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 »  Abstract
 » Introduction
 » Materials
 » Methods
 » Results
 » Discussion
 » Conclusion
 »  References
 »  Article Figures

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 Table of Contents    
Year : 2023  |  Volume : 55  |  Issue : 5  |  Page : 307-314

Study of fingolimod, nitric oxide inhibitor, and P-glycoprotein inhibitor in modulating the P-glycoprotein expression via an endothelin–sphingolipid pathway in an animal model of pharmacoresistant epilepsy

1 Department of Pharmacology, Post Graduate Institute of Medical Education and Research (PGIMER), Chandigarh, Haryana, India
2 Department of Pharmacology, Post Graduate Institute of Medical Education and Research (PGIMER), Chandigarh; Department of Pharmacology, MM Institute of Medical Sciences and Research, Maharishi Markandeshwar (Deemed to be university), Mullana, Ambala, Haryana, India
3 Department of Experimental Medicine and Biotechnology, Post Graduate Institute of Medical Education and Research (PGIMER), Chandigarh, Haryana, India
4 Department of Pharmacology, Post Graduate Institute of Medical Education and Research (PGIMER), Chandigarh; Department of Pharmacology, MM College of Pharmacy, Maharishi Markandeshwar (Deemed to be university), Mullana, Ambala, Haryana, India
5 Department of Immunopathology, Post Graduate Institute of Medical Education and Research (PGIMER), Chandigarh, Haryana, India
6 Department of Neurology, Post Graduate Institute of Medical Education and Research (PGIMER), Chandigarh, Haryana, India

Date of Submission23-Feb-2023
Date of Decision29-Aug-2023
Date of Acceptance30-Aug-2023
Date of Web Publication02-Nov-2023

Correspondence Address:
Bikash Medhi
Department of Pharmacology, Post Graduate Institute of Medical Education and Research, Chandigarh
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/ijp.ijp_100_23

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 » Abstract 

BACKGROUND: The overexpression of P-glycoprotein (P-gp) contributes to drug resistance in patients with epilepsy, and the change of P-gp expression located at the blood–brain barrier alienates the anti-seizure effects of P-gp substrates. Thus, the present study explored the effect of fingolimod (FTY720) acting through an endothelin–sphingolipid pathway on P-gp-induced pentylenetetrazol (PTZ)-kindled phenobarbital (PB)-resistant rats.
MATERIALS AND METHODS: PTZ kindling (30 mg/kg; i.p.) and PB (40 mg/kg; orally) were used to develop an animal model of refractory epilepsy. The effect of Fingolimod on seizure score (Racine scale), plasma and brain levels of PB (high-performance liquid chromatography), and blood–brain barrier permeability (Evans blue dye) was determined. Further, Fingolimod's neuroprotective effect was determined by measuring the levels of various inflammatory cytokines, oxidative stress parameters, and neurotrophic factors in rat brain homogenate. The Fingolimod's effect on P-gp expression was estimated by reverse transcriptase–polymerase chain reaction and immunohistochemistry in rat brain. The H and E staining was done to determine the neuronal injury.
RESULTS: Fingolimod significantly (P < 0.001) reduced the seizure score in a dose-dependent manner and alleviated the blood–brain barrier permeability. It decreased the P-gp expression, which further increased the brain PB concentration. Fingolimod significantly (P < 0.01) reduced oxidative stress as well as inflammation. Moreover, it attenuated the raised neuronal injury score in a resistant model of epilepsy.
CONCLUSION: The modulation of the P-gp expression by Fingolimod improved drug delivery to the brain in an animal model of refractory epilepsy. Therefore, S1P signaling could serve as an additional therapeutic target to overcome refractoriness.

