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Table of Contents    
ORIGINAL ARTICLE
Year : 2020  |  Volume : 68  |  Issue : 3  |  Page : 588-592

Aneurysmal Subarachnoid Hemorrhage: Impact on Phenytoin Permeability across the Blood–Brain Barrier


1 Department of Pharmacology, Postgraduate Institute of Medical Education and Research (PGIMER), Chandigarh, India
2 Department of Pediatrics, Postgraduate Institute of Medical Education and Research (PGIMER), Chandigarh, India
3 Department of Neurosurgery, Postgraduate Institute of Medical Education and Research (PGIMER), Chandigarh, India

Date of Web Publication6-Jul-2020

Correspondence Address:
Sandeep Mohindra
37, 5th Floor, Nehru Block, Department of Neurosurgery, Post Graduate Institute of Medical Education and Research, Chandigarh - 160 012
India
Dr. Smita Pattanaik
4015, P N Chhuttani Block, Department of Pharmacology, Post Graduate Institute of Medical Education and Research, Chandigarh - 160 012
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0028-3886.288987

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 » Abstract 


Background: Phenytoin (PHT) is a routinely prescribed prophylactic antiepileptic following aneurysmal subarachnoid hemorrhage (aSAH). However, its prophylactic use in aSAH is controversial as emerging evidence suggests worsening of the neurological and functional outcomes. In addition, there is profound damage to the blood–brain barrier (BBB) in aSAH, posing uncertainty about the permeability of PHT across BBB in these patients. This pilot study was designed to evaluate the alteration in PHT permeability across BBB in aSAH patients.
Materials and Methods: For conducting the study, 20 patients (control n = 10; aSAH (grade 3 or 4) n = 10) were recruited from a tertiary care hospital. The patients undergoing cranial surgery for pathology with intracerebral mass lesions on MRI were chosen as control for aSAH group. Both groups were administered PHT loading dose (20 mg/kg), infused in 5% dextrose, at a rate not more than 50 mg/min, followed by a maintenance dose (5 mg/kg). Quantification of PHT concentration was performed in brain tissue, plasma, and cerebrospinal fluid (CSF) by LC-MS/MS.
Results: The median PHT concentration in brain was found to be significantly decreased (64.8%) in aSAH group (3.78 μg/g) as compared to control (10.73 μg/g), P = 0.010. Similarly, median PHT brain concentration as fraction of plasma was significantly decreased in aSAH group (36.72%) compared to that of control (89.55%), P = 0.003. There was no significant difference in PHT concentration in plasma, CSF, and CSF as a fraction of plasma between both the groups.
Conclusion: There is a definite decrease in the penetration of PHT to the brain in patients with grade 3 and 4 aSAH.


Keywords: Aneurysmal subarachnoid hemorrhage, antiepileptic, aSAH, brain tumor, human brain, LC-MS/MS, phenytoin
Key Messages: This is a pilot study to evaluate the impact of subarachnoid hemorrhage (SAH) on the PHT permeability across BBB in aSAH patients. The study was conducted in a tertiary care hospital in India, and it revealed that there is a definite decrease in free PHT concentration in brain tissue in case of aSAH compared to those with tumor having no apparent BBB disruption.


How to cite this article:
Dhir N, Attri SV, Pattanaik S, Kumar M P, Gill NK, Patial A, Rathore N, Saha L, Mohindra S. Aneurysmal Subarachnoid Hemorrhage: Impact on Phenytoin Permeability across the Blood–Brain Barrier. Neurol India 2020;68:588-92

How to cite this URL:
Dhir N, Attri SV, Pattanaik S, Kumar M P, Gill NK, Patial A, Rathore N, Saha L, Mohindra S. Aneurysmal Subarachnoid Hemorrhage: Impact on Phenytoin Permeability across the Blood–Brain Barrier. Neurol India [serial online] 2020 [cited 2020 Aug 12];68:588-92. Available from: http://www.neurologyindia.com/text.asp?2020/68/3/588/288987

Neha Dhir and Savita Verma Attri have equal contribution.
Corresponding author: Smita Pattanaik




