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. 2023 Sep-Oct;55(5):307-314.
doi: 10.4103/ijp.ijp_100_23.

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

Nitika Garg  1 , Rupa Joshi  2 , Alka Bhatia  3 , Seema Bansal  4 , Amitava Chakrabarti  1 , Ajay Prakash  1 , Biman Saikia  5 , Manish Modi  6 , Bikash Medhi  1
Affiliations

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

Nitika Garg et al. Indian J Pharmacol. 2023 Sep-Oct.

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; P-glycoprotein; neuroprotection; phenobarbital resistance; refractory epilepsy.

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Conflict of interest statement

None

Figures

Figure 1
Figure 1
Effect of different treatments on pentylenetetrazol-induced seizures in resistant rats. PTZ: Pentylenetetrazol, PB: Phenobarbital
Figure 2
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
Figure 3
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
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
Figure 5
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
Figure 6
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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