Keywords: Fingolimod, neuroprotection, P-glycoprotein, phenobarbital resistance, refractory epilepsy

How to cite this article:
Garg N, Joshi R, Bhatia A, Bansal S, Chakrabarti A, Prakash A, Saikia B, Modi M, Medhi B. Study of fingolimod, nitric oxide inhibitor, and P-glycoprotein inhibitor in modulating the P-glycoprotein expression via an endothelin–sphingolipid pathway in an animal model of pharmacoresistant epilepsy. Indian J Pharmacol 2023;55:307-14

How to cite this URL:
Garg N, Joshi R, Bhatia A, Bansal S, Chakrabarti A, Prakash A, Saikia B, Modi M, Medhi B. Study of fingolimod, nitric oxide inhibitor, and P-glycoprotein inhibitor in modulating the P-glycoprotein expression via an endothelin–sphingolipid pathway in an animal model of pharmacoresistant epilepsy. Indian J Pharmacol [serial online] 2023 [cited 2023 Nov 28];55:307-14. Available from: https://www.ijp-online.com/text.asp?2023/55/5/307/389230

 » Introduction Top

Epilepsy is a chronic disease that involves the dysfunction of the central nervous system (CNS) and leads to excessive neuronal firing and damage. It accounts for 1% of the world's diseases. Drug treatment resistance is an emerging problem as more than 30% of the patients show refractoriness to the treatment of epilepsy. It is said that once the individual develops resistance to antiepileptic drugs (AEDs), the likelihood of seizure freedom is only 5%–10%. The various factors such as brain insult, brain inflammation, neuronal cell death, blood–brain barrier (BBB) damage, oxidative stress, neuronal rewiring, and mossy fiber sprouting initiate and propagate significant alterations in the brain and serve as an emerging treatment target in epilepsy.[1] The central challenge in the treatment of CNS diseases like epilepsy is the transportation of drugs through BBB. It has been observed that almost all the lipophilic drugs are transported across the BBB through the P-glycoprotein (P-gp) transporter. Moreover, a large number of AEDs also act as a P-gp substrate.[2] The basis of the present study lies in the principle of the transporter hypothesis, which postulates that the augmented expression of the P-gp efflux transporter at the BBB leads to reduced therapeutic concentration of AEDs in the brain, thus leading to refractoriness.[3] It has been found out that knocking out P-gp or inhibiting its expression through P-gp inhibitors is not a valid and safe treatment option owing to increased systemic toxicity.[4],[5] In addition, the BBB and associated neurovascular unit deal with maintaining the homeostasis of the brain, essential for the efficient functioning of the neurons. Hence, there arises an urgent need to discover alternatives to the transport inhibitors so that the defensive function of the BBB can be maintained. It was reported that the endothelin-stimulated signaling pathway that involved sphingolipid activation is associated with the regulation of P-gp.[6],[7] The biological effects of sphingosine-1-phosphate were carried by five specific G-protein-coupled receptors. Later studies supported the involvement of the endothelin–sphingolipid pathway in increased uptake of P-gp substrate, without hampering the tight junction permeability.[8] Consequently, the present study has been designed to target the endothelin–sphingolipid pathway through sphingosine-1-phosphate agonist, fingolimod also known as FTY720 in modulating the epileptogenic process and P-gp expression in the phenobarbital (PB)-resistant pentylenetetrazol (PTZ)-kindled refractory model of epilepsy in rats. Fingolimod is a prodrug that gets activated in phosphorylated form and acts as a nonselective S1PR agonist. It is a clinically relevant option as presently, it is in use for relapsing multiple sclerosis.[9] The possible involvement of the endothelin–sphingolipid pathway in modulating P-gp expression in the refractory model of epilepsy was planned to be studied through a mechanistic approach using the inhibitor of inducible nitric oxide, i.e. L-NAME. The role of Fingolimod on P-gp expression, seizure severity, various inflammatory cytokine levels, oxidative stress, BBB permeability, and neuronal injury has been investigated. A comparison of the efficacy of an S1P agonist Fingolimod with the known P-gp inhibitor verapamil has also been studied.

