Evaluation of Brain Pharmacokinetic and Neuropharmacodynamic

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Evaluation of brain pharmacokinetic and neuropharmacodynamic attributes of antiepileptic drug, lacosamide, in hepatic and renal impairment: preclinical evidence Baldeep Kumar, Manish Modi, Biman Saikia, and Bikash Medhi ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.7b00084 • Publication Date (Web): 21 Mar 2017 Downloaded from http://pubs.acs.org on March 26, 2017

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ACS Chemical Neuroscience

Evaluation of brain pharmacokinetic and neuropharmacodynamic attributes of antiepileptic drug, lacosamide, in hepatic and renal impairment: preclinical evidence Authors: Baldeep Kumar1, Manish Modi2, Biman Saikia3, Bikash Medhi1*

Running head: PK/PD Attributes of lacosamide in seizures

*Corresponding Author Prof. Bikash Medhi, Department of Pharmacology, Research Block-B, Postgraduate Institute of Medical Education and Research (PGIMER), Chandigarh 160012, INDIA Phone: 0172-2755250; Fax: 01722744043; Email: [email protected] 1

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Department of Pharmacology, Postgraduate Institute of Medical Education and Research, Chandigarh 160012, INDIA 2 Department of Neurology, Postgraduate Institute of Medical Education and Research, Chandigarh 160012, INDIA 3 Department of Immunopathology, Postgraduate Institute of Medical Education and Research, Chandigarh 160012, INDIA

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For Table of Contents Use Only Title: Evaluation of brain pharmacokinetic and neuropharmacodynamic attributes of antiepileptic drug, lacosamide, in hepatic and renal impairment: A preclinical evidence Authors: Baldeep Kumar, Manish Modi, Biman Saikia, Bikash Medhi


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Abstract The knowledge of pharmacokinetic and pharmacodynamic properties of antiepileptic drugs is helpful in optimizing drug therapy for epilepsy. This study was designed to evaluate the pharmacokinetic and pharmacodynamic properties of lacosamide in experimentally induced hepatic and renal impairment in seizure animals. Hepatic or renal impairment was induced by injection of carbon tetrachloride or diclofenac sodium, respectively. After induction, the animals were administered with a single dose of lacosamide. At different time points, MES seizure recordings were done followed by isolation of plasma and brain samples for drug quantification and pharmacodynamic measurements. Our results showed a significant increase in area under curve (AUC) of lacosamide in hepatic and renal impairment groups. Reduced clearance of lacosamide was observed in animals with renal impairment. Along with pharmacokinetic alterations, the changes in pharmacodynamic effects of lacosamide were also observed in all the groups. Lacosamide showed a significant protection against MES induced seizures, oxidative stress and neuroinflammatory cytokines. The present findings revealed that the experimentally induced hepatic or renal impairment could alter the pharmacokinetic as well as pharmacodynamic properties of lacosamide. Hence, these conditions may affect the safety and efficacy of lacosamide. Keywords: Lacosamide, Seizure, Renal failure, Pharmacokinetic, Pharmacodynamic.


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INTRODUCTION Epilepsy is defined as a neurological disease characterized by any of the following criteria: At least two unprovoked (or reflex) seizures occurring >24 h apart; One unprovoked (or reflex) seizure and a probability of further seizures similar to the general recurrence risk (at least 60%) after two unprovoked seizures, occurring over the next 10 years; diagnosis of an epilepsy syndrome [1]. The purpose of therapeutic treatment is to maximize the seizure control with minimum undesirable drug effects or adverse effects, thus improving the quality of life of a patient. The integration of pharmacokinetic/pharmacodynamic concepts can accelerate the process of drug discovery and early clinical drug development. The underlying principle for PK-PD studies is to link the pharmacokinetics & therapeutic effects of a drug in view of establishing dose-concentration-effect relationships [2,3]. PK/PD correlation plays an important role in several aspects of drug development and clinical practice such as evaluation of safety & efficacy, identification of factors contributing to variability of drug response among the patients, dose optimization and monitoring [4]. The patients with epilepsy may suffer from concurrent renal or hepatic diseases that in turn might modify the PK and PD profiles of antiepileptic drugs. Knowledge of PK/PD properties of antiepileptic drugs in epileptic patients with other co-morbidities is essential to ensure safety and efficacy of therapy in this special population. Lacosamide is a novel drug approved for therapeutic treatment of partial epilepsy. In clinical practice, it is used as adjunctive treatment for partial onset seizures with or without secondary generalization in epileptic patients. Following oral administration, lacosamide is rapidly absorbed. Lacosamide is primarily eliminated from body by metabolism and renal excretion. It has an elimination half-life of approximately 13h [5]. The long elimination half-life of these drugs makes them more vulnerable to get accumulated inside the body in patients with altered metabolism and excretion. Hepatic or renal dysfunction can lead to altered pharmacokinetics properties of these drugs which may


