Methylated flavonoids as anti-seizure agents: Naringenin 4′,7-dimethyl ether attenuates epileptic seizures in zebrafish and mouse models

Highlights

• Methylated flavonoids are proposed as a source for anti-seizure drug discovery.

• Methylated flavanones are highly active against seizures in zebrafish larvae.

• Naringenin 4′,7-dimethyl ether is highly active against seizures in zebrafish larvae.

• Naringenin 4′,7-dimethyl ether is active against drug-resistant seizures in mice.

• Naringenin 4′,7-dimethyl ether is proposed as a lead anti-seizure compound.

Abstract

Epilepsy is a neurological disease that affects more than 70 million people worldwide and is characterized by the presence of spontaneous unprovoked recurrent seizures. Existing anti-seizure drugs (ASDs) have side effects and fail to control seizures in 30% of patients due to drug resistance. Hence, safer and more efficacious drugs are sorely needed. Flavonoids are polyphenolic structures naturally present in most plants and consumed daily with no adverse effects reported. These structures have shown activity in several seizure and epilepsy animal models through allosteric modulation of GABA_A receptors, but also via potent anti-inflammatory action in the brain. As such, dietary flavonoids offer an interesting source for ASD and anti-epileptogenic drug (AED) discovery, but their pharmaceutical potential is often hampered by metabolic instability and low oral bioavailability. It has been argued that their drug-likeness can be improved via methylation of the free hydroxyl groups, thereby dramatically enhancing metabolic stability and membrane transport, facilitating absorption and highly increasing bioavailability. Since no scientific data is available regarding the use of methylated flavonoids in the fight against epilepsy, we studied naringenin (NRG), kaempferol (KFL), and three methylated derivatives, i.e., naringenin 7-O-methyl ether (NRG-M), naringenin 4′,7-dimethyl ether (NRG-DM), and kaempferide (4′-O-methyl kaempferol) (KFD) in the zebrafish pentylenetetrazole (PTZ) seizure model. We demonstrate that the methylated flavanones NRG-DM and NRG-M are highly effective against PTZ-induced seizures in larval zebrafish, whereas NRG and the flavonols KFL and KFD possess only a limited activity. Moreover, we show that NRG-DM is active in two standard acute mouse seizure models, i.e., the timed i.v. PTZ seizure model and the 6-Hz psychomotor seizure model. Based on these results, NRG-DM is proposed as a lead compound that is worth further investigation for the treatment of generalized seizures and drug-resistant focal seizures. Our data therefore highlights the potential of methylated flavonoids in the search for new and improved ASDs.

Introduction

Epilepsy is one of the most common neurological diseases affecting more than 70 million people worldwide (Ngugi et al., 2010, Singh and Trevick, 2016). It is characterized by the presence of spontaneous unprovoked recurrent seizures and involves risks of comorbidities, anxiety, depression and increased mortality. Moreover, patients suffer from social stigma and the stress of living with an unpredictable disease that can lead to loss of autonomy in daily life (Moshé et al., 2015). Even though over 25 anti-seizure drugs (ASDs) are on the market, seizures cannot be controlled in 30% of patients due to drug resistance (Dalic and Cook, 2016, Franco et al., 2016, Moshé et al., 2015). In fact, the probability of achieving seizure freedom did not lower since the 70s, despite the availability of new-generation ASDs (Franco et al., 2016). In addition, existing ASDs have many adverse effects, ranging from gastrointestinal problems to hepatotoxicity and even cognitive impairment (Löscher et al., 2013, Moshé et al., 2015). Hence, there is an unmet need for the development of new and safe ASDs that can control seizures effectively and prevent seizure-induced neurological damage (Löscher, 2017, Łukawski et al., 2016).

Significantly, inflammatory mediators such as cytokines and prostaglandins, released from brain cells and peripheral immune cells are involved in the origin of seizures, as well as in epileptogenesis (de Vries et al., 2016, Dey et al., 2016, Löscher et al., 2013, Vezzani et al., 2013). Thus, in the search for more effective ASDs with novel mechanisms of action, anti-inflammatory agents have been proposed not only as potential ASDs but also as candidate anti-epileptogenic drugs (AEDs) that can prevent or favorably modify the development of epilepsy (Dey et al., 2016, Vezzani, 2015).