Experimental animals

The present study was conducted in the Experimental Pharmacology Laboratory, PGIMER, Chandigarh. The male Wistar rats were made to acclimatize at a controlled temperature (24°C) with a 12-h light-dark cycle for a minimum of 1 week before the start of experiments. The procedure for animal care as well as drug treatment was accepted and sanctioned by the Institutional Animal Ethics Committee (81/IAEC/505) and Institutional Biosafety Committee (482/IBC/2015).

 » Materials Top

PTZ acquired from Sigma, India, and PB as a humble gift from Harman Finochem Pvt. Ltd., Aurangabad. Moreover, the purchase of Fingolimod and verapamil was done from Sigma-Aldrich, India. The resistance was induced by the standard drug of choice, i.e. PB. PTZ in saline was administered intraperitoneally, whereas the suspension of PB in carboxymethylcellulose was administered orally. Fingolimod (0.5, 1 mg/kg), L-NAME (10 mg/kg), and verapamil (10 mg/kg) were dissolved in saline and administered intraperitoneally. The drug solutions were constituted fresh on each day of experimentation and were given orally by gavage at a maximum limit of 1.0 mL/100 g body weight.

 » Methods Top

The experimental model of pentylenetetrazol-induced phenobarbital-resistant epilepsy in rats was performed as follows:

  1. The subconvulsive dose of PTZ, i.e. 30 mg/kg, was administered through an intraperitoneal route for 28 days on a daily basis, to generate chemical kindling in the rats
  2. The seizure scoring was done 30 min after each PTZ injection according to the Racine scale[10]
  3. The animals showing successive Stage 2 for 5 days, consecutive Stage 3 for 3 days, or consecutive Stage 4/5 for 2 days were considered kindled
  4. The kindled rats were given PB (40 mg/kg) orally and other treatments for 21 days followed by PTZ (30 mg/kg)
  5. Seizure scoring was done, and the rats that continued to experience consecutive Stage 2 for 5 days/consecutive Stage 3 for 3 days/consecutive Stage 4/5 for 2 days after PB administration were considered refractory to PB.

Estimation of phenobarbital levels

The C-18 column was used for the chromatographic separation studies. The mobile phase constitutes acetonitrile: methanol: phosphate buffer in the ratio of 2:4:4, which gave good resolution and adequate peak parameters. The flow rate of PB elution was maintained at 3.0 mL/min. Acetonitrile and phosphate buffer (19/81 by Vol.) constituted the mobile phase. The absorption of PB occurred at 195 nm, and the respective peak heights were anticipated to measure the quantity. The 10 μL sample was injected, and the temperature of the column was retained at 50°C. The calculated time for retention peak for PB was 5.00 ± 0.02 min.

Evans blue extravasation test

This test was employed for testing the BBB permeability of the rat. The blood-barrier disruption leads to the permeability of albumin into the brain tissue. Evans, which would have bound to the albumin, also gets diffused through the BBB. The processing of the brain tissue leads to the estimation of the Evans, which would indirectly serve as a variable for assessing the BBB permeability.

Biochemical estimation

The spectrophotometric assay was done to determine the presence of free carbonyl protein groups as the biomarker for oxidative stress in the brain. The rat brain homogenate was employed to assess the various antioxidant enzymes.

Estimation of inflammatory cytokines

The pro-inflammatory cytokines such as interleukin (IL)-1 β, IL-6, and tumor necrosis factor-alpha (TNF-α) were estimated by utilizing enzyme-linked immunosorbent assay (ELISA) kits from Boster Biological Technology Co., Ltd.

Estimation of brain-derived neurotrophic factor and nuclear factor kappa B

The estimation of levels of BDNF and NF-κb was done by ELISA kits from Elabscience Biotechnology Co. Ltd., in the rat serum and rat brain homogenate, respectively.