Subarachnoid hemorrhage (SAH) is a devastating neurological emergency that leads to significant mortality.[1],[2],[3],[4] The rupture of the intracranial arterial aneurysm is the most common cause for nontraumatic subarachnoid bleed.[1] Seizures are well-recognized complications following aneurysmal subarachnoid hemorrhage (aSAH), occurring in 20% of aSAH-affected patients especially within the first 24 hours of ictus.[2],[5],[6],[7],[8] Seizures result in increased intracranial pressure causing hemodynamic instability, increased metabolic demand, and diminished oxygen supply precipitating to delayed neuronal injury.[9],[10] Thus, the use of antiepileptic drugs (AEDs) as prophylaxis in these patients is a common neurosurgical practice, especially when transcranial route is used for management.[2],[5],[11],[12],[13] Various AEDs have been tried in such patients that include valproate, phenytoin (PHT), phenobarbital, carbamazepine, zonisamide, levetiracetam, and topiramate.[6],[14],[15],[16] Among these AEDs, PHT is the frequently prescribed AED followed by phenobarbital and carbamazepine.[14] However, the clinical practice of use of PHT in aSAH falls in a customary or narrative-based fashion, rather than evidence based.[6],[15] Emerging evidence also suggests that, use of PHT is associated with worsening of both neurologic and functional recovery in these patients.[17],[18],[19]

Experimental and clinical studies have revealed that in aSAH there is profound damage to both capillary basement membrane and endothelial cells of blood–brain barrier (BBB). This is followed by the BBB disruption that leads to changes in its permeability, affecting the penetration of drugs and other substances across this barrier [20],[21] which (the penetration of drugs), may be decreased, increased, or even unchanged.[22],[23],[24],[25],[26],[27] In the available scientific literature, there is no corroboration to suggest the penetration of PHT through BBB in aSAH patients. Thus, the current study aimed to evaluate the alteration in PHT permeability across the BBB in aSAH patients as compared to those with brain tumors (no evidence of BBB permeability breach), which are selected as control.


 » Materials and Methods Top


Patient enrollment

A total number of 20 subjects (control = 10, cases = 10) were enrolled into the study from the Department of Neurosurgery at a tertiary care Hospital. The inclusion–exclusion criteria and demographic details for both aSAH (grade 3 or 4) and control groups are described in [Table 1] and [Table 2], respectively. In cases of anterior communicating artery ( ACoM) aneurysms (n = 5), gyrus resection was required and the brain tissue was subjected to PHT estimation. In cases of middle cerebral artery (MCA) and posterior communicating artery (PCoM) aneurysms, only those cases that warranted temporal lobe hematoma evacuation along with aneurysm clipping were included. For internal carotid artery bifurcation (ICA (b)) aneurysms, both patients had frontal lobe hematomas requiring evacuation along with aneurysm clipping. The brain tissue taken en-route for hematoma evacuation was collected for PHT estimation. Before enrollment, the purpose and nature of the study was explained to all the subjects. The willingness to participate was documented as written informed consent. The study was initiated after getting approval from the institutional ethics committee (NK/1791/Study/2173).
Table 1: Inclusion and Exclusion criteria for both control and aSAH groups

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Table 2: Demographic data of patients in control and aSAH group

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Sample collection and PHT estimation in the brain, plasma, and CSF

Both cases and controls, were administered a loading dose of PHT (20 mg/kg), infused in 5% dextrose at a rate, not more than 50 mg/min, and followed by a maintenance dose of 5 mg/kg. During surgical procedure, simultaneous samples of blood, CSF, and brain tissue were harvested for PHT estimation. Samples were stored at −80°C until analysis. For PHT quantification, respective samples were processed and PHT extraction was done using MassTox® TDM BASIC kit-A (Chromsystems Instruments and Chemicals GmbH, Germany; Order no. 92111/200, a commercially available kit) as per the manufacturer's instructions. QTRAP® 4500 series liquid chromatography-mass spectrometry (LC-MS/MS) system (AB Sciex Pvt. Ltd, USA) was used for PHT estimation in all three biological matrices. The entire system was connected to a computer where all data were collected and analyzed using the Analyst® Software 1.6.2 (AB Sciex Pvt. Ltd, USA).

Statistical analysis

Data were expressed as range and median. The Shapiro–Wilk test was performed for checking the normality. Based on normality, either Wilcoxon rank-sum test (Mann–Whitney U test) or unpaired t-test was used to compare the PHT concentration in all three matrices. P ≤ 0.05 was considered statistical significant. The R software (version 3.4.4)[28] was used for statistical computation. The additional packages i.e., ggplot2[29] and ggsignif [30] were also used.

