- Email: [email protected]
Altered plasma prostaglandin E2 levels in epilepsy and in response to antiepileptic drug monotherapy
Prostaglandins, Leukotrienes and Essential Fatty Acids 153 (2020) 102056
Contents lists available at ScienceDirect
Prostaglandins, Leukotrienes and Essential Fatty Acids journal homepage: www.elsevier.com/locate/plefa
Original research article
Altered plasma prostaglandin E2 levels in epilepsy and in response to antiepileptic drug monotherapy
T
Chitra Rawata,b, Shivangia,c, Suman Kushwahad, Sangeeta Sharmad, Achal K Srivastavae, ⁎ Ritushree Kukretia,b, a
Institute of Genomics and Integrative Biology (IGIB), Council of Scientific and Industrial Research (CSIR), Mall Road, Delhi 110007, India Academy of Scientific and Innovative Research (AcSIR), Council of Scientific and Industrial Research (CSIR), Delhi 110007, India c Department of Biotechnology, Delhi Technological University, Shahbad Daulatpur, Main Bawana Road, Delhi 110042, India d Institute of Human Behavior & Allied Sciences (IHBAS), Dilshad Garden, Delhi 110095, India e Department of Neurology, All India Institute of Medical Sciences, Ansari Nagar, Delhi 110029, India b
A R T I C LE I N FO
A B S T R A C T
Keywords: Arachidonic acid Carbamazepine Epilepsy Phenytoin Prostaglandin E2 (PGE2) Valproate
Prostaglandin E2 (PGE2), a physiologically active lipid compound, is increased in several diseases characterized by chronic inflammation. To determine its significance in epilepsy-associated inflammation and response to antiepileptic drug (AED), we evaluated the plasma PGE2 (median, pg/ml) levels in drug-free patients with epilepsy (N = 34) and patients receiving AED monotherapy (N = 55) in addition to that in healthy controls (N = 34). When compared to controls, plasma PGE2 levels were significantly elevated in all drug-free patients independent of the type of epilepsy (137.2 versus 475.7 pg/ml, p < 0.0001). Among the patients receiving AED monotherapy, only valproate responders showed a significant decrease compared to both drug-free patients (232.1 versus 475.7 pg/ml, p < 0.01) as well as valproate non-responders (232.1 versus 611.9 pg/ml, p < 0.0001). Both responders and non-responders on phenytoin or carbamazepine monotherapy had elevated PGE2 levels similar to drug-free patients. In addition, no difference was observed in plasma profiles of PGE2 precursor, arachidonic acid among the groups. Our work presents the clinical evidence of the association between plasma PGE2 levels and valproate efficacy in patients with epilepsy.
AA, AED, AUC, BBB, BMI, CBZ, COX-2, CSF, ELISA, FDR, HETE, ILAE, LC-MS, LOX, NSAID, OTC, PGE2,
arachidonic acid; antiepileptic drug; area under the curve; blood-brain barrier; body mass index; carbamazepine; cyclooxygenase-2; cerebrospinal fluid; enzyme-linked immunosorbent assay; false discovery rate; hydroxyeicosatetraenoic acids; International League Against Epilepsy; liquid chromatography-tandem mass spectrometry; lipoxygenase; non-steroidal anti-inflammatory drug; over-the-counter; prostaglandin E2;
PHT, PWE, ROC, TDM, VA,
phenytoin; patients with epilepsy; receiver operating characteristic; therapeutic drug monitoring; valproate
1. Introduction Epilepsy, a common neurological disease characterized by recurrent seizures, affects around 69 million people worldwide [1] including 6–7 million cases from India [2]. The currently available symptomatic antiepileptic drug (AED) therapy shows significant inter-patient variability in response towards it with 30% of the cases being refractory [3]. One reason behind this refractoriness is the lack of knowledge of the underlying pathophysiology of the disease progression. Studies investigating the molecular mechanisms behind epileptogenesis revealed inflammation as a contributing factor as well as a consequence of
⁎ Corresponding author at Genomics and Molecular Medicine Unit, Institute of Genomics and Integrative Biology (IGIB), Council of Scientific and Industrial Research (CSIR), Mall Road, Delhi 110007, India. E-mail addresses: [email protected], [email protected] (R. Kukreti).
https://doi.org/10.1016/j.plefa.2020.102056 Received 19 August 2019; Received in revised form 18 January 2020; Accepted 21 January 2020 0952-3278/ © 2020 Elsevier Ltd. All rights reserved.
Prostaglandins, Leukotrienes and Essential Fatty Acids 153 (2020) 102056
C. Rawat, et al.
epilepsy. These patients were further followed-up at 2nd, 4th, 8th, and 12th months from the date of enrolment and were evaluated for seizure control, compliance to medications, side effects, and therapeutic drug monitoring (TDM) which included monitoring of drug, dose and serum drug levels. Patients who remained seizure-free on AED monotherapy in the previous 10 months of one-year study duration, after attaining steady state drug levels in the initial two months of study were categorized as responders and those who had at least 3 seizures in the same duration were categorized as non-responders. Besides, 34 newly-diagnosed drug-free PWE were also enrolled with similar ethnicity and selection criteria [14]. For seizure diagnosis, seizure types, and epilepsy classification, guidelines of International League Against Epilepsy (ILAE) 1981 and 1989 were followed [15,16]. The study protocol was approved by institutional ethics committee. Diagnosis and treatment were performed by experienced neurologists. A cohort of 34 ethnicitymatched unrelated healthy individuals falling within the same age range, with no seizure history or any other chronic medical ailment, was also enrolled for the study after obtaining informed consent. The study design also ensured exclusion of otherwise healthy subjects on any medication including Over-The-Counter (OTC) drugs.
seizure and epilepsy development [4]. Release of several inflammatory molecules such as cytokines, chemokines, prostaglandins, etc. by brain, brain endothelial cells and peripheral blood cells were often found to affect neuronal function and excitability [5]. Brain accumulation of arachidonic acid (AA), a polyunsaturated fatty acid, as a result of seizure activity in in vivo seizure models is widely established in the literature [6]. AA is metabolized by two pathways: lipoxygenase (LOX) pathway which synthesises the hydroxyeicosatetraenoic acids (HETEs) and leukotrienes, and cyclooxygenase (COX) pathway which synthesises the prostaglandins and thromboxanes. While the leukotriene metabolites of the LOX pathway were not detectable in the brain tissue samples from drug-resistant patients with epilepsy (PWE) [7], the HETE molecules of the same pathway were found to remain unaltered in the plasma and hippocampus following seizures in kainic acid-induced epilepsy models [8]. In contrast, the COX pathway metabolite, prostaglandin E2 (PGE2), derived by the enzyme COX-2, has received much attention over the past two decades due to its involvement in seizure generation and epilepsy development [9]. Such evidences indicate a specific role of COX-2-mediated inflammation in epilepsy. Increased expression of COX-2 has been reported in different cells within the brain of rodent models following a convulsive challenge leading to increased production of prostaglandins, majorly PGE2, further aggravating seizure severity [10,11]. Mounting evidences also reported that increased biosynthesis of PGE2 following seizure may upregulate the multidrug efflux transporter, P-glycoprotein (P-gp) at the blood-brain barrier (BBB) causing increased efflux of the prescribed AEDs into the blood stream, therefore leading to poor efficacy [12]. Blocking this undesired increase in PGE2 levels may, therefore, be beneficial to treat epilepsy and to increase AED efficacy. It is possible that the prescribed AEDs have a potential effect on the biosynthesis of PGE2, therefore, may reduce neuroinflammation in PWE. Since neuroinflammation comprises the activation of not only the brain cells but also the circulating peripheral immune cells, evaluating plasma PGE2 levels in epilepsy as a biomarker of AED response may, therefore, help in understanding the mechanism of action of these drugs and their efficacy. The current study is designed to examine the concentration of PGE2 in the plasma of PWE. We compared the plasma PGE2 profiles of newlydiagnosed drug-free patients with that of healthy subjects. Furthermore, we examined the PGE2 profiles of patients receiving three different AED monotherapies, phenytoin (PHT), carbamazepine (CBZ) and valproate (VA) and compared them with that of healthy subjects as well as drugfree patients. In addition, the plasma levels of the PGE2 precursor, AA were also measured in the subjects from different groups for a better understanding and identification of the responsible biological pathway.
2.2. Serum drug quantitation Serum drug levels of the prescribed AEDs were assessed in Autoanalyzer from Logitech Pvt. Ltd. (Model Echo) using CEDIA ® II assay kits by Microgenics Corporation, Fremont, USA. The serum drug levels of each patient at different follow-ups were averaged over a period before correlating with response.
2.3. Plasma prostaglandin E2 (PGE2) and arachidonic acid (AA) measurement Approximately 6–8 ml of whole blood samples were collected from all the subjects in Vacuette EDTA Tubes (Greiner Bio-One, USA). Samples from patients receiving AED monotherapy were collected after the completion of one-year follow-up period while that from drug-free patients were collected prior to initiation of any AED treatment regimen. All the samples were collected in predetermined daytime hours and from only those patients who experienced their last seizure at least 72 h before the time of collection to avoid interference of recent seizures on PGE2 levels so that the outcome of AED therapy can be studied. Plasma was separated from the whole blood sample using centrifugation at 1400 rpm for 6 min and collected in fresh tubes. In order to measure the plasma PGE2 levels of the enrolled subjects, a PGE2 measurement assay was performed on the collected plasma samples using DetectX Prostaglandin E2 Enzyme Immunoassay kit (Arbor Assays, Ann Arbor, MI, USA) according to manufacturer's instructions. For measuring plasma AA levels, Human Arachidonic Acid (AA) ELISA kit (Abbkine Scientific Co., Ltd., China) was used as per the directed protocol.