Histopathology of rat brain

The hippocampus sections were taken and stained with hematoxylin and eosin dye. The neuronal injury score was assessed in the bright-field images of the slides taken on Olympus upright microscope.

Isolation of RNA and real-time polymerase chain reaction for P-glycoprotein expression

The reverse transcriptase-polymerase chain reaction (RT-PCR) was performed on applied biosystem step-up machine. The cDNA was diluted in nuclease-free water in a ratio of 1:1000 and was gently vortexed and centrifuged. To the PCR tubes, 2 μL PCR mix, 0.25 μL forward primer, 0.25 μL reverse primer, 1 μL cDNA, and 6 μL water were added. A total of 10 μL reaction mixture was made and was carried out in duplicate. The no-template negative control was run to assess for any contamination present. The positive control of the housekeeping β-actin gene was run. The melting temperatures of 60°C were selected for Mdr-1a and 66°C for Mdr-1b gene.

Immunohistochemical staining

The brain sections mounted on poly-L-lysine-coated slides were first deparaffinized and then rehydrated and washed. Post the antigen retrieval and sequestration of endogenous peroxidase activity, each section was incubated with an anti-P-gp antibody, followed by Horse-Raddish peroxidase HRP-labeled secondary P-gp. The staining was done with 0.05% diaminobenzidine tetrahydrochloride in phosphate-buffered saline containing H2O2 (1 μL/mL) for 1 min and then counterstained with hematoxylin and eosin.

Statistical parameters

The mean and standard deviation for each piece of data were displayed. To evaluate the quantitative parameters of seizure score and oxidative stress, the one-way ANOVA was carried out, after which the Bonferroni post hoc analysis was performed. Using an unpaired Student's t-test, the plasma PB concentration was examined at various doses. It was determined that P = 0.05 was statistically significant.

 » Results Top

Seizure score

PB 40 mg/kg was selected for the resistant model of epilepsy. The effect of Fingolimod was examined at two doses, i.e. 0.5 and 1 mg/kg. At week 6, Fingolimod treatment at 1 mg/kg dose displayed a significant decline in seizure score (P < 0.001) when compared with PTZ + PB-resistant rats as shown in [Figure 1]. At the end of week 7, both doses reduced the seizure score significantly (P < 0.001). The verapamil treatment, which is a known P-gp inhibitor, also reduced the seizure score by the end of week 7 when compared with the PTZ + PB-resistant group (P < 0.001). The treatment with the combination of L-NAME and Fingolimod reduced the seizure score in comparison with PTZ + PB resistant (P < 0.05); however, the seizure score was found to be increased in the Fingolimod 1 mg/kg group (P < 0.01).
Figure 1: Effect of different treatments on pentylenetetrazol-induced seizures in resistant rats. PTZ: Pentylenetetrazol, PB: Phenobarbital

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Phenobarbital levels in the brain and plasma

The brain–plasma ratio of PB levels was found to be decreased in the PB-resistant group [Figure 2]. Fingolimod at 1 mg/kg dose elevated the brain-to-plasma ratio of PB significantly in comparison to resistant rats (P < 0.05). However, the Fingolimod 0.5 mg/kg treatment group, as well as the combination treatment group of L-NAME (10 mg/kg) and Fingolimod (1 mg/kg), did not show any significant rise in the PB brain–plasma ratio. The treatment of verapamil at 10 mg/kg dose markedly increases (P < 0.001) the brain–plasma concentration of PB in comparison to the PB resistant group.
Figure 2: Estimation of phenobarbital levels in the brain and plasma samples of pentylenetetrazol-kindled rats by high-performance liquid chromatography. *P < 0.05, ***P < 0.001 in comparison to phenobarbital-resistant group. Data represented as mean ± standard error of the mean

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Effect of different treatments on blood–brain barrier permeability

