there was 11.19 % release of KT from formulation G7 after 0.5 h, and 84.43 % after 8 h, and the drug release continued to rise thereafter.

Following comparison with other formulations, G7 (based on 1.2 % alginate/0.4 % HPMC) showed better ability to retain KT and hence was chosen for further studies. Additionally, the chosen formulation demonstrated analgesic as well as anti-inflammatory activity. Taken together, results of this study suggest that the alginate/HPMC aqueous system show potential as an in situ gel-forming system for intrathecal delivery in VF.

This work involved the development of alginate-based in situ gels for the delivery of KT. The gels demonstrated high drug encapsulation efficiency and can be employed for continuous and extended release of KT. The gels also exhibit analgesic and anti-inflammatory activity in vivo thereby indicating the potential for round-the-clock management of pain and inflammation in patients with VF. However, further studies are required to determine the suitability of the formulation in patients with VF.