2. Materials and methods 2.1. Study subjects The study involved 55 PWE of North Indian ethnicity receiving AED monotherapy who were enrolled and followed-up at the Outpatient Department of Neurology, Institute of Human Behaviour and Allied Sciences (IHBAS), Delhi, India after obtaining informed consent. The inclusion criteria were as follows: the patients were >5 years of age and were receiving one of the three AEDs, i.e., PHT, CBZ or VA monotherapy. The exclusion criteria were as follows: patients having regular consumption of the COX-2-inhibiting drugs, nonsteroidal antiinflammatory drugs (NSAIDs) or performing regular vigorous physical activity defined by the Centers for Disease Control and Prevention [13]; patients with gross neurological deficits such as mental retardation and/or motor deficits; with imaging abnormalities including the presence of a tumour, tuberculoma, multiple neurocysticercosis, vascular malformations, and atrophic lesions; with severe hepatic or renal disorders and diabetes mellitus; and the cases of pregnant women with
2.4. Statistical analysis Data was presented as median (interquartile range). Kruskal-Wallis with post-hoc Dunn test adjusted by the Benjamini-Hochberg false discovery rate (FDR) method was used to compare the PGE2 and AA levels between test groups and control groups. Boxplot construction and statistical analyses were performed using GraphPad Prism 8. p < 0.05 was considered statistically significant. Additionally, receiver operating characteristic (ROC) analysis was performed to assess the diagnostic performance of plasma PGE2 levels using the pROC package for R.
2
Prostaglandins, Leukotrienes and Essential Fatty Acids 153 (2020) 102056
C. Rawat, et al.
Table 1 Demographic and clinical characteristics of patients receiving AED monotherapy. Demographic and clinical characteristics
PHT responders
PHT non-responders
CBZ responders
CBZ non-responders
VA responders
VA non-responders
N Male/female ratio
7 5/2
6 5/1
10 5/5
9 4/5
15 9/6
8 4/4
Age (years), median (IQR) Age at onset (years), median (IQR) BMI (kg/m2), median (IQR) Seizure type (N) Focal Generalized
22.0 (17.5–34.0) 16.0 (13.5–18.5) 22.0 (19.3–26.0)
22.5 (17.5–23.8) 9.5 (2.5–14.3) 20.9 (19.5–22.9)
23.0 (22.3–28.3) 15.5 (11.3–20.0) 20.1 (18.9–23.7)
21.0 (18.0–22.0) 9.0 (3.8–11.8) 20.8 (19.0–20.9)
23.0 (19.5–26.0) 13.0 (12.0–16.0) 21.9 (19.1–25.4)
20.0 (17.0–28.0) 13.0 (7.0–17.0) 19.2 (18.0–22.1)
5 2
3 3
8 2
6 3
– 15
– 8
Epilepsy type (N) Idiopathic epilepsy Symptomatic epilepsy Cryptogenic epilepsy
1 1 5
– 1 5
2 – 8
2 – 7
15 – –
8 – –
Drug dose [mg/day] Median (IQR)
200 (200–275)
275 (250–300)
600 (425–800)
750 (475–800)
800 (400–1000)
800 (800–1050)
4.9 (4.3–5.9)
5.0 (4.4–5.6)
12.8 (8.6–13.1)
13.8 (13.3–16.2)
16.3 (11.4–17.5)
16.7 (15.8–20.4)
14.6 (12.3–14.2)
15.5 (14.9–21.9)
7.0 (6.7–7.3)
7.0 (5.9–8.2)
83.7 (76.1–103.4)
83.0 (72.2–96.2)
2.7 (2.2–2.7)
2.9 (2.8–4.8)
0.6 (0.6–0.8)
0.5 (0.4–0.5)
5.3 (4.9–10.1)
5.3 (4.6–6.5)
Dosage [(mg/day)/kg] Median (IQR) Serum drug level [mg/L] Median (IQR) Dose corrected serum drug level [(mg/L)/[(mg/ day)/kg]] Median (IQR)
N: number of samples; IQR: interquartile range; BMI: body mass index; PHT: phenytoin; CBZ: carbamazepine; VA: valproate.
3. Results
high diagnostic performance (Supplementary Fig. 1a).
3.1. Subject characteristics
3.3. Plasma PGE2 levels: drug response
A total of 123 subjects including 34 healthy individuals referred to as “Healthy controls” and 89 PWE, aged 10–48 years, were recruited in the study. Of the 89 patients, 34 newly-diagnosed drug-free patients were assigned to the “Epilepsy_All” group who were further categorized into different epilepsy subtypes, idiopathic epilepsy (N = 13), symptomatic epilepsy (N = 12) and cryptogenic epilepsy (N = 9) (Supplementary Table 1). The rest 55 patients were receiving different AED monotherapies and were segregated on the basis of drug and drug response: “PHT responders” (N = 7), “PHT non-responders” (N = 6), “CBZ responders” (N = 10), “CBZ non-responders” (N = 9), “VA responders” (N = 15), and “VA non-responders” (N = 8) (Table 1). There were no significant differences in drug dose and serum drug levels between the responders and the non-responders. PGE2 levels were measured in the plasma samples of all the subjects and were compared with their demographic characteristics. Gender, age and body mass index (BMI) appeared to have no effect on PGE2 levels.
After the completion of one-year follow-up period, patients receiving AED monotherapy were segregated on the basis of their differential drug response and there plasma PGE2 levels were compared with that of “Healthy controls” and the drug-free “Epilepsy_All” group (Table 2). Responders and non-responders of PHT and CBZ had significantly higher PGE2 levels than the “Healthy controls”, however, no difference was observed when compared with the “Epilepsy_All” group (Fig. 2A and B). In contrast, “VA responders” were found to have significantly lower PGE2 levels than the “Epilepsy_All” (232.1 pg/ml versus 475.7 pg/ml, p < 0.01) and the “VA non-responders” (232.1 pg/ ml versus 611.9 pg/ml, p < 0.0001) (Fig. 2C). “VA responders” and “Healthy controls” had no statistically significant difference between their PGE2 levels. The comparisons of “VA responders vs Drug-free” and “VA responders vs VA non-responders” using ROC analysis displayed an AUC of 0.847 and 0.933, respectively (Supplementary Fig. 1b and c), again showing a high diagnostic performance of plasma PGE2 levels in PWE.
3.2. Plasma PGE2 levels: epilepsy versus controls 3.4. Plasma AA levels Plasma PGE2 levels of drug-free patients of the “Epilepsy_All” group were compared with that of “Healthy controls” (Table 2). The PGE2 levels were significantly higher in “Epilepsy_All” than in “Healthy controls” (475.7 pg/ml versus 137.2 pg/ml, p < 0.0001) (Fig. 1A). Upon segregation of “Epilepsy_All” to different epilepsy subtypes, PGE2 levels remained significantly elevated (p < 0.001) in each of the three groups, idiopathic, symptomatic and cryptogenic epilepsy (Fig. 1B). There were no statistically significant differences among the patients of these subtypes and therefore, further analysis was performed without the segregation of the “Epilepsy_All” group into the epilepsy subtypes. The ROC analysis between the “Epilepsy_All” and “Healthy controls” groups showed an area under the curve (AUC) of 0.942 indicating a
For measuring the plasma AA levels, a subset of 16 subjects from the “Healthy controls” and 16 subjects from the drug-free “Epilepsy_All” were randomly selected along with the 55 patients receiving different AED treatments. The ELISA assay did not reveal any significant difference in AA levels among the different groups (Supplementary Table 2). 4. Discussion Currently, clinical evidences suggest the involvement of various drug-metabolizing enzymes (CYPs), drug transporters (ABC transporters) and drug target genes (ion-channels in brain) in determining AED 3
Prostaglandins, Leukotrienes and Essential Fatty Acids 153 (2020) 102056
C. Rawat, et al.
difference was observed among the three epilepsy subtypes, idiopathic, symptomatic and cryptogenic epilepsy. Drug response was found to have no association with the plasma PGE2 levels of patients receiving PHT or CBZ monotherapy. Both responders and non-responders receiving PHT or CBZ monotherapy had elevated levels of PGE2 than healthy subjects. However, “VA responders” had significantly lower PGE2 levels than “VA non-responders” as well as drug-free patients. Upon measuring the plasma levels of the PGE2 precursor molecule, AA, no difference was observed among the groups. To examine the association of epilepsy with PGE2 levels, we compared the plasma PGE2 levels in drug-free PWE of the “Epilepsy_All” group with that of “Healthy controls” and found a significant increase in the patients. The increase was independent of the type of epilepsy. Several studies reported a key role of prostaglandins in causing seizuremediated neuroinflammation. Such reports demonstrated induction of COX-2 mRNA and protein expression in different rodent brain tissues such as hippocampus, cerebral cortex, striatum, brain stem, cerebellum, and nucleus basalis following seizure induction [18–21]. Such studies reported that COX-2 induction following seizure activates the prostaglandin signalling, consequently triggering secondary damage to the brain and aggravating the disease severity. It was proposed that elevated levels of PGE2 bind to the G protein-coupled receptor, EP1, leading to increased influx of calcium ions, in turn enhancing glutamate release presynaptically and causing seizure generation [22,23]. PGE2 production following seizures was also observed to stimulate neuronal loss in in vivo models [24,25]. Furthermore, co-administration of PGE2 with subconvulsant dose of pentylenetetrazol-generated seizures whereas administration of PGE2 antibodies attenuated the seizures in rats, suggesting the involvement of PGE2 in triggering seizure and governing its threshold [11]. Our study also assessed the plasma PGE2 levels of patients receiving AED monotherapy. “VA responders” were observed to have significantly lower levels of PGE2 than “VA non-responders” as well as the drug-free “Epilepsy_All” group. Our findings corresponds to a previous report by Tang et al. [26] which reported downregulation of the COX-2 mRNA levels in a small population of children with epilepsy responding to VA compared to drug-free patients, however, no difference was observed in the non-responders. VA, a broad spectrum AED, had also been reported to downregulate COX-2 in in vitro and in vivo studies further causing reduced biosynthesis of PGE2 [27–29]. Rao et al. [28] and Chuang et al. [29] reported that the VA-mediated COX-2 downregulation is attributed to the reduced binding of the transcription factor, NF-κB to COX-2 promoter, ultimately reducing PGE2 production in the brain. However, our study found VA-mediated PGE2 reduction to be associated with drug efficacy. COX-2 regulates the multidrug efflux transporter, P-gp by producing increased levels of PGE2, thereby causing increased efflux of the prescribed AED/s resulting in poor
Table 2 Plasma PGE2 levels in healthy controls, drug-free patients and patients receiving AED monotherapy. Sample groups
N
PGE2 levels (pg/ml) median (IQR)
pValue w.r.t. healthy controls
pValue w.r.t. Epilepsy_All
Healthy controls
34
137.2 (106.5–175.6)
–
<0.0001
Drug-free Epilepsy_All
34
<0.0001
–
Idiopathic epilepsy
13
<0.0001
–
Symptomatic epilepsy Cryptogenic epilepsy
12
475.7 (331.2–621.0) 489.3 (369.8–567.4) 412.2 (343.8–578.3) 473.3 (319.4–923.9)
<0.0001
–
0.0002
–
0.0001
0.8502
0.0016
0.9742
0.0011
0.9072
0.0011
0.9072
0.0827
0.0048
<0.0001
0.3566
9
PHT Responders
7
Non-responders
6
CBZ Responders
10
Non-responders
9
VA Responders
15
Non-responders
8
579.7 (358.2–793.3) 432.7 (353.8–610.7)
521.9 (257.1–813.0) 491.0 (336.2–605.4)
232.1 (190.2–281.6) 611.9 (509.0–789.1)
N: number of samples; IQR: interquartile range; w.r.t.: with respect to; PHT: phenytoin; CBZ: carbamazepine; VA: valproate. Bold signifies p < 0.05. pvalue was calculated using Kruskal-Wallis with post-hoc Dunn test adjusted by the Benjamini-Hochberg FDR method.