The brains of saline control rats showed no concentration of Evans blue signifying the intact BBB. The PB-resistant rats showed a significant high concentration of Evans blue (P < 0.01) when compared with normal saline as shown in [Figure 3]. Fingolimod treatment at 1 mg/kg considerably (P < 0.01) alleviated the BBB permeability in comparison to PB-resistant rats. Treatment with direct P-gp inhibitor verapamil also noticeably (P < 0.05) protected the BBB disruption in contrast to PB-resistant rats. The treatment of L-NAME given before the Fingolimod treatment reversed the effect of Fingolimod by increasing the BBB disruption compared to PB-resistant rats.
Figure 3: Effect of different treatments on blood–brain barrier permeability in rat brain. Data represented as mean ± standard error of the mean. **P < 0.01; in comparison to control; #P < 0.05; ##P < 0.01 as compared to pentylenetetrazol–phenobarbital group. PTZ + PB: Pentylenetetrazol–phenobarbital

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Inflammatory markers

The effect of different treatments was studied on various inflammatory markers such as IL-1β, IL-6, and TNF-α. The PB-resistant group showed a significant augmentation in inflammatory markers such as IL-1β (P < 0.001), IL-6 (P < 0.01), and TNF-α (P < 0.01) compared to the saline control group. The treatment with Fingolimod alleviates inflammation at both the doses of 0.5 and 1 mg/kg (IL-1β [P < 0.001], IL-6 [P < 0.01], and TNF-α [P < 0.05]) in comparison with the PB-resistant group. The 10 mg/kg verapamil treatment also reduced the IL-1β levels significantly (IL-1β [P < 0.05], IL-6 [P < 0.01], and TNF-α [P < 0.05]) when compared with PB-resistant rats. However, no significant decrease in the levels of IL-1β, IL-6, and TNF-α was observed with L-NAME treatment as described in [Figure 4].
Figure 4: Effect of various therapies on the levels of interleukin-1, interleukin-6, tumor necrosis factor, nuclear factor kappa-B, brain-derived neurotrophic factor, and matrix metalloproteinase-9, as well as GSH, superoxide dismutase, and catalase in rat brain. The data are shown as mean standard error of the mean. *P < 0.05; **P < 0.01; ***P < 0.001 versus the control group of rats; #P < 0.05; ##P < 0.01; ###P < 0.001 versus the phenobarbital-resistant group; and $P < 0.05, versus the pentylenetetrazol group. @P < 0.05 as compared to the fingolimod 1 mg/kg group. PTZ: Pentylenetetrazol, PB: Phenobarbital, CMC: Carboxymethylcellulose, GSH: Reduced Glutathione

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Membrane metalloproteinase-9

The PB-resistant group showed elevated (P < 0.01) matrix metalloproteinase (MMP-9) levels when compared to the saline group. The treatment with Fingolimod reduced (P < 0.001) the MMP-9 levels at both doses of 0.5 mg/kg and 1 mg/kg when compared with the PB-resistant group. The treatment with verapamil did not show any significant decrease in the MMP-9 levels. The L-NAME and Fingolimod groups showed increased levels of MMP-9 as shown in [Figure 4].

Nuclear factor kappa-B levels

The PB-resistant group showed elevated (P < 0.01) NF-κB levels in contrast to the saline group. The treatment with Fingolimod 1 mg/kg reduces the NF-κB levels in contrast to the PTZ group (P < 0.05) and resistant group (P < 0.05). A significant (P < 0.05) reduction in the NF-κB levels was also observed with verapamil treatment in comparison with the PTZ group as shown in [Figure 4].

Brain-derived neurotrophic factor levels

The serum BDNF levels were significantly (P < 0.001) reduced in the PTZ-kindled rats, followed by the vehicle and PB-resistant group in comparison to the saline control group. Fingolimod significantly increased the reduced BDNF levels at both 0.5 mg/kg (P < 0.05) and 1 mg/kg doses (P < 0.01) when compared to the PB-resistant group. However, no significant increase in the BDNF levels was seen in verapamil and the combination of the L-NAME and Fingolimod groups as shown in [Figure 4].

