(T1330-03-17) Formulating Adenosine for Localized Delivery

Purpose: Adenosine (Ado) is an endogenously produced metabolite and an FDA approved imaging agent and used for treatment of cardiac conditions. Studies have shown that adenosine has immunomodulatory effects on multiple types of cells, including macrophages and dendritic cells that function as drivers in inflammatory conditions. (1) There have been efforts in repurposing adenosine for hyperinflammatory conditions such as acute respiratory distress syndrome (ARDS). The application of adenosine in organ-specific treatments is limited by the drug’s short half-life ( < 10 seconds) in plasma and extracellular fluids. As such, high doses are needed to attain pharmacological actions, but in certain anatomical sites only limited volume can be administered (i.e., < 5 mL in intra-tracheal space). Thus, an unmet need is a formulation in which highly concentrated adenosine can be delivered in pharmacologically relevant doses locally in tissues. Herein, we propose a crystalline Ado formulation (cADO) in which 2X of saturating concentration of the drug can be administered. To our knowledge, this is the first report of Ado crystals generated using a facile, rapid, and solvent-free process.

Methods: The method of generating crystalline Ado involved a two-step process in which the compound is dissolved in distilled water at a supersaturated concentration through heating followed by incubation in aqueous media to allow nucleation and growth. The crystals formed were collected, centrifuged, resuspended in water, and refrigerated till further use. The samples were characterized using X ray diffraction (XRD), brightfield, polarized light, and scanning electron microscopy (SEM). Crystallinity was further investigated using thermal techniques such as differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), and hot stage polarized light microscopy (PLM). The biological effects of the formulation were studied in mice by analyzing immune cells in lymph nodes drained from a subcutaneous injection site.

Results: Ado crystals yielded from said process were characterized using XRD and microscopic studies (Fig 1) and were found distinct from crystals procured from a commercial vendor. TGA analysis indicates cADO exists as a monohydrate form as evidenced by the stoichiometry of loss of water upon heating (Fig 2). DSC analysis of both crystals shows distinct peaks corresponding to the drug melting in both the crystal samples while the cADO formulation shows an additional exothermic peak indicative of recrystallization or change in form upon heating (Fig 3). Immune cells in lymph nodes drained from footpad injected with cADO exhibit suppressed inflammatory response to LPS (data not shown).

Conclusion: The crystalline Ado generated using the facile and solvent-free method is a promising strategy for repurposing the nucleoside as a local immunomodulator.

References: 1. Haskó G, Pacher P. Regulation of macrophage function by adenosine. Arteriosclerosis, thrombosis, and vascular biology. 2012;32(4):865-9.


Fig 1. SEM images of the developed crystalline Ado (A) and crystals procured from the vendor (B)


Fig 2 . XRD and TGA analysis of formulated crystals(Ado_water) and crystals obtained from the vendor(Ado_raw) indicating distinct crystal forms and a monohydrate crystal form as evidenced by loss of water stoichiometrically equivalent to monohydrate form of crystal


Fig 3 . DSC analysis of formulated crystals(A) and crystals obtained from the vendor(B) indicating distinct peaks corresponding to the melting of drug with an additional exothermic peak indicative of transformations or recrystallization upon heating observed in formulated crystals