efficacy [17]. However, such studies remained inconclusive in addressing pharmacoresistance in epilepsy indicating a gap in the current pharmacological research. Inflammation, a crucial factor involved in epilepsy, is often overlooked as a therapeutic target for managing the disease. Therefore, this study evaluated the levels of a key inflammatory mediator, PGE2 in the plasma samples of drug-free PWE and in patients receiving AED monotherapy and compared them with that of healthy subjects. Drug-free PWE were observed to have significantly higher levels of plasma PGE2 than the healthy subjects. No
Fig 1.. Comparison of plasma PGE2 levels (***p < 0.001): A. “Healthy controls” vs all newly-diagnosed drugfree patients with epilepsy; B. “Healthy controls” vs newly-diagnosed drug-free patients with different epilepsy subtypes. p value was calculated using Kruskal-Wallis with post-hoc Dunn test adjusted by the BenjaminiHochberg FDR method.
4
Prostaglandins, Leukotrienes and Essential Fatty Acids 153 (2020) 102056
C. Rawat, et al.
efficacy [12]. Inhibiting COX-2 may, therefore, be helpful in achieving enhanced efficacy of prescribed AEDs. In vivo and in vitro data revealed prevention of glutamate- or status epilepticus-induced P-gp upregulation by COX-2 inhibitors in rodent and human brain capillary endothelial cells [30–32]. Furthermore, sub-chronic COX-2 inhibition decreased Pgp expression by reducing PGE2 synthesis in chronic epileptic rats enhancing uptake of PHT in brain [33]. To investigate the effect of COX-2 inhibition on drug efficacy, Schlichtiger et al. studied the treatment outcome of the selective COX-2 inhibitor, celecoxib in phenobarbitaltreated responder and non-responder epileptic rats [34]. Celecoxib treatment significantly diminished P-gp expression as well as seizure frequency in both responders and non-responders revealing the potential adjunctive role of COX-2 inhibitors in increasing AED efficacy via Pgp downregulation. Other NSAIDs such as rofecoxib, aspirin, ibuprofen, indomethacin, metamizole, paracetamol and piroxicam also potentiated the anticonvulsant activity of AEDs such as tiagabine, VA and PHT in mice [35,36]. Clinical studies also reported seizure reduction in patients receiving AED therapy when were prescribed the NSAID, aspirin in adjunction [37,38]. Such reports indicate that production of PGE2 by COX-2 plays a key role in deciding the AED treatment outcome. Since, “VA responders” in our study were found to have reduced plasma PGE2 levels, it suggests that lower biosynthesis of PGE2 by VA may cause downregulation of P-gp leading to enhanced efficacy in the responders. In regard to the differential response to the other two AEDs, PHT and CBZ, our study observed no variation in the plasma PGE2 levels between the responder and non-responder groups. Both the responders as well as non-responders on the two AED monotherapies had elevated plasma PGE2 levels compared to “Healthy Controls” suggesting no effect of the two drugs on PGE2 production. In addition to PGE2, we also measured the plasma levels of its precursor molecule, AA in the same patients to determine if the changes in PGE2 levels are COX-2 mediated or are the result of alterations in the upstream AA concentrations. Contrary to the preclinical in vivo reports revealing increased brain and plasma AA levels following seizure induction [8,39], we observed no difference between the PWE and healthy individuals. The failure to replicate the preclinical findings in the clinical samples suggests that the increase in the plasma PGE2 levels of the PWE is COX-2 mediated and is independent of any change in AA levels. However, it is also likely that the change in AA levels may be an acute phase event and may be observed for a narrow time duration. Our study recruited patients who experienced their last seizure at least 72 h prior to the sample collection to investigate the plasma profiles of patients on AED therapy independent of any recent seizure activity. This could be a probable reason for no variation in the AA levels among the groups. For a better understanding, plasma profiles of AA in the patients need to be investigated at different time-intervals following a seizure. Our study has some limitations. First, it is limited by a small sample size. The study is a pilot investigation for which a small sample size would yield greater scientific value relative to the expense of the immunoassays. However, to establish the robustness of the findings, future studies will require larger sample sizes. Secondly, our study lacks the data on the relevance of altered peripheral PGE2 levels for indicating the changes in the central levels (brain or cerebrospinal fluid, CSF levels) of the prostaglandin. In this regard, previous preclinical studies have reported contradictory findings. While Davidson et al. [40] reported increased PGE2 levels in both plasma and CSF in response to cytokines and their inducers and suggested enhanced cerebral entry of PGE2 from the peripheral circulation, Eskilsson et al. [41] found no significant relationship between the levels of PGE2 in plasma and in CSF suggesting that peripherally and centrally produced PGE2 are of distinct sources of origin. Therefore, future studies are further required to examine the extent of correlation between the peripheral and central PGE2 levels in clinical samples. Third, our study does not provide the data regarding the selectivity of altered plasma PGE2 in PWE. Though PGE2 is the major prostaglandin produced during inflammation [42],
Fig 2.. Comparison of plasma PGE2 levels between healthy controls, drug-free patients and patients receiving AED monotherapy (**p < 0.01, ***p < 0.001). A. Patients receiving phenytoin (PHT) monotherapy; B. Patients receiving carbamazepine (CBZ) monotherapy; C. Patients receiving valproate (VA) monotherapy. p value was calculated using Kruskal-Wallis with post-hoc Dunn test adjusted by the Benjamini-Hochberg FDR method. 5
Prostaglandins, Leukotrienes and Essential Fatty Acids 153 (2020) 102056
C. Rawat, et al.
References
COX pathway synthesises other eicosanoids, viz., PGI2, PGD2, PGF2, and TXA2 as well. To determine PGE2 selectivity, the profiles of these eicosanoids also need to be assessed in future. Fourth, we used enzymelinked immunosorbent assay (ELISA) instead of the high-throughput liquid chromatography-tandem mass spectrometry (LC-MS) technique. Though, LC-MS is a highly sensitive and specific analytical method, previous comparative studies have demonstrated similar findings from the two [43,44]. However, for the confirmation of the current findings, the same should be replicated using LC-MS in future. The strength of our study is the stringent selection criteria for recruiting patient samples and the presence of two control groups, viz., healthy subjects and drug-free patients for investigating the effect of the prescribed drug in patients receiving AED monotherapy. In conclusion, our study suggests that elevated plasma PGE2 is associated with epilepsy and its reduced levels are associated with response to VA. Therefore, plasma PGE2 levels may serve as a potential biomarker for determining drug response. To the best of our knowledge, our study is the first to report the clinical evidence of the association between plasma PGE2 levels and VA efficacy in PWE. Future work may emphasize on validating the findings in large sample-size population. Furthermore, molecular mechanisms underlying the role of COX-2 or PGE2 in AED efficacy needs to be explored to establish a cause-effect relationship between the two. In addition, investigating the effect of COX-2 inhibitors in adjunction to prescribed AEDs on drug efficacy in large-sample, randomized, controlled trials may provide information on the clinical application of COX-2 inhibitors for epilepsy management. Our work delivers preliminary data on plasma PGE2 levels as the potential biomarker of valproate efficacy in PWE.
