To study new target-oriented molecules that are active against rheumatoid arthritis-dependent pain, new dual inhibitors incorporating both a carbonic anhydrase (CA)-binding moiety and a cyclooxygenase inhibitor (NSAID) were tested in a rat model of rheumatoid arthritis induced by CFA intra-articular (i.a.) injection. A comparison between a repeated per os treatment and a single i.a. injection was performed. CFA (50 µL) was injected in the tibiotarsal joint, and the effect of per os repeated treatment (1 mg kg⁻¹) or single i.a injection (1 mg mL⁻¹, 50 µL) with NSAIDs-CAIs hybrid molecules, named 4 and 5, was evaluated. The molecules 4 and 5, which were administered daily for 14 days, significantly prevented CFA-induced hypersensitivity to mechanical noxious (Paw pressure test) and non-noxious stimuli (von Frey test), the postural unbalance related to spontaneous pain (Incapacitance test) and motor alterations (Beam balance test). Moreover, to study a possible localized activity, 4 and 5 were formulated in liposomes (lipo 4 and lipo 5, both 1 mg mL⁻¹) and directly administered by a single i.a. injection seven days after CFA injection. Lipo 5 decreased the mechanical hypersensitivity to noxious and non-noxious stimuli and improved motor coordination. Oral and i.a. treatments did not rescue the joint, as shown by the histological analysis. This new class of potent molecules, which is able to inhibit at the same time CA and cyclooxygenase, shows high activity in a preclinical condition of rheumatoid arthritis, strongly suggesting a novel attractive pharmacodynamic profile.

Pulmonary fibrosis is a severe lung disease with progressive worsening of dyspnea, characterized by chronic inflammation and remodeling of lung parenchyma. Carbonic anhydrases are a family of zinc-metallo-enzymes that catalyze the reversible interconversion of carbon-dioxide and water to bicarbonate and protons. Carbonic Anhydrase Inhibitor (CAI) exhibited anti-inflammatory effects in animals with permanent-middle-cerebral artery occlusion, arthritis and neuropathic pain. The pharmacological profile of a new class of hybrid compounds constituted by a CAI connected to a Nonsteroidal-Anti-Inflammatory Drug (NSAID) was studied in the modulation of inflammation and fibrosis. In-vitro tests were performed to assess their effects on cyclo-oxygenase enzyme (COX)-1 and COX-2, namely inhibition of platelet aggregation and thromboxane B2 production in the human-platelet-rich plasma, and reduction of Prostaglandin-E2 production in lipopolysaccharide-treated-RAW-264.7 macrophage cell line. The activity of compound 3, one of the most active, was studied in a model of bleomycin-induced lung fibrosis in C57BL/6 mice. The hybrid compounds showed a higher potency in inhibiting PGE₂ production, but not in modifying the platelet aggregation and the TXB₂ production in comparison to the reference molecules, indicating an increased activity in COX-2 inhibition. In the in-vivo murine model, the compound 3 was more effective in decreasing inflammation, lung stiffness and oxidative stress in comparison to the reference drugs given alone or in association. In conclusion, these CAI-NSAID hybrid compounds are promising new anti-inflammatory drugs for the treatment of lung chronic inflammatory diseases.