Effect of different treatments on P-glycoprotein (mdr-1a and mdr-1b) expression

The overall fold change in P-gp (mdr-1a and mdr-1b) expression was found to be higher in the PB resistant as well as the Fingolimod groups in contrast to the normal saline group as shown in [Figure 5]. The marked rise in P-gp (mdr-1a and mdr-1b) expression was seen in the PTZ and PB-resistant groups (P < 0.001). The 1 mg/kg Fingolimod treatment reduced the P-gp expression significantly(mdr-1a [P < 0.05] and mdr-1b [P < 0.001]) in comparison to the normal saline as well as the PTZ group. The L-NAME reduced the P-gp expression significantly (mdr-1a [P < 0.01] and mdr-1b [P < 0.001]) in contrast to the PB-resistant group. Verapamil caused a noteworthy reduction in the P-gp (mdr-1a [P < 0.01] and mdr-1b [P < 0.001]) expression when compared to the PTZ group.
Figure 5: The effect of various therapies on P-glycoprotein (Mdr1a and Mdr1b) expression and distribution in the rat brain. The data are shown as mean ± standard error of the mean. ***P < 0.001, and **P < 0.01, in comparison to control rats; #P < 0.05 and ###P < 0.001 in comparison to pentylenetetrazol–phenobarbital group. P-glycoprotein expression is seen in A and B: Large blood vessels on the endothelium lining at the blood–brain barrier in the saline control group. P-glycoprotein expression is seen in A: large arteries and B: neurons in the pentylenetetrazol-phenobarbital group. The fingolimod-treated group shows P-glycoprotein distribution in A: Endothelial small blood vessels B: Endothelial large blood vessels. The combination treatment group of L-NAME and fingolimod shows P-glycoprotein distribution in A: Endothelial blood vessels B: Perineuronal membrane. The verapamil-treated group shows P-glycoprotein distribution in A: Endothelial blood vessels; B: Neurons. PTZ: Pentylenetetrazol, PB: Phenobarbital, CMC: Carboxymethylcellulose, Magnification-40X

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Distribution of P-glycoprotein in the brain

The present study investigated a heterogeneous P-gp distribution within the epileptic brain tissue. The P-gp-positive cells were observed in rat capillary blood vessels on the endothelial lining at BBB in the normal control rats. In contrast, in PB-resistant epileptic rat brain tissue, the P-gp was found to be diffusely distributed in small and large blood vessels of endothelial cells and membrane of neurons signifying high inflammation. No neuronal P-gp expression was seen with Fingolimod treatment in comparison with the L-NAME and verapamil groups, suggesting better regulation of P-gp with Fingolimod [Figure 5].

Oxidative stress parameters

The oxidative stress markers such as superoxide dismutase (SOD), reduced glutathione, and catalase were studied. The PTZ-PB-resistant group showed a significant decrease in these parameters when compared with normal saline-treated rats (P < 0.001). The treatment with Fingolimod reversed the decrease in SOD levels in a dose-dependent fashion with a highly significant increase at 1 mg/kg dose (P < 0.01), followed by 0.5 mg/kg (P < 0.05) in contrast to the PB-resistant group. However, the reduced glutathione levels were significantly reversed by Fingolimod (1 mg/kg), but not at 0.5 mg/kg. Both L-NAME and Fingolimod increased the SOD levels significantly (P < 0.01). The 1 mg/kg Fingolimod treatment significantly reversed the catalase activity in contrast to PTZ (P < 0.01) and PB-resistant rats (P < 0.05). The L-NAME reversed the defensive effect of Fingolimod by lowering the catalase activity in comparison with Fingolimod 1 mg/kg (P < 0.05). There were no considerable changes in SOD and reduced glutathione levels in the verapamil-treated group. The Fingolimod treatment (0.5 mg/kg) and the verapamil treatment led to increased catalase activity (P < 0.05) in contrast to PTZ-treated rats, as shown in [Figure 4].