[1] A.K. Ngugi, C. Bottomley, I. Kleinschmidt, J.W. Sander, C.R Newton, Estimation of the burden of active and life-time epilepsy: a meta-analytic approach, Epilepsia 51 (2010) 883–890. [2] M. Gourie-Devi, Epidemiology of neurological disorders in India: review of background, prevalence and incidence of epilepsy, stroke, Parkinson's disease and tremors, Neurol. India 62 (2014) 588–598. [3] S Herman, Intractable epilepsy: relapsing, remitting, or progressive? Epilepsy Curr. 10 (2010) 146–148. [4] A. Vezzani, J. French, T. Bartfai, T.Z Baram, The role of inflammation in epilepsy, Nat. Rev. Neurol. 7 (2011) 31–40. [5] A. Vezzani, B. Viviani, Neuromodulatory properties of inflammatory cytokines and their impact on neuronal excitability, Neuropharmacology 96 (2015) 70–82. [6] B.K. Siesjö, C.D. Agardh, F. Bengtsson, M.L Smith, Arachidonic acid metabolism in seizures, Ann. N Y Acad. Sci. 559 (1989) 323–339. [7] J. Rumià, F. Marmol, J. Sanchez, M. Carreño, N. Bargalló, T. Boget, L. Pintor, X. Setoain, E. Bailles, A. Donaire, E. Ferrer, P Puig-Parellada, Eicosanoid levels in the neocortex of drug-resistant epileptic patients submitted to epilepsy surgery, Epilepsy Res. 99 (2012) 127–131. [8] J.M. Post, S. Loch, R. Lerner, F. Remmers, E. Lomazzo, B. Lutz, L Bindila, Antiepileptogenic effect of subchronic palmitoylethanolamide treatment in a mouse model of acute epilepsy, Front. Mol. Neurosci. 11 (2018) 67. [9] C. Rawat, S. Kukal, U.R. Dahiya, R Kukreti, Cyclooxygenase-2 (COX-2) inhibitors: future therapeutic strategies for epilepsy management, J. Neuroinflamm. 16 (2019) 197. [10] T. Takemiya, K. Suzuki, H. Sugiura, S. Yasuda, K. Yamagata, Y. Kawakami, E Maru, Inducible brain COX-2 facilitates the recurrence of hippocampal seizures in mouse rapid kindling, Prostaglandins Other Lipid Mediat. 71 (2003) 205–216. [11] M.S. Oliveira, A.F. Furian, L.F. Royes, M.R. Fighera, N.G. Fiorenza, M. Castelli, P. Machado, D. Bohrer, M. Veiga, J. Ferreira, E.A. Cavalheiro, C.F Mello, Cyclooxygenase-2/PGE2 pathway facilitates pentylenetetrazol-induced seizures, Epilepsy Res. 79 (2008) 14–21. [12] H Potschka, Modulating P-glycoprotein regulation: future perspectives for pharmacoresistant epilepsies? Epilepsia 51 (2010) 1333–1347. [13] Centers for Disease Control and Prevention, Physical Activity, (2015) https://www. cdc.gov/physicalactivity/basics/adults/. [14] C. Rawat, S. Kushwaha, A.K. Srivastava, R Kukreti, Peripheral blood gene expression signatures associated with epilepsy and its etiologic classification, Genomics (2019). [15] Commission on Classification and Terminology of the International League Against Epilepsy, Proposal for revised classification of epilepsies and epileptic syndromes, Epilepsia 30 (1989) 389–399. [16] Commission on Classification and Terminology of the International League Against Epilepsy, Proposal for revised clinical and electroencephalographic classification of epileptic seizures, Epilepsia 22 (1981) 489–501. [17] W. Löscher, U. Klotz, F. Zimprich, D Schmidt, The clinical impact of pharmacogenetics on the treatment of epilepsy, Epilepsia 50 (2009) 1–23. [18] K. Yamagata, K.I. Andreasson, W.E. Kaufmann, C.A. Barnes, P.F Worley, Expression of a mitogen-inducible cyclooxygenase in brain neurons: regulation by synaptic activity and glucocorticoids, Neuron 11 (1993) 371–386. [19] J. Chen, T. Marsh, J.S. Zhang, S.H Graham, Expression of cyclo-oxygenase 2 in rat brain following kainate treatment, Neuroreport 6 (1995) 245–248. [20] J. Adams, Y. Collaço-Moraes, J de Belleroche, Cyclooxygenase-2 induction in cerebral cortex: an intracellular response to synaptic excitation, J. Neurochem. 66 (1996) 6–13. [21] V.L. Marcheselli, N.G. Bazan, Sustained induction of prostaglandin endoperoxide synthase-2 by seizures in hippocampus. Inhibition by a platelet-activating factor antagonist, J. Biol. Chem. 271 (1996) 24794–24799. [22] I. Nishihara, T. Minami, Y. Watanabe, S. Ito, O Hayaishi, Prostaglandin E2 stimulates glutamate release from synaptosomes of rat spinal cord, Neurosci. Lett. 196 (1995) 57–60. [23] N. Sang, J. Zhang, V. Marcheselli, N.G. Bazan, C Chen, Postsynaptically synthesized prostaglandin E2 (PGE2) modulates hippocampal synaptic transmission via a presynaptic PGE2 EP2 receptor, J. Neurosci. 25 (2005) 9858–9870. [24] K. Kawaguchi, R.W. Hickey, M.E. Rose, L. Zhu, J. Chen, S.H Graham, Cyclooxygenase-2 expression is induced in rat brain after kainate-induced seizures and promotes neuronal death in CA3 hippocampus, Brain Res. 1050 (2005) 130–137. [25] T. Takemiya, M. Maehara, K. Matsumura, S. Yasuda, H. Sugiura, K Yamagata, Prostaglandin E2 produced by late induced COX-2 stimulates hippocampal neuron loss after seizure in the CA3 region, Neurosci. Res. 56 (2006) 103–110. [26] Y. Tang, T.A. Glauser, D.L. Gilbert, et al., Valproic acid blood genomic expression patterns in children with epilepsy—a pilot study, Acta. Neurol. Scand. 109 (2004) 159–168. [27] F. Bosetti, G.R. Weerasinghe, T.A. Rosenberger, S.I Rapoport, Valproic acid downregulates the conversion of arachidonic acid to eicosanoids via cyclooxygenase-1 and -2 in rat brain, J. Neurochem. 85 (2003) 690–696. [28] J.S. Rao, R.P. Bazinet, S.I. Rapoport, H.J Lee, Chronic treatment of rats with sodium valproate downregulates frontal cortex NF-kappab DNA binding activity and COX-2 mRNA, Bipolar Disord. 9 (2007) 513–520. [29] Y.F. Chuang, H.Y. Yang, T.L. Ko, et al., Valproic acid suppresses lipopolysaccharideinduced cyclooxygenase-2 expression via MKP-1 in murine brain microvascular endothelial cells, Biochem. Pharmacol. 88 (2014) 372–383. [30] B. Bauer, A.M. Hartz, A. Pekcec, K. Toellner, D.S. Miller, H Potschka, Seizure-
Funding Subject recruitment was funded by Indian Council of Medical Research (ICMR), GAP0091 and the immunoassay experiments were funded by Council of Scientific and Industrial Research (CSIR), MLP1804. CRediT authorship contribution statement Chitra Rawat: Conceptualization, Writing - original draft, Methodology. : Methodology. Suman Kushwaha: Validation. Sangeeta Sharma: Resources. Achal K Srivastava: Validation. Ritushree Kukreti: Conceptualization, Writing - original draft, Data curation. Declaration of Competing Interest None of the authors has any competing interest to disclose. Acknowledgements The authors express their thanks to patients and their family members as well as healthy subjects for participating in the study. They gratefully acknowledge valuable scientific discussions and suggestions by Prof. Samir K. Brahmachari (CSIR-IGIB) and Prof. M. Gourie-Devi (Sir Ganga Ram Hospital). They are thankful to Dr. Nimesh G. Desai, Director, IHBAS and Dr. Anurag Agrawal, Director, IGIB for their support during project implementation. CR acknowledges University Grants Commission (UGC) for providing fellowship. Supplementary materials Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.plefa.2020.102056. 6
Prostaglandins, Leukotrienes and Essential Fatty Acids 153 (2020) 102056
C. Rawat, et al.
[31]
[32]
[33]
[34]
[35]
[36]
[37]
in Sturge-Weber syndrome, J. Child. Neurol. 26 (2011) 692–702. [38] E.I. Lance, A.K. Sreenivasan, T.A. Zabel, E.H. Kossoff, A.M Comi, Aspirin use in Sturge-Weber syndrome: side effects and clinical outcomes, J. Child. Neurol. 28 (2013) 213–218. [39] N.G. Bazan, D.L. Birkle, W. Tang, T.S Reddy, The accumulation of free arachidonic acid, diacylglycerols, prostaglandins, and lipoxygenase reaction products in the brain during experimental epilepsy, Adv. Neurol. 44 (1986) 879–902. [40] J. Davidson, H.T. Abul, A.S. Milton, D Rotondo, Cytokines and cytokine inducers stimulate prostaglandin E2 entry into the brain, Pflugers Arch. 442 (2001) 526–533. [41] A. Eskilsson, T. Matsuwaki, K. Shionoya, E. Mirrasekhian, J. Zajdel, M. Schwaninger, D. Engblom, A Blomqvist, Immune-Induced fever is dependent on local but not generalized prostaglandin E2 synthesis in the brain, J. Neurosci. 37 (2017) 5035–5044. [42] P. Ciceri, Y. Zhang, A.F. Shaffer, K.M. Leahy, M.B. Woerner, W.G. Smith, K. Seibert, P.C Isakson, Pharmacology of celecoxib in rat brain after kainate administration, J. Pharmacol. Exp. Ther. 302 (2002) 846–852. [43] C.E. Staatz, P.J. Taylor, S.E Tett, Comparison of an Elisa and an LC/MS/MS method for measuring tacrolimus concentrations and making dosage decisions in transplant recipients, Ther. Drug. Monit. 24 (2002) 607–615. [44] A.S. Gandhi, D. Budac, T. Khayrullina, R. Staal, G Chandrasena, Quantitative analysis of lipids: a higher-throughput LC-MS/MS-based method and its comparison to Elisa, Future Sci. OA 3 (2017) FSO157.
