Discussion
MS is a chronic disease of the CNS characterized by autoimmune inflammation, demyelination, and axonal damage(Comabella & Khoury, 2012; Reich, Lucchinetti & Calabresi, 2018). The underlying immunological disorders in MS result in a variety of neurological and autoimmune symptoms. A number of studies have revealed a critical role of T cells in the pathogenesis of EAE and MS makes them a promising target for drug discovery(Fletcher, Lalor, Sweeney, Tubridy & Mills, 2010). Pyrimidine nucleotides are critical for DNA and RNA synthesis and are indispensable for the development and survival of mature T lymphocytes(Quemeneur, Gerland, Flacher, Ffrench, Revillard & Genestier, 2003). Human DHODH catalyzes the fourth committed step in the de novo pyrimidine nucleotide biosynthetic pathway(Liu, Neidhardt, Grossman, Ocain & Clardy, 2000a). Pharmacological intervention of pyrimidine biosynthesis by selectively inhibition of DHODH has been explored for the development of new therapeutic agents against MS. Synthetic small molecule A771726 have been approved for the treatment of MS as an immunomodulatory drug. However, the long elimination half-life of A771726 is associated with its observed hepatotoxicity in clinical and potential teratogenicity in animal study(Garnock-Jones, 2013). More safe and effective therapeutic agents targeting DHODH are needed to benefit patient with MS.
Black pepper was widely used in traditional medicine as a natural herb for the therapy of a variety of diseases including rheumatism, pains, and fevers(Gorgani, Mohammadi, Najafpour & Nikzad, 2017). Piperine is the predominant active constituent of black pepper and also is the most abundant alkaloid in pepper (approximately 98%). Previous research has revealed that piperine has widely pharmacological activities, including anti-inflammatory, antiarthritic effects, antioxidant and chemopreventive activities(Bang et al., 2009; Manayi, Nabavi, Setzer & Jafari, 2017). To the best of our knowledge, this is the first study of revealing piperine’s potent protective activity in autoimmune demyelinating disease.
Piperine was widely used as a flavoring ingredient in foods and beverages manufacturers with well-documented safety since it is first isolated from pepper in 1819(Gorgani, Mohammadi, Najafpour & Nikzad, 2017). Using modern drug safety evaluation system, Bastaki M et al organized a 90-day GLP compliant toxicity study in sprague-dawley rats with multiple doses of piperine (5-50 mg/kg bw/day) to evaluate the safety of daily uptake of piperine in foods. Notably, no adverse effects were observed following piperine ingestion even at the highest dosage (50 mg/kg bw/day)(Bastaki et al., 2018). Herein, we present data that piperine has the ability to interact with DHODH and reduce T cell proliferation via a DHODH dependent manner. We further show that piperine effectively alleviates autoimmune response in MOG-induced EAE mouse model without obvious toxicity following the oral administration of piperine (30 mg/kg). As piperine exerts its pharmacological effects in preclinical MS model through DHODH inhibition, and as piperine is a clinical stage compound with excellent safety(Quijia & Chorilli, 2020), drug repurposing of piperine may be a valuable manipulation to benefit patients with MS.
According to the MS immunopathogenesis hypothesis, activated T cells can cross the BBB and reactive with CNS proteins, including myelin basic protein [MBP] and myelin oligodendrocyte glycoprotein [MOG](Bar-Or, Pachner, Menguy-Vacheron, Kaplan & Wiendl, 2014). The BBB penetration of a small molecule may facilitate targeting activated T cells in the brain lesion. Intriguingly, piperine is capable of crossing the BBB as detected in an in vitro parallel artificial membrane permeability assay. Furthermore, in vivo tissue distribution assay indicated that piperine can penetrate BBB in rat (Liu et al., 2013). So, the BBB penetration of piperine may lay a foundation for its superior pharmacological activity in nervous system.
Piperine was identified as a less potent agonist of TRPV1 (EC50=37.9 μM)(McNamara, Randall & Gunthorpe, 2005). Through a bioassay-guided isolation of the ethanol extract from the fruits of Piper longum, Ro’s group have identified piperine as a moderate dual MAO-A and MAO-B inhibitor with IC50 values of 20.9 μM and 7.0 μM, respectively(Munier-Lehmann, Vidalain, Tangy & Janin, 2013). Herein, we demonstrate that piperine is a potent, direct DHODH inhibitor with an IC50 value of 0.88 ± 0.04 μM. Meanwhile, piperine effectively reduced T cell proliferation through DHODH inhibition in vitro , suggesting a potential role of piperine in immune function modulating. Furthermore, piperine treatment significantly reduce immune cell infiltration in central nervous tissue and resultant remission of EAE symptoms. We thereby speculate that DHODH is a main functional target of pepirine in EAE, although it remains to be investigated in the future whether other proteins were involved in the therapeutic effect of piperine in EAE.
In summary, we demonstrate that piperine regulates the activation of T lymphocytes through via pharmacological inhibiting DHODH and blocking de novo pyrimidine biosynthesis. Importantly, EAE disease progression was observably alleviated following oral administration of piperine in preventive and therapeutic regimens. These compelling preclinical data presented here suggest that piperine is a potential therapeutic agent for MS with a novel mechanism-of-action, and further demonstrate that DHODH is a promising therapeutic target for the treatment of MS.