Neuronal injury score

The neuronal injury was seen in the dentate gyrus of the brain [Figure 6]. The control rats showed regular neurons with no injury or rare isolated apoptotic neurons having a neuronal injury score of 0.5. The PTZ and PB-resistant groups showed a diffuse neuronal injury score of 3.5–4. The treatment with Fingolimod reduced the neuronal injury score in a dose-dependent approach. A highly significant reduction was observed at 1 mg/kg dose (P < 0.001), followed by 0.5 mg/kg dose (P < 0.05), while differentiating to PB-resistant rats. The direct P-gp inhibitor also reduced the neuronal injury score (P < 0.05) in comparison with the PB-resistant group. However, an increased neuronal injury score was observed in the combination group of L-NAME and Fingolimod (P < 0.01).
Figure 6: Effect of different treatments on neuronal damage score *P<0.05, **P < 0.01, ***P < 0.001 versus control rats; #P < 0.05, ###P<0.001 versus pentylenetetrazol–phenobarbital group. PTZ: Pentylenetetrazol, PB: Phenobarbital, Magnification - 40X

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 » Discussion Top

The problem of drug resistance is a major challenge for the treating physicians as well as patients with epilepsy despite the availability of newer AEDs. To understand the underlying mechanism of drug resistance, we validated an adapted protocol of drug-resistant epilepsy which compromises both seizure and drug effect to cause drug resistance in rodents.[11] This model could be used for the prevention of drug-resistant epilepsy and to screen for novel AEDs. PB has been extensively used for the management of tonic–clonic seizures in developing countries. The main reason behind PB tolerance is pharmacokinetics, owing to its fast metabolism rate.[12]

It is known that P-gp upregulation in pharmacoresistant epilepsy results in a decreased therapeutic concentration of AED in the brain. Fingolimod, a sphingosine-1-phosphate agonist, has been known for its effective anti-inflammatory properties in patients with multiple sclerosis and animal models of neurodegenerative disease.[13],[14] Therefore, it provides a suitable target to study its effect on P-gp regulation and, hence, AED concentration in the brain of PB-resistant epileptic rats. A study showed that add-on treatment of verapamil (a known P-gp transporter) can reduce seizure frequency in children with drug-resistant epilepsy.[15] Another study suggested that L-NAME decreased the anti-seizure effect of aripiprazole in a PTZ as well as an electroshock model of epilepsy.[16]

The histologic studies confirmed BBB damage due to extended seizures through the accumulation of Evan's blue dye in the rat brain.[17] Fingolimod was found to enhance BBB stability by stimulating the astrocytic release of granulocyte and macrophage colony-stimulating factor.[18]

The upregulation of cytokines receptors activates the intracellular signaling pathways, leading to the propagation of seizures through activating NMDA receptor, inhibiting the reuptake of glutamate by astrocytes, and increasing TNF-a production.[19],[20],[21] Fingolimod treatment was found to suppress cytokine-induced inflammation.[22]

Temporal lobe epilepsy is associated with disturbed BDNF signaling, which contributes to autonomic dysfunction and impaired cerebral autoregulation.[23] The serum BDNF is more often found to be higher in patients with epilepsy.[24] A study showed that the administration of Fingolimod in seizure-prone rats promoted the release of neurotrophic factors.[25]

The present study shows the elevated distribution of P-gp in the endothelial lining of BBB and neurons supported by both experimental and clinical evidence.[26],[27] The endothelin–sphingolipid pathway regulates the P-gp activity and helps in improving the drug deliverance to the brain; therefore, Fingolimod was the suitable drug of choice.