induced up-regulation of P-glycoprotein at the blood-brain barrier through glutamate and cyclooxygenase-2 signaling, Mol. Pharmacol. 73 (2008) 1444–1453. J. Avemary, J.D. Salvamoser, A. Peraud, J. Rémi, S. Noachtar, G. Fricker, H Potschka, Dynamic regulation of P-glycoprotein in human brain capillaries, Mol. Pharm. 10 (2013) 3333–3341. G. Zibell, B. Unkrüer, A. Pekcec, A.M. Hartz, B. Bauer, D.S. Miller, H Potschka, Prevention of seizure-induced up-regulation of endothelial P-glycoprotein by COX-2 inhibition, Neuropharmacology 56 (2009) 849–855. E.A. van Vliet, G. Zibell, A. Pekcec, J. Schlichtiger, P.M. Edelbroek, L. Holtman, E. Aronica, J.A. Gorter, H Potschka, COX-2 inhibition controls P-glycoprotein expression and promotes brain delivery of phenytoin in chronic epileptic rats, Neuropharmacology 58 (2010) 404–412. J. Schlichtiger, A. Pekcec, H. Bartmann, P. Winter, C. Fuest, J. Soerensen, H Potschka, Celecoxib treatment restores pharmacosensitivity in a rat model of pharmacoresistant epilepsy, Br. J. Pharmacol. 160 (2010) 1062–1071. A. Dhir, S.K. Kulkarni, Rofecoxib, a selective cyclooxygenase-2 (COX-2) inhibitor potentiates the anticonvulsant activity of tiagabine against pentylenetetrazol-induced convulsions in mice, Inflammopharmacology 14 (2006) 222–225. R. Kaminski, M. Kozicka, J. Parada-turska, M. Dziki, Z. Kleinrok, W.A. Turski, S.J Czuczwar, Effect of non-steroidal anti-inflammatory drugs on the anticonvulsive activity of valproate and diphenylhydantoin against maximal electroshock-induced seizures in mice, Pharmacol. Res. 37 (1998) 375–381. M.J. Bay, E.H. Kossoff, C.U. Lehmann, T.A. Zabel, A.M Comi, Survey of aspirin use
7
Contents lists available at ScienceDirect
Prostaglandins, Leukotrienes and Essential Fatty Acids journal homepage: www.elsevier.com/locate/plefa
Original research article
Altered plasma prostaglandin E2 levels in epilepsy and in response to antiepileptic drug monotherapy
T
Chitra Rawata,b, Shivangia,c, Suman Kushwahad, Sangeeta Sharmad, Achal K Srivastavae, ⁎ Ritushree Kukretia,b, a
Institute of Genomics and Integrative Biology (IGIB), Council of Scientific and Industrial Research (CSIR), Mall Road, Delhi 110007, India Academy of Scientific and Innovative Research (AcSIR), Council of Scientific and Industrial Research (CSIR), Delhi 110007, India c Department of Biotechnology, Delhi Technological University, Shahbad Daulatpur, Main Bawana Road, Delhi 110042, India d Institute of Human Behavior & Allied Sciences (IHBAS), Dilshad Garden, Delhi 110095, India e Department of Neurology, All India Institute of Medical Sciences, Ansari Nagar, Delhi 110029, India b
A R T I C LE I N FO
A B S T R A C T
Keywords: Arachidonic acid Carbamazepine Epilepsy Phenytoin Prostaglandin E2 (PGE2) Valproate
Prostaglandin E2 (PGE2), a physiologically active lipid compound, is increased in several diseases characterized by chronic inflammation. To determine its significance in epilepsy-associated inflammation and response to antiepileptic drug (AED), we evaluated the plasma PGE2 (median, pg/ml) levels in drug-free patients with epilepsy (N = 34) and patients receiving AED monotherapy (N = 55) in addition to that in healthy controls (N = 34). When compared to controls, plasma PGE2 levels were significantly elevated in all drug-free patients independent of the type of epilepsy (137.2 versus 475.7 pg/ml, p < 0.0001). Among the patients receiving AED monotherapy, only valproate responders showed a significant decrease compared to both drug-free patients (232.1 versus 475.7 pg/ml, p < 0.01) as well as valproate non-responders (232.1 versus 611.9 pg/ml, p < 0.0001). Both responders and non-responders on phenytoin or carbamazepine monotherapy had elevated PGE2 levels similar to drug-free patients. In addition, no difference was observed in plasma profiles of PGE2 precursor, arachidonic acid among the groups. Our work presents the clinical evidence of the association between plasma PGE2 levels and valproate efficacy in patients with epilepsy.
AA, AED, AUC, BBB, BMI, CBZ, COX-2, CSF, ELISA, FDR, HETE, ILAE, LC-MS, LOX, NSAID, OTC, PGE2,
arachidonic acid; antiepileptic drug; area under the curve; blood-brain barrier; body mass index; carbamazepine; cyclooxygenase-2; cerebrospinal fluid; enzyme-linked immunosorbent assay; false discovery rate; hydroxyeicosatetraenoic acids; International League Against Epilepsy; liquid chromatography-tandem mass spectrometry; lipoxygenase; non-steroidal anti-inflammatory drug; over-the-counter; prostaglandin E2;
PHT, PWE, ROC, TDM, VA,
phenytoin; patients with epilepsy; receiver operating characteristic; therapeutic drug monitoring; valproate
1. Introduction Epilepsy, a common neurological disease characterized by recurrent seizures, affects around 69 million people worldwide [1] including 6–7 million cases from India [2]. The currently available symptomatic antiepileptic drug (AED) therapy shows significant inter-patient variability in response towards it with 30% of the cases being refractory [3]. One reason behind this refractoriness is the lack of knowledge of the underlying pathophysiology of the disease progression. Studies investigating the molecular mechanisms behind epileptogenesis revealed inflammation as a contributing factor as well as a consequence of
⁎ Corresponding author at Genomics and Molecular Medicine Unit, Institute of Genomics and Integrative Biology (IGIB), Council of Scientific and Industrial Research (CSIR), Mall Road, Delhi 110007, India. E-mail addresses: [email protected], [email protected] (R. Kukreti).
https://doi.org/10.1016/j.plefa.2020.102056 Received 19 August 2019; Received in revised form 18 January 2020; Accepted 21 January 2020 0952-3278/ © 2020 Elsevier Ltd. All rights reserved.
Prostaglandins, Leukotrienes and Essential Fatty Acids 153 (2020) 102056
C. Rawat, et al.
epilepsy. These patients were further followed-up at 2nd, 4th, 8th, and 12th months from the date of enrolment and were evaluated for seizure control, compliance to medications, side effects, and therapeutic drug monitoring (TDM) which included monitoring of drug, dose and serum drug levels. Patients who remained seizure-free on AED monotherapy in the previous 10 months of one-year study duration, after attaining steady state drug levels in the initial two months of study were categorized as responders and those who had at least 3 seizures in the same duration were categorized as non-responders. Besides, 34 newly-diagnosed drug-free PWE were also enrolled with similar ethnicity and selection criteria [14]. For seizure diagnosis, seizure types, and epilepsy classification, guidelines of International League Against Epilepsy (ILAE) 1981 and 1989 were followed [15,16]. The study protocol was approved by institutional ethics committee. Diagnosis and treatment were performed by experienced neurologists. A cohort of 34 ethnicitymatched unrelated healthy individuals falling within the same age range, with no seizure history or any other chronic medical ailment, was also enrolled for the study after obtaining informed consent. The study design also ensured exclusion of otherwise healthy subjects on any medication including Over-The-Counter (OTC) drugs.
seizure and epilepsy development [4]. Release of several inflammatory molecules such as cytokines, chemokines, prostaglandins, etc. by brain, brain endothelial cells and peripheral blood cells were often found to affect neuronal function and excitability [5]. Brain accumulation of arachidonic acid (AA), a polyunsaturated fatty acid, as a result of seizure activity in in vivo seizure models is widely established in the literature [6]. AA is metabolized by two pathways: lipoxygenase (LOX) pathway which synthesises the hydroxyeicosatetraenoic acids (HETEs) and leukotrienes, and cyclooxygenase (COX) pathway which synthesises the prostaglandins and thromboxanes. While the leukotriene metabolites of the LOX pathway were not detectable in the brain tissue samples from drug-resistant patients with epilepsy (PWE) [7], the HETE molecules of the same pathway were found to remain unaltered in the plasma and hippocampus following seizures in kainic acid-induced epilepsy models [8]. In contrast, the COX pathway metabolite, prostaglandin E2 (PGE2), derived by the enzyme COX-2, has received much attention over the past two decades due to its involvement in seizure generation and epilepsy development [9]. Such evidences indicate a specific role of COX-2-mediated inflammation in epilepsy. Increased expression of COX-2 has been reported in different cells within the brain of rodent models following a convulsive challenge leading to increased production of prostaglandins, majorly PGE2, further aggravating seizure severity [10,11]. Mounting evidences also reported that increased biosynthesis of PGE2 following seizure may upregulate the multidrug efflux transporter, P-glycoprotein (P-gp) at the blood-brain barrier (BBB) causing increased efflux of the prescribed AEDs into the blood stream, therefore leading to poor efficacy [12]. Blocking this undesired increase in PGE2 levels may, therefore, be beneficial to treat epilepsy and to increase AED efficacy. It is possible that the prescribed AEDs have a potential effect on the biosynthesis of PGE2, therefore, may reduce neuroinflammation in PWE. Since neuroinflammation comprises the activation of not only the brain cells but also the circulating peripheral immune cells, evaluating plasma PGE2 levels in epilepsy as a biomarker of AED response may, therefore, help in understanding the mechanism of action of these drugs and their efficacy. The current study is designed to examine the concentration of PGE2 in the plasma of PWE. We compared the plasma PGE2 profiles of newlydiagnosed drug-free patients with that of healthy subjects. Furthermore, we examined the PGE2 profiles of patients receiving three different AED monotherapies, phenytoin (PHT), carbamazepine (CBZ) and valproate (VA) and compared them with that of healthy subjects as well as drugfree patients. In addition, the plasma levels of the PGE2 precursor, AA were also measured in the subjects from different groups for a better understanding and identification of the responsible biological pathway.
2.2. Serum drug quantitation Serum drug levels of the prescribed AEDs were assessed in Autoanalyzer from Logitech Pvt. Ltd. (Model Echo) using CEDIA ® II assay kits by Microgenics Corporation, Fremont, USA. The serum drug levels of each patient at different follow-ups were averaged over a period before correlating with response.
2.3. Plasma prostaglandin E2 (PGE2) and arachidonic acid (AA) measurement Approximately 6–8 ml of whole blood samples were collected from all the subjects in Vacuette EDTA Tubes (Greiner Bio-One, USA). Samples from patients receiving AED monotherapy were collected after the completion of one-year follow-up period while that from drug-free patients were collected prior to initiation of any AED treatment regimen. All the samples were collected in predetermined daytime hours and from only those patients who experienced their last seizure at least 72 h before the time of collection to avoid interference of recent seizures on PGE2 levels so that the outcome of AED therapy can be studied. Plasma was separated from the whole blood sample using centrifugation at 1400 rpm for 6 min and collected in fresh tubes. In order to measure the plasma PGE2 levels of the enrolled subjects, a PGE2 measurement assay was performed on the collected plasma samples using DetectX Prostaglandin E2 Enzyme Immunoassay kit (Arbor Assays, Ann Arbor, MI, USA) according to manufacturer's instructions. For measuring plasma AA levels, Human Arachidonic Acid (AA) ELISA kit (Abbkine Scientific Co., Ltd., China) was used as per the directed protocol.