Outcomes

In both control and aSAH groups, the median PHT concentration in all three matrices was calculated for comparison. In addition, the brain and CSF PHT concentrations were computed as a fraction of plasma PHT concentration using the following formula. Hypothetically, 1 ml of plasma was equated to that of 1 μg of brain tissue for calculation.

Brain PHT concentration as fraction of plasma = (PHT concentration in brain tissue/PHT concentration in plasma) * 100

CSF PHT concentration as fraction of plasma = (PHT concentration in CSF/PHT concentration in plasma) * 100


 » Results Top


This is a pilot study comparing the alteration in PHT permeability across BBB in aSAH grade 3 or 4 (with apparent BBB disruption) versus the control (tumor) with no radiological evidence of BBB disruption. There were 10 subjects enrolled in each group (control and aSAH). As the PHT concentration in brain tissue in one of the subjects in aSAH group was found to be an outlier (29.94 μg/g) as assessed by boxplot, the subject value was excluded for result analysis from all matrices. The final analysis was done between control (n = 10) and aSAH group (n = 9). For control group, the median concentration of PHT was found to be as follows:

  1. Plasma - 11.59 μg/ml (interquartile range [IQR] = 16.43; range = 3.6 to 38.47 μg/ml)
  2. CSF - 1.07 μg/ml (IQR = 0.65; range = 0.31 to 1.94 μg/ml)
  3. Brain tissue - 10.73 μg/g (IQR = 9.01; range = 2.75 to 27.83 μg/g)
  4. CSF as fraction of plasma - 7.37% (IQR = 2.44, range = 2.82 to 10.85%)
  5. Brain tissue as a fraction of plasma - 89.55% (IQR = 40.47, range = 23.81 to 144.05%)


For aSAH group, the median concentration of PHT was found to be as follows

  1. Plasma - 10.82 μg/ml (IQR = 3.92; range = 3.38 to 36.89 μg/ml)
  2. CSF - 0.45 μg/ml (IQR = 0.54; range = 0.02 to 1.27 μg/ml)
  3. Brain tissue - 3.78 μg/g (IQR = 2.49; range = 1.41 to 8.36 μg/g)
  4. CSF as fraction of plasma - 4.34% (IQR = 7.13, range = 0.11 to 23.58%)
  5. Brain tissue as a fraction of plasma - 36.72% (IQR = 8.84, range = 12.67 to 54.75%)


The distribution of data for some matrices was found to be nonparametric (as assessed by Shapiro–Wilk test; P < 0.05). The Mann–Whitney U test was used for the comparison of results between control and aSAH group. Median PHT concentration in plasma for control (11.59μg/ml) and aSAH (10.82μg/ml) was not significantly different, P = 0.661 [Figure 1]. Median PHT concentration in CSF for control (1.07μg/ml) and aSAH (0.45μg/ml) was not significantly different, P = 0.4377 [Figure 1]. Median PHT concentration in brain tissue was significantly higher in the control (10.73 μg/g) than in the aSAH group (3.78 μg/g), P = 0.010 [Figure 1]. Median PHT concentration as assessed by the CSF concentration as fraction of plasma for control (7.37 μg/g) and aSAH (4.34 μg/g) was not significantly different, P = 0.4967 [Figure 2]. Median PHT concentration as assessed by the brain concentration as fraction of plasma was significantly higher in control (89.55 %) as compared to aSAH group (36.72 %), P = 0.003 [Figure 2].
Figure 1: Phenytoin concentration among control and aSAH group in three biological matrices (plasma, cerebrospinal fluid, and brain tissue). The phenytoin concentration in plasma and cerebrospinal fluid is expressed as μg/ml while in brain tissue as μ g/gm. ▴ represents outlier; *P ≤ 0.05

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Figure 2: Phenytoin concentration in brain tissue and cerebrospinal fluid as a fraction of plasma among control and aSAH groups. ▴ represents outlier; *P ≤ 0.05

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 » Discussion Top


The demographic parameters were similar for the aSAH and the control group. Subjects having aneurysmal subarachnoid bleed of grade 3 and 4 (different sites) were enrolled in aSAH group. To evaluate the alteration in BBB permeability of PHT in the aSAH group, we compared them with the control group consisting of subjects with astrocytoma, having no obvious disruption of BBB [31] evidenced by noncontrast-enhancing intracerebral mass lesions. The results were computed as median PHT concentration in all three matrices (plasma, CSF, and brain tissue) in both groups [Figure 1]. In addition, the PHT concentration in brain and CSF was computed as a fraction of plasma to standardize PHT concentration in these matrices according to the variation observed in the plasma [Figure 2].