The increased oxidative stress can be one of many causes for altered P-gp expression in resistant rats. A recent study also indicated the antioxidant role of Fingolimod in Parkinson's disease.[28] Thus, Fingolimod provided a useful intervention strategy by reducing oxidative stress-induced cell injury. A marked neuron loss is pragmatic in the murine model of temporal lobe epilepsy.[29] Fingolimod is considered a therapeutic option in neuropsychiatric conditions since it helps in hippocampal neurogenesis.[30]

 » Conclusion Top

The augmented P-gp expression contributes to resistance toward AEDs in patients with epilepsy. Therefore, a novel strategy targeting the regulation of P-gp may give way to enhanced penetration of AEDs in the brain. However, the need of specific data to clearly understand the functionality of P-gp-mediated transport through BBB and prediction of pharmacokinetic–pharmacodynamic relationships of AEDs, which are P-gp substrates, should be recognized.

Financial support and sponsorship

The authors are thankful to the Indian Council of Medical Research, New Delhi, India, for providing financial assistance as a Junior Research Fellowship (3/1/3/JRF-2014/HRD-49) to Nitika Garg under the guidance of Prof. Bikash Medhi, for carrying out this research work.

Conflicts of interest

There are no conflicts of interest.

 » References Top

Sharma S, Puttachary S, Thippeswamy T. Glial source of nitric oxide in epileptogenesis: A target for disease modification in epilepsy. J Neurosci Res 2019;97:1363-77.  Back to cited text no. 1
Miller DS. Regulation of P-glycoprotein and other ABC drug transporters at the blood-brain barrier. Trends Pharmacol Sci 2010;31:246-54.  Back to cited text no. 2
Sisodiya SM, Martinian L, Scheffer GL, van der Valk P, Scheper RJ, Harding BN, et al. Vascular colocalization of P-glycoprotein, multidrug-resistance associated protein 1, breast cancer resistance protein and major vault protein in human epileptogenic pathologies. Neuropathol Appl Neurobiol 2006;32:51-63.  Back to cited text no. 3
Krishna R, St-Louis M, Mayer LD. Increased intracellular drug accumulation and complete chemosensitization achieved in multidrug-resistant solid tumors by co-administering valspodar (PSC 833) with sterically stabilized liposomal doxorubicin. Int J Cancer 2000;85:131-41.  Back to cited text no. 4
Liang XJ, Aszalos A. Multidrug transporters as drug targets. Curr Drug Targets 2006;7:911-21.  Back to cited text no. 5
Hartz AM, Bauer B, Fricker G, Miller DS. Rapid regulation of P-glycoprotein at the blood-brain barrier by endothelin-1. Mol Pharmacol 2004;66:387-94.  Back to cited text no. 6
Wang X, Hawkins BT, Miller DS. Activating PKC-β1 at the blood-brain barrier reverses induction of P-glycoprotein activity by dioxin and restores drug delivery to the CNS. J Cereb Blood Flow Metab 2011;31:1371-5.  Back to cited text no. 7
Cannon RE, Peart JC, Hawkins BT, Campos CR, Miller DS. Targeting blood-brain barrier sphingolipid signaling reduces basal P-glycoprotein activity and improves drug delivery to the brain. Proc Natl Acad Sci U S A 2012;109:15930-5.  Back to cited text no. 8
Killestein J, Rudick RA, Polman CH. Oral treatment for multiple sclerosis. Lancet Neurol 2011;10:1026-34.  Back to cited text no. 9
De Sarro A, Naccari F, De Sarro G. Enhanced susceptibility of pentylenetetrazole kindled mice to quinolone effects. Int J Antimicrob Agents 1999;12:239-44.  Back to cited text no. 10
Jing X, Liu X, Wen T, Xie S, Yao D, Liu X, et al. Combined effects of epileptic seizure and phenobarbital induced overexpression of P-glycoprotein in brain of chemically kindled rats. Br J Pharmacol 2010;159:1511-22.  Back to cited text no. 11
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  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]


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