2. Materials and methods 2.1. Study subjects The study involved 55 PWE of North Indian ethnicity receiving AED monotherapy who were enrolled and followed-up at the Outpatient Department of Neurology, Institute of Human Behaviour and Allied Sciences (IHBAS), Delhi, India after obtaining informed consent. The inclusion criteria were as follows: the patients were >5 years of age and were receiving one of the three AEDs, i.e., PHT, CBZ or VA monotherapy. The exclusion criteria were as follows: patients having regular consumption of the COX-2-inhibiting drugs, nonsteroidal antiinflammatory drugs (NSAIDs) or performing regular vigorous physical activity defined by the Centers for Disease Control and Prevention [13]; patients with gross neurological deficits such as mental retardation and/or motor deficits; with imaging abnormalities including the presence of a tumour, tuberculoma, multiple neurocysticercosis, vascular malformations, and atrophic lesions; with severe hepatic or renal disorders and diabetes mellitus; and the cases of pregnant women with
2.4. Statistical analysis Data was presented as median (interquartile range). Kruskal-Wallis with post-hoc Dunn test adjusted by the Benjamini-Hochberg false discovery rate (FDR) method was used to compare the PGE2 and AA levels between test groups and control groups. Boxplot construction and statistical analyses were performed using GraphPad Prism 8. p < 0.05 was considered statistically significant. Additionally, receiver operating characteristic (ROC) analysis was performed to assess the diagnostic performance of plasma PGE2 levels using the pROC package for R.
2
Prostaglandins, Leukotrienes and Essential Fatty Acids 153 (2020) 102056
C. Rawat, et al.
Table 1 Demographic and clinical characteristics of patients receiving AED monotherapy. Demographic and clinical characteristics
PHT responders
PHT non-responders
CBZ responders
CBZ non-responders
VA responders
VA non-responders
N Male/female ratio
7 5/2
6 5/1
10 5/5
9 4/5
15 9/6
8 4/4
Age (years), median (IQR) Age at onset (years), median (IQR) BMI (kg/m2), median (IQR) Seizure type (N) Focal Generalized
22.0 (17.5–34.0) 16.0 (13.5–18.5) 22.0 (19.3–26.0)
22.5 (17.5–23.8) 9.5 (2.5–14.3) 20.9 (19.5–22.9)
23.0 (22.3–28.3) 15.5 (11.3–20.0) 20.1 (18.9–23.7)
21.0 (18.0–22.0) 9.0 (3.8–11.8) 20.8 (19.0–20.9)
23.0 (19.5–26.0) 13.0 (12.0–16.0) 21.9 (19.1–25.4)
20.0 (17.0–28.0) 13.0 (7.0–17.0) 19.2 (18.0–22.1)
5 2
3 3
8 2
6 3
– 15
– 8
Epilepsy type (N) Idiopathic epilepsy Symptomatic epilepsy Cryptogenic epilepsy
1 1 5
– 1 5
2 – 8
2 – 7
15 – –
8 – –
Drug dose [mg/day] Median (IQR)
200 (200–275)
275 (250–300)
600 (425–800)
750 (475–800)
800 (400–1000)
800 (800–1050)
4.9 (4.3–5.9)
5.0 (4.4–5.6)
12.8 (8.6–13.1)
13.8 (13.3–16.2)
16.3 (11.4–17.5)
16.7 (15.8–20.4)
14.6 (12.3–14.2)
15.5 (14.9–21.9)
7.0 (6.7–7.3)
7.0 (5.9–8.2)
83.7 (76.1–103.4)
83.0 (72.2–96.2)
2.7 (2.2–2.7)
2.9 (2.8–4.8)
0.6 (0.6–0.8)
0.5 (0.4–0.5)
5.3 (4.9–10.1)
5.3 (4.6–6.5)
Dosage [(mg/day)/kg] Median (IQR) Serum drug level [mg/L] Median (IQR) Dose corrected serum drug level [(mg/L)/[(mg/ day)/kg]] Median (IQR)
N: number of samples; IQR: interquartile range; BMI: body mass index; PHT: phenytoin; CBZ: carbamazepine; VA: valproate.
3. Results
high diagnostic performance (Supplementary Fig. 1a).
3.1. Subject characteristics
3.3. Plasma PGE2 levels: drug response
A total of 123 subjects including 34 healthy individuals referred to as “Healthy controls” and 89 PWE, aged 10–48 years, were recruited in the study. Of the 89 patients, 34 newly-diagnosed drug-free patients were assigned to the “Epilepsy_All” group who were further categorized into different epilepsy subtypes, idiopathic epilepsy (N = 13), symptomatic epilepsy (N = 12) and cryptogenic epilepsy (N = 9) (Supplementary Table 1). The rest 55 patients were receiving different AED monotherapies and were segregated on the basis of drug and drug response: “PHT responders” (N = 7), “PHT non-responders” (N = 6), “CBZ responders” (N = 10), “CBZ non-responders” (N = 9), “VA responders” (N = 15), and “VA non-responders” (N = 8) (Table 1). There were no significant differences in drug dose and serum drug levels between the responders and the non-responders. PGE2 levels were measured in the plasma samples of all the subjects and were compared with their demographic characteristics. Gender, age and body mass index (BMI) appeared to have no effect on PGE2 levels.
After the completion of one-year follow-up period, patients receiving AED monotherapy were segregated on the basis of their differential drug response and there plasma PGE2 levels were compared with that of “Healthy controls” and the drug-free “Epilepsy_All” group (Table 2). Responders and non-responders of PHT and CBZ had significantly higher PGE2 levels than the “Healthy controls”, however, no difference was observed when compared with the “Epilepsy_All” group (Fig. 2A and B). In contrast, “VA responders” were found to have significantly lower PGE2 levels than the “Epilepsy_All” (232.1 pg/ml versus 475.7 pg/ml, p < 0.01) and the “VA non-responders” (232.1 pg/ ml versus 611.9 pg/ml, p < 0.0001) (Fig. 2C). “VA responders” and “Healthy controls” had no statistically significant difference between their PGE2 levels. The comparisons of “VA responders vs Drug-free” and “VA responders vs VA non-responders” using ROC analysis displayed an AUC of 0.847 and 0.933, respectively (Supplementary Fig. 1b and c), again showing a high diagnostic performance of plasma PGE2 levels in PWE.
3.2. Plasma PGE2 levels: epilepsy versus controls 3.4. Plasma AA levels Plasma PGE2 levels of drug-free patients of the “Epilepsy_All” group were compared with that of “Healthy controls” (Table 2). The PGE2 levels were significantly higher in “Epilepsy_All” than in “Healthy controls” (475.7 pg/ml versus 137.2 pg/ml, p < 0.0001) (Fig. 1A). Upon segregation of “Epilepsy_All” to different epilepsy subtypes, PGE2 levels remained significantly elevated (p < 0.001) in each of the three groups, idiopathic, symptomatic and cryptogenic epilepsy (Fig. 1B). There were no statistically significant differences among the patients of these subtypes and therefore, further analysis was performed without the segregation of the “Epilepsy_All” group into the epilepsy subtypes. The ROC analysis between the “Epilepsy_All” and “Healthy controls” groups showed an area under the curve (AUC) of 0.942 indicating a
For measuring the plasma AA levels, a subset of 16 subjects from the “Healthy controls” and 16 subjects from the drug-free “Epilepsy_All” were randomly selected along with the 55 patients receiving different AED treatments. The ELISA assay did not reveal any significant difference in AA levels among the different groups (Supplementary Table 2). 4. Discussion Currently, clinical evidences suggest the involvement of various drug-metabolizing enzymes (CYPs), drug transporters (ABC transporters) and drug target genes (ion-channels in brain) in determining AED 3
Prostaglandins, Leukotrienes and Essential Fatty Acids 153 (2020) 102056
C. Rawat, et al.
difference was observed among the three epilepsy subtypes, idiopathic, symptomatic and cryptogenic epilepsy. Drug response was found to have no association with the plasma PGE2 levels of patients receiving PHT or CBZ monotherapy. Both responders and non-responders receiving PHT or CBZ monotherapy had elevated levels of PGE2 than healthy subjects. However, “VA responders” had significantly lower PGE2 levels than “VA non-responders” as well as drug-free patients. Upon measuring the plasma levels of the PGE2 precursor molecule, AA, no difference was observed among the groups. To examine the association of epilepsy with PGE2 levels, we compared the plasma PGE2 levels in drug-free PWE of the “Epilepsy_All” group with that of “Healthy controls” and found a significant increase in the patients. The increase was independent of the type of epilepsy. Several studies reported a key role of prostaglandins in causing seizuremediated neuroinflammation. Such reports demonstrated induction of COX-2 mRNA and protein expression in different rodent brain tissues such as hippocampus, cerebral cortex, striatum, brain stem, cerebellum, and nucleus basalis following seizure induction [18–21]. Such studies reported that COX-2 induction following seizure activates the prostaglandin signalling, consequently triggering secondary damage to the brain and aggravating the disease severity. It was proposed that elevated levels of PGE2 bind to the G protein-coupled receptor, EP1, leading to increased influx of calcium ions, in turn enhancing glutamate release presynaptically and causing seizure generation [22,23]. PGE2 production following seizures was also observed to stimulate neuronal loss in in vivo models [24,25]. Furthermore, co-administration of PGE2 with subconvulsant dose of pentylenetetrazol-generated seizures whereas administration of PGE2 antibodies attenuated the seizures in rats, suggesting the involvement of PGE2 in triggering seizure and governing its threshold [11]. Our study also assessed the plasma PGE2 levels of patients receiving AED monotherapy. “VA responders” were observed to have significantly lower levels of PGE2 than “VA non-responders” as well as the drug-free “Epilepsy_All” group. Our findings corresponds to a previous report by Tang et al. [26] which reported downregulation of the COX-2 mRNA levels in a small population of children with epilepsy responding to VA compared to drug-free patients, however, no difference was observed in the non-responders. VA, a broad spectrum AED, had also been reported to downregulate COX-2 in in vitro and in vivo studies further causing reduced biosynthesis of PGE2 [27–29]. Rao et al. [28] and Chuang et al. [29] reported that the VA-mediated COX-2 downregulation is attributed to the reduced binding of the transcription factor, NF-κB to COX-2 promoter, ultimately reducing PGE2 production in the brain. However, our study found VA-mediated PGE2 reduction to be associated with drug efficacy. COX-2 regulates the multidrug efflux transporter, P-gp by producing increased levels of PGE2, thereby causing increased efflux of the prescribed AED/s resulting in poor
Table 2 Plasma PGE2 levels in healthy controls, drug-free patients and patients receiving AED monotherapy. Sample groups
N
PGE2 levels (pg/ml) median (IQR)
pValue w.r.t. healthy controls
pValue w.r.t. Epilepsy_All
Healthy controls
34
137.2 (106.5–175.6)
–
<0.0001
Drug-free Epilepsy_All
34
<0.0001
–
Idiopathic epilepsy
13
<0.0001
–
Symptomatic epilepsy Cryptogenic epilepsy
12
475.7 (331.2–621.0) 489.3 (369.8–567.4) 412.2 (343.8–578.3) 473.3 (319.4–923.9)
<0.0001
–
0.0002
–
0.0001
0.8502
0.0016
0.9742
0.0011
0.9072
0.0011
0.9072
0.0827
0.0048
<0.0001
0.3566
9
PHT Responders
7
Non-responders
6
CBZ Responders
10
Non-responders
9
VA Responders
15
Non-responders
8
579.7 (358.2–793.3) 432.7 (353.8–610.7)
521.9 (257.1–813.0) 491.0 (336.2–605.4)
232.1 (190.2–281.6) 611.9 (509.0–789.1)
N: number of samples; IQR: interquartile range; w.r.t.: with respect to; PHT: phenytoin; CBZ: carbamazepine; VA: valproate. Bold signifies p < 0.05. pvalue was calculated using Kruskal-Wallis with post-hoc Dunn test adjusted by the Benjamini-Hochberg FDR method.
