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.