No significant difference was found in plasma median PHT concentration in the aSAH group as compared to control group. The estimated median PHT concentration in plasma of control and aSAH were found to be 11.59 μg/ml and 10.82 μg/g, respectively, and the values were in accordance with available literature.[32],[33] Lower PHT concentrations were observed in CSF as compared to plasma and brain tissue [Figure 1]; this is well known and is due to the lower content of protein in the CSF. On comparing median PHT concentration in brain of aSAH with that of controls, PHT was found to be significantly decreased in aSAH group as compared to the control group (64.8%, P = 0.010) [Figure 1]. Similarly, when PHT concentration in brain tissue (calculated as a fraction of plasma) was compared between both control and aSAH groups, there was a significant decrease in PHT concentration in aSAH group (59%, P = 0.003) [Figure 2]. This clearly demonstrates that the concentration of PHT penetrating the brain following aSAH is hampered and there is a significant decrease in its permeability across BBB after aSAH in grade 3 and 4. Hence, the utility of PHT administration as a prophylactic antiepileptic in cases of aSAH seems questionable. Further, the electrolyte abnormalities in aSAH could contribute to the seizurogenic activity rather than cortical damage and hence the correction of imbalances should gain priority in clinical management.

This is the first study that demonstrates the proof with laboratory evidence of decreased penetration of PHT across BBB in aSAH of grade 3 and 4 patients. The results are in agreement with previous study conducted in rodents,[20] where similar findings were obtained in brains after experimental BBB disruption. The previous literature revealed that damaged BBB affects the distribution of lipophilic drugs like that of PHT where the concentration of drugs reaching into the brain is decreased.[20] In our study, we also found that a definite reduction in PHT concentration reaching the brain parenchyma in subjects with aSAH compared to those with cerebral tumors, where there is no obvious BBB disruption.

Cerebral vasospasm is one of the important events following aSAH that accounts for poor prognosis in these patients.[34] In the current study, the brain tissue obtained was peripathological in both tumors (controls) and aSAH (cases); thus, the effect of edema on the BBB permeability and its effects on drug penetration was not evaluated in this study and remains a question for further research. Moreover, the reliability of the results can be tested in prospective studies correlating the radiological parameters and differential distribution of the drug in edematous and nonedematous areas of the brain.

Limitations of the study

  1. This is a pragmatic study of its own kind to investigate the clinical conundrum that has been lingering for a long time in neurosurgical practice. The sample size of the study is small, although relatively homogenous. Therefore, confirmation of these findings is desirable in larger studies.
  2. Though this study estimated intracerebral PHT concentration, it cannot conclude about the therapeutic adequacy of the same, as there are no defined therapeutic concentrations of AEDs for the brain. Hence, plasma concentration has been considered as a validated surrogate.
  3. We also did not evaluate the pharmacodynamics endpoint in terms of seizure control to correlate with brain-plasma PHT concentration levels, which would require a larger sample size.
  4. In aSAH cases, performing detailed MRI sequences to label the brain parenchyma as ischemic or edematous is not warranted. Therefore, the effect of edema or ischemia on PHT permeability across BBB was not evaluated in this study and remains a query to be tested and needs further research.



 » Conclusion Top


We conclude that there is a definite decrease in PHT permeability across BBB following aSAH grade 3 and 4. This decreased penetration makes us wary of continued use of prophylactic PHT in in aSAH management, especially when the said drug has been notorious for long-term morbidity, including chest infection and cerebral infarcts. Further studies incorporating a larger sample size are warranted to support or refute the role of altered BBB permeability of PHT in aSAH.

Ethical conduct of research

The approval of the Institute Ethics Committee (NK/1791/Study/2173) was obtained before conducting the study.

Declaration of patient consent

The authors certify that they have obtained all appropriate patient consent forms. In the form, the patient (s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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