efficacy [17]. However, such studies remained inconclusive in addressing pharmacoresistance in epilepsy indicating a gap in the current pharmacological research. Inflammation, a crucial factor involved in epilepsy, is often overlooked as a therapeutic target for managing the disease. Therefore, this study evaluated the levels of a key inflammatory mediator, PGE2 in the plasma samples of drug-free PWE and in patients receiving AED monotherapy and compared them with that of healthy subjects. Drug-free PWE were observed to have significantly higher levels of plasma PGE2 than the healthy subjects. No
Fig 1.. Comparison of plasma PGE2 levels (***p < 0.001): A. “Healthy controls” vs all newly-diagnosed drugfree patients with epilepsy; B. “Healthy controls” vs newly-diagnosed drug-free patients with different epilepsy subtypes. p value was calculated using Kruskal-Wallis with post-hoc Dunn test adjusted by the BenjaminiHochberg FDR method.
4
Prostaglandins, Leukotrienes and Essential Fatty Acids 153 (2020) 102056
C. Rawat, et al.
efficacy [12]. Inhibiting COX-2 may, therefore, be helpful in achieving enhanced efficacy of prescribed AEDs. In vivo and in vitro data revealed prevention of glutamate- or status epilepticus-induced P-gp upregulation by COX-2 inhibitors in rodent and human brain capillary endothelial cells [30–32]. Furthermore, sub-chronic COX-2 inhibition decreased Pgp expression by reducing PGE2 synthesis in chronic epileptic rats enhancing uptake of PHT in brain [33]. To investigate the effect of COX-2 inhibition on drug efficacy, Schlichtiger et al. studied the treatment outcome of the selective COX-2 inhibitor, celecoxib in phenobarbitaltreated responder and non-responder epileptic rats [34]. Celecoxib treatment significantly diminished P-gp expression as well as seizure frequency in both responders and non-responders revealing the potential adjunctive role of COX-2 inhibitors in increasing AED efficacy via Pgp downregulation. Other NSAIDs such as rofecoxib, aspirin, ibuprofen, indomethacin, metamizole, paracetamol and piroxicam also potentiated the anticonvulsant activity of AEDs such as tiagabine, VA and PHT in mice [35,36]. Clinical studies also reported seizure reduction in patients receiving AED therapy when were prescribed the NSAID, aspirin in adjunction [37,38]. Such reports indicate that production of PGE2 by COX-2 plays a key role in deciding the AED treatment outcome. Since, “VA responders” in our study were found to have reduced plasma PGE2 levels, it suggests that lower biosynthesis of PGE2 by VA may cause downregulation of P-gp leading to enhanced efficacy in the responders. In regard to the differential response to the other two AEDs, PHT and CBZ, our study observed no variation in the plasma PGE2 levels between the responder and non-responder groups. Both the responders as well as non-responders on the two AED monotherapies had elevated plasma PGE2 levels compared to “Healthy Controls” suggesting no effect of the two drugs on PGE2 production. In addition to PGE2, we also measured the plasma levels of its precursor molecule, AA in the same patients to determine if the changes in PGE2 levels are COX-2 mediated or are the result of alterations in the upstream AA concentrations. Contrary to the preclinical in vivo reports revealing increased brain and plasma AA levels following seizure induction [8,39], we observed no difference between the PWE and healthy individuals. The failure to replicate the preclinical findings in the clinical samples suggests that the increase in the plasma PGE2 levels of the PWE is COX-2 mediated and is independent of any change in AA levels. However, it is also likely that the change in AA levels may be an acute phase event and may be observed for a narrow time duration. Our study recruited patients who experienced their last seizure at least 72 h prior to the sample collection to investigate the plasma profiles of patients on AED therapy independent of any recent seizure activity. This could be a probable reason for no variation in the AA levels among the groups. For a better understanding, plasma profiles of AA in the patients need to be investigated at different time-intervals following a seizure. Our study has some limitations. First, it is limited by a small sample size. The study is a pilot investigation for which a small sample size would yield greater scientific value relative to the expense of the immunoassays. However, to establish the robustness of the findings, future studies will require larger sample sizes. Secondly, our study lacks the data on the relevance of altered peripheral PGE2 levels for indicating the changes in the central levels (brain or cerebrospinal fluid, CSF levels) of the prostaglandin. In this regard, previous preclinical studies have reported contradictory findings. While Davidson et al. [40] reported increased PGE2 levels in both plasma and CSF in response to cytokines and their inducers and suggested enhanced cerebral entry of PGE2 from the peripheral circulation, Eskilsson et al. [41] found no significant relationship between the levels of PGE2 in plasma and in CSF suggesting that peripherally and centrally produced PGE2 are of distinct sources of origin. Therefore, future studies are further required to examine the extent of correlation between the peripheral and central PGE2 levels in clinical samples. Third, our study does not provide the data regarding the selectivity of altered plasma PGE2 in PWE. Though PGE2 is the major prostaglandin produced during inflammation [42],
Fig 2.. Comparison of plasma PGE2 levels between healthy controls, drug-free patients and patients receiving AED monotherapy (**p < 0.01, ***p < 0.001). A. Patients receiving phenytoin (PHT) monotherapy; B. Patients receiving carbamazepine (CBZ) monotherapy; C. Patients receiving valproate (VA) monotherapy. p value was calculated using Kruskal-Wallis with post-hoc Dunn test adjusted by the Benjamini-Hochberg FDR method. 5
Prostaglandins, Leukotrienes and Essential Fatty Acids 153 (2020) 102056
C. Rawat, et al.
References
COX pathway synthesises other eicosanoids, viz., PGI2, PGD2, PGF2, and TXA2 as well. To determine PGE2 selectivity, the profiles of these eicosanoids also need to be assessed in future. Fourth, we used enzymelinked immunosorbent assay (ELISA) instead of the high-throughput liquid chromatography-tandem mass spectrometry (LC-MS) technique. Though, LC-MS is a highly sensitive and specific analytical method, previous comparative studies have demonstrated similar findings from the two [43,44]. However, for the confirmation of the current findings, the same should be replicated using LC-MS in future. The strength of our study is the stringent selection criteria for recruiting patient samples and the presence of two control groups, viz., healthy subjects and drug-free patients for investigating the effect of the prescribed drug in patients receiving AED monotherapy. In conclusion, our study suggests that elevated plasma PGE2 is associated with epilepsy and its reduced levels are associated with response to VA. Therefore, plasma PGE2 levels may serve as a potential biomarker for determining drug response. To the best of our knowledge, our study is the first to report the clinical evidence of the association between plasma PGE2 levels and VA efficacy in PWE. Future work may emphasize on validating the findings in large sample-size population. Furthermore, molecular mechanisms underlying the role of COX-2 or PGE2 in AED efficacy needs to be explored to establish a cause-effect relationship between the two. In addition, investigating the effect of COX-2 inhibitors in adjunction to prescribed AEDs on drug efficacy in large-sample, randomized, controlled trials may provide information on the clinical application of COX-2 inhibitors for epilepsy management. Our work delivers preliminary data on plasma PGE2 levels as the potential biomarker of valproate efficacy in PWE.