Of interest, flavonoids and their glycosides have been shown to exert mild to potent activity in several seizure and epilepsy animal models (Sucher and Carles, 2015, Zhu et al., 2014). Investigating the underlying mechanism of action, it was found that these structures modulate allosterically GABA_A receptors by binding to the benzodiazepine receptor site (Hanrahan et al., 2011, Johnston, 2015). However, flavonoids are also known to exert potent anti-inflammatory effects in the brain by means of free-radical-scavenging activity (Diniz et al., 2015), or by directly modulating key components of the neuroinflammatory cascade (Spencer et al., 2012). Accordingly, this neuroprotective activity has been invoked to explain their anticonvulsant effects as well (Diniz et al., 2015, Golechha et al., 2014, Golechha et al., 2011, Lin et al., 2015).

Flavonoids are polyphenolic structures that are daily consumed due to their widespread distribution in almost all terrestrial plants with no adverse effects reported (Jäger and Saaby, 2011, Tapas et al., 2008). As such, dietary flavonoids and chemical derivatives thereof offer a possibly interesting source for ASD and AED chemical discovery work (Jäger and Saaby, 2011, Marder and Paladini, 2002, Singh et al., 2014). Although their pharmaceutical potential is often hampered by metabolic instability and low oral bioavailability, it has been argued that their drug-likeness can be improved via methylation of the free hydroxyl groups, dramatically enhancing the metabolic stability and membrane transport, facilitating absorption and highly increasing the bioavailability (Koirala, 2016, Koirala et al., 2016). Of importance, this methylation also abrogates their chemical anti-oxidant capacity, but leaves the general protective activity of the compounds against oxidative stress intact (Deng et al., 2006).

Since to the best of our knowledge no scientific data is available regarding the use of methylated flavonoids in the fight against epilepsy, we studied the anti-seizure activity of naringenin (NRG) and kaempferol (KFL), and of some of their common methylated derivatives, i.e., naringenin 7-O-methyl ether (NRG-M), naringenin 4′,7-dimethyl ether (NRG-DM), and kaempferide (4′-O-methyl kaempferol) (KFD) (Fig. 1A).

KFL belongs to the chemical subgroup of the flavonols that feature a double bond in the heterocyclic benzopyran moiety (C ring) resulting in planar A (fused aromatic ring) and C rings (Hanrahan et al., 2011). Interestingly, KFL is a key active constituent of the extract of Crinum jagus L. that is used in traditional Cameroonian medicine as an anti-epileptic remedy. After isolation, the compound was found to improve convulsions-induced by pentylenetetrazole (PTZ) and to protect mice against PTZ-induced kindling development (Taiwe et al., 2016).

NRG on the other hand belongs to the flavanones (Ferreira et al., 2014), a subgroup with a puckered conformation of the C ring due to the lack of the double bond (Hanrahan et al., 2011). NRG is found in citrus fruits and has strong anti-inflammatory and anti-oxidant activities (Alam et al., 2014, Miler et al., 2016, Patel et al., 2014, Renugadevi and Prabu, 2009). It can pass the blood brain barrier, which enables the compound to exert neuroactive properties (Patel et al., 2014, Youdim et al., 2003). Recently, NRG was shown to delay seizure onset, to ameliorate induced morphological brain alterations, and to reduce microglia-derived neuro-inflammation in a kainic acid-induced mouse seizure model (Park et al., 2016). Of interest, NRG and KFL feature a highly similar hydroxylation pattern (Fig. 1A), allowing for a better comparison between the biological activity of the two different flavonoid subgroups.

To evaluate the anti-seizure activity of the compounds, we used a zebrafish PTZ seizure model (Afrikanova et al., 2013, Baraban et al., 2005), a standard assay in preliminary ASD discovery due to the high level of translation to rodents (Afrikanova et al., 2013, Buenafe et al., 2013, Orellana-Paucar et al., 2012). Zebrafish are recognized as an important organism for modeling human diseases with respect to the 3R principle (Doke and Dhawale, 2015, Grone and Baraban, 2015) because it is a lower vertebrate model that is highly conserved with regard to the human reference genome (Howe et al., 2013), physiology, and pharmacology (MacRae and Peterson, 2015).

In this study, we show that the methylated NRG-M and NRG-DM are highly effective against PTZ-induced seizures in larval zebrafish, whereas NRG, KFL, and KFD possess only a limited activity. NRG-DM was further investigated in standard acute rodent seizure models, i.e., the mouse timed i.v. PTZ seizure model and the mouse 6-Hz psychomotor seizure model, and showed dose-dependent seizure reduction in both. Thus, highlighting the potential of methylated flavonoids as anti-seizure agents.