[1] A.K. Ngugi, C. Bottomley, I. Kleinschmidt, J.W. Sander, C.R Newton, Estimation of the burden of active and life-time epilepsy: a meta-analytic approach, Epilepsia 51 (2010) 883–890. [2] M. Gourie-Devi, Epidemiology of neurological disorders in India: review of background, prevalence and incidence of epilepsy, stroke, Parkinson's disease and tremors, Neurol. India 62 (2014) 588–598. [3] S Herman, Intractable epilepsy: relapsing, remitting, or progressive? Epilepsy Curr. 10 (2010) 146–148. [4] A. Vezzani, J. French, T. Bartfai, T.Z Baram, The role of inflammation in epilepsy, Nat. Rev. Neurol. 7 (2011) 31–40. [5] A. Vezzani, B. Viviani, Neuromodulatory properties of inflammatory cytokines and their impact on neuronal excitability, Neuropharmacology 96 (2015) 70–82. [6] B.K. Siesjö, C.D. Agardh, F. Bengtsson, M.L Smith, Arachidonic acid metabolism in seizures, Ann. N Y Acad. Sci. 559 (1989) 323–339. [7] J. Rumià, F. Marmol, J. Sanchez, M. Carreño, N. Bargalló, T. Boget, L. Pintor, X. Setoain, E. Bailles, A. Donaire, E. Ferrer, P Puig-Parellada, Eicosanoid levels in the neocortex of drug-resistant epileptic patients submitted to epilepsy surgery, Epilepsy Res. 99 (2012) 127–131. [8] J.M. Post, S. Loch, R. Lerner, F. Remmers, E. Lomazzo, B. Lutz, L Bindila, Antiepileptogenic effect of subchronic palmitoylethanolamide treatment in a mouse model of acute epilepsy, Front. Mol. Neurosci. 11 (2018) 67. [9] C. Rawat, S. Kukal, U.R. Dahiya, R Kukreti, Cyclooxygenase-2 (COX-2) inhibitors: future therapeutic strategies for epilepsy management, J. Neuroinflamm. 16 (2019) 197. [10] T. Takemiya, K. Suzuki, H. Sugiura, S. Yasuda, K. Yamagata, Y. Kawakami, E Maru, Inducible brain COX-2 facilitates the recurrence of hippocampal seizures in mouse rapid kindling, Prostaglandins Other Lipid Mediat. 71 (2003) 205–216. [11] M.S. Oliveira, A.F. Furian, L.F. Royes, M.R. Fighera, N.G. Fiorenza, M. Castelli, P. Machado, D. Bohrer, M. Veiga, J. Ferreira, E.A. Cavalheiro, C.F Mello, Cyclooxygenase-2/PGE2 pathway facilitates pentylenetetrazol-induced seizures, Epilepsy Res. 79 (2008) 14–21. [12] H Potschka, Modulating P-glycoprotein regulation: future perspectives for pharmacoresistant epilepsies? Epilepsia 51 (2010) 1333–1347. [13] Centers for Disease Control and Prevention, Physical Activity, (2015) https://www. cdc.gov/physicalactivity/basics/adults/. [14] C. Rawat, S. Kushwaha, A.K. Srivastava, R Kukreti, Peripheral blood gene expression signatures associated with epilepsy and its etiologic classification, Genomics (2019). [15] Commission on Classification and Terminology of the International League Against Epilepsy, Proposal for revised classification of epilepsies and epileptic syndromes, Epilepsia 30 (1989) 389–399. [16] Commission on Classification and Terminology of the International League Against Epilepsy, Proposal for revised clinical and electroencephalographic classification of epileptic seizures, Epilepsia 22 (1981) 489–501. [17] W. Löscher, U. Klotz, F. Zimprich, D Schmidt, The clinical impact of pharmacogenetics on the treatment of epilepsy, Epilepsia 50 (2009) 1–23. [18] K. Yamagata, K.I. Andreasson, W.E. Kaufmann, C.A. Barnes, P.F Worley, Expression of a mitogen-inducible cyclooxygenase in brain neurons: regulation by synaptic activity and glucocorticoids, Neuron 11 (1993) 371–386. [19] J. Chen, T. Marsh, J.S. Zhang, S.H Graham, Expression of cyclo-oxygenase 2 in rat brain following kainate treatment, Neuroreport 6 (1995) 245–248. [20] J. Adams, Y. Collaço-Moraes, J de Belleroche, Cyclooxygenase-2 induction in cerebral cortex: an intracellular response to synaptic excitation, J. Neurochem. 66 (1996) 6–13. [21] V.L. Marcheselli, N.G. Bazan, Sustained induction of prostaglandin endoperoxide synthase-2 by seizures in hippocampus. Inhibition by a platelet-activating factor antagonist, J. Biol. Chem. 271 (1996) 24794–24799. [22] I. Nishihara, T. Minami, Y. Watanabe, S. Ito, O Hayaishi, Prostaglandin E2 stimulates glutamate release from synaptosomes of rat spinal cord, Neurosci. Lett. 196 (1995) 57–60. [23] N. Sang, J. Zhang, V. Marcheselli, N.G. Bazan, C Chen, Postsynaptically synthesized prostaglandin E2 (PGE2) modulates hippocampal synaptic transmission via a presynaptic PGE2 EP2 receptor, J. Neurosci. 25 (2005) 9858–9870. [24] K. Kawaguchi, R.W. Hickey, M.E. Rose, L. Zhu, J. Chen, S.H Graham, Cyclooxygenase-2 expression is induced in rat brain after kainate-induced seizures and promotes neuronal death in CA3 hippocampus, Brain Res. 1050 (2005) 130–137. [25] T. Takemiya, M. Maehara, K. Matsumura, S. Yasuda, H. Sugiura, K Yamagata, Prostaglandin E2 produced by late induced COX-2 stimulates hippocampal neuron loss after seizure in the CA3 region, Neurosci. Res. 56 (2006) 103–110. [26] Y. Tang, T.A. Glauser, D.L. Gilbert, et al., Valproic acid blood genomic expression patterns in children with epilepsy—a pilot study, Acta. Neurol. Scand. 109 (2004) 159–168. [27] F. Bosetti, G.R. Weerasinghe, T.A. Rosenberger, S.I Rapoport, Valproic acid downregulates the conversion of arachidonic acid to eicosanoids via cyclooxygenase-1 and -2 in rat brain, J. Neurochem. 85 (2003) 690–696. [28] J.S. Rao, R.P. Bazinet, S.I. Rapoport, H.J Lee, Chronic treatment of rats with sodium valproate downregulates frontal cortex NF-kappab DNA binding activity and COX-2 mRNA, Bipolar Disord. 9 (2007) 513–520. [29] Y.F. Chuang, H.Y. Yang, T.L. Ko, et al., Valproic acid suppresses lipopolysaccharideinduced cyclooxygenase-2 expression via MKP-1 in murine brain microvascular endothelial cells, Biochem. Pharmacol. 88 (2014) 372–383. [30] B. Bauer, A.M. Hartz, A. Pekcec, K. Toellner, D.S. Miller, H Potschka, Seizure-
Funding Subject recruitment was funded by Indian Council of Medical Research (ICMR), GAP0091 and the immunoassay experiments were funded by Council of Scientific and Industrial Research (CSIR), MLP1804. CRediT authorship contribution statement Chitra Rawat: Conceptualization, Writing - original draft, Methodology. : Methodology. Suman Kushwaha: Validation. Sangeeta Sharma: Resources. Achal K Srivastava: Validation. Ritushree Kukreti: Conceptualization, Writing - original draft, Data curation. Declaration of Competing Interest None of the authors has any competing interest to disclose. Acknowledgements The authors express their thanks to patients and their family members as well as healthy subjects for participating in the study. They gratefully acknowledge valuable scientific discussions and suggestions by Prof. Samir K. Brahmachari (CSIR-IGIB) and Prof. M. Gourie-Devi (Sir Ganga Ram Hospital). They are thankful to Dr. Nimesh G. Desai, Director, IHBAS and Dr. Anurag Agrawal, Director, IGIB for their support during project implementation. CR acknowledges University Grants Commission (UGC) for providing fellowship. Supplementary materials Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.plefa.2020.102056. 6
Prostaglandins, Leukotrienes and Essential Fatty Acids 153 (2020) 102056
C. Rawat, et al.
[31]
[32]
[33]
[34]
[35]
[36]
[37]
in Sturge-Weber syndrome, J. Child. Neurol. 26 (2011) 692–702. [38] E.I. Lance, A.K. Sreenivasan, T.A. Zabel, E.H. Kossoff, A.M Comi, Aspirin use in Sturge-Weber syndrome: side effects and clinical outcomes, J. Child. Neurol. 28 (2013) 213–218. [39] N.G. Bazan, D.L. Birkle, W. Tang, T.S Reddy, The accumulation of free arachidonic acid, diacylglycerols, prostaglandins, and lipoxygenase reaction products in the brain during experimental epilepsy, Adv. Neurol. 44 (1986) 879–902. [40] J. Davidson, H.T. Abul, A.S. Milton, D Rotondo, Cytokines and cytokine inducers stimulate prostaglandin E2 entry into the brain, Pflugers Arch. 442 (2001) 526–533. [41] A. Eskilsson, T. Matsuwaki, K. Shionoya, E. Mirrasekhian, J. Zajdel, M. Schwaninger, D. Engblom, A Blomqvist, Immune-Induced fever is dependent on local but not generalized prostaglandin E2 synthesis in the brain, J. Neurosci. 37 (2017) 5035–5044. [42] P. Ciceri, Y. Zhang, A.F. Shaffer, K.M. Leahy, M.B. Woerner, W.G. Smith, K. Seibert, P.C Isakson, Pharmacology of celecoxib in rat brain after kainate administration, J. Pharmacol. Exp. Ther. 302 (2002) 846–852. [43] C.E. Staatz, P.J. Taylor, S.E Tett, Comparison of an Elisa and an LC/MS/MS method for measuring tacrolimus concentrations and making dosage decisions in transplant recipients, Ther. Drug. Monit. 24 (2002) 607–615. [44] A.S. Gandhi, D. Budac, T. Khayrullina, R. Staal, G Chandrasena, Quantitative analysis of lipids: a higher-throughput LC-MS/MS-based method and its comparison to Elisa, Future Sci. OA 3 (2017) FSO157.
induced up-regulation of P-glycoprotein at the blood-brain barrier through glutamate and cyclooxygenase-2 signaling, Mol. Pharmacol. 73 (2008) 1444–1453. J. Avemary, J.D. Salvamoser, A. Peraud, J. Rémi, S. Noachtar, G. Fricker, H Potschka, Dynamic regulation of P-glycoprotein in human brain capillaries, Mol. Pharm. 10 (2013) 3333–3341. G. Zibell, B. Unkrüer, A. Pekcec, A.M. Hartz, B. Bauer, D.S. Miller, H Potschka, Prevention of seizure-induced up-regulation of endothelial P-glycoprotein by COX-2 inhibition, Neuropharmacology 56 (2009) 849–855. E.A. van Vliet, G. Zibell, A. Pekcec, J. Schlichtiger, P.M. Edelbroek, L. Holtman, E. Aronica, J.A. Gorter, H Potschka, COX-2 inhibition controls P-glycoprotein expression and promotes brain delivery of phenytoin in chronic epileptic rats, Neuropharmacology 58 (2010) 404–412. J. Schlichtiger, A. Pekcec, H. Bartmann, P. Winter, C. Fuest, J. Soerensen, H Potschka, Celecoxib treatment restores pharmacosensitivity in a rat model of pharmacoresistant epilepsy, Br. J. Pharmacol. 160 (2010) 1062–1071. A. Dhir, S.K. Kulkarni, Rofecoxib, a selective cyclooxygenase-2 (COX-2) inhibitor potentiates the anticonvulsant activity of tiagabine against pentylenetetrazol-induced convulsions in mice, Inflammopharmacology 14 (2006) 222–225. R. Kaminski, M. Kozicka, J. Parada-turska, M. Dziki, Z. Kleinrok, W.A. Turski, S.J Czuczwar, Effect of non-steroidal anti-inflammatory drugs on the anticonvulsive activity of valproate and diphenylhydantoin against maximal electroshock-induced seizures in mice, Pharmacol. Res. 37 (1998) 375–381. M.J. Bay, E.H. Kossoff, C.U. Lehmann, T.A. Zabel, A.M Comi, Survey of aspirin use
7