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further lead to increased drug exposure and drug residence time, hence, increases the risk of adverse effects in these patients. Therefore, the present study was aimed to evaluate the

pharmacokinetic and neuropharmacodynamic changes of Lacosamide in altered pathological conditions (hepatic and renal impairment) in maximal electroshock (MES) induced seizure in rats. RESULT & DISCUSSION To the best of our knowledge, this is the first study to examine the effect of hepatic or renal impairment on pharmacokinetic and pharmacodynamic effects of lacosamide. As our data demonstrated that the experimentally induced hepatic and renal impairment significantly modified the pharmacokinetic profile of lacosamide and resulted in decreased drug clearance and prolonged elimination half-life. Liver or kidney dysfunction can affect the pharmacokinetic and related pharmacodynamic behaviour of antiepileptic drugs. These alterations are more important in case of drugs with longer elimination half life. As kidney is the major route of drug elimination, renal dysfunction dramatically influences the pharmacokinetic profile of a drug and its metabolites which in turn may affect pharmacodynamic response to an administered dose. Lacosamide at all three doses (12.5, 18, 25mg kg-1) significantly protected the rats (n=5 per dose) against MES induced seizures (Fig. 1). Lacosamide (25mg kg-1) was found to be more effective dose in reducing seizure development as compared to the lowest (12.5mg kg-1) dose and further studies were carried out with this effective dose (25 mg kg-1). - Figure 1 The mean plasma & brain tissue concentration-time profiles of lacosamide obtained after oral administration are shown in Fig. 2. From graph, it can be considered that the peak plasma as well as brain tissue concentration was achieved at 1.5h post-dose in control, MES and hepatic impairment group. However, in renal impairment group, it came around 4h. A


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monoexponential fall has been observed both in lacosamide plasma and brain tissue concentrations. The plasma and brain tissue concentration–time course of lacosamide was better analyzed with a biexponential model, wherein the first exponential represents the absorption process and the second represents an elimination process. The plasma and brain level data was fitted into pharmacokinetic modeling software (WinNonlin®, Pharsight Corporation) choosing one-compartment body model with first order elimination. Taking into consideration the weighted sum of squared residuals, correlation, the Akaike’s information criterion (AIC), Bayesian information criterion (BIC or SBC) and visual inspection of observed and predicted values, the one-compartment body model with first order elimination was found to be best pharmacokinetic model to fit plasma and brain tissue drug concentration-time data. Among various possible compartment models and weighting schemes, the best fit 1-Compartment model was found to be 1/Ŷ weighting. -Figure 2Therefore, the pharmacokinetic parameters were calculated using the one-compartment firstorder model for concentration time data obtained from all the four groups (Table 1). Hepatic as well as renal impairment in animals significantly increase the peak plasma concentration (Cmax) as compared to control and MES seizure groups. The higher Tmax value in renal impairment group indicated the slow absorption. The drug clearance was significantly reduced in animals with renal impairment which in turn resulted in increased elimination half life, almost twice that of control and seizure groups. A significant increase in drug exposure has been observed in both hepatic and renal impairment groups as evident by higher values of AUC in plasma as well as brain tissue. -Table 1In present study, our findings demonstrated that single dose administration of lacosamide to animals with experimentally induced hepatic or renal impairment significantly


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altered the pharmacokinetic properties of lacosamide. This significant increase in Cmax and AUC in carbon tetrachloride induced hepatic impairment group could be due to reduced hepatic clearance of lacosamide. The physiological changes associated with hepatic impairment can alter the drug disposition and; the reduced drug metabolism due to hepatic dysfunction might lead to increased oral bioavailability [6]. Due to renal impairment, the clearance (CL) of lacosamide was significantly altered in renal impairment group as compared to seizure group. This resulted in increased elimination half-life of drug (t1/2) as compared to seizure group. The increased AUC and reduced clearance of lacosamide could lead to toxic effects. Similar to our findings, a study reported by Matar and Tayem evaluated the effect of hepatic and renal failure on single-dose pharmacokinetics of AED, topiramate (t1/2 ~20h; primarily eliminated through kidney). The investigators showed that hepatic and renal impairment resulted in significant prolongation of elimination half-life and reduced clearance of topiramate [7]. In another study, Glue et al. (1997) found a decrease in total body clearance and increased half-life and AUC of antiepileptic drug felbamate in subjects with renal dysfunction [8]. Increased AUC and reduced clearance indicate the drug accumulation which can further cause toxicity. Simultaneously, the pharmacodynamic effects of lacosamide were evaluated. The pharmacodynamic effect of lacosamide was evaluated at different time points in MES induced seizures in animals with hepatic impairment or renal impairment. Lacosamide administration (25 mg kg-1) significantly protected (~66%) the animals against MES induced tonic hind limb extension (THLE) at 0.5 to 4h post-dose in seizure group and the protective effect was extended up to 12h in hepatic impairment as well as renal impairment groups (Fig. 3) which might be due to reduced clearance of drug from body. Hence, the drug was available at site of action for longer time. Accordingly, the pharmacokinetic changes in patients with renal impairment can significantly impact the pharmacodynamic responses also as it may


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alter the drug concentration at site of action. It is demonstrated that the effect of renal impairment on drug pharmacokinetics is real and clinically significant in case of epileptic patients because of increased risk of adverse drug effects. -Figure 3Various experimental and clinical studies indicated the increased burden of oxidative stress in pathophysiology of epilepsy [9-11]. We have also evaluated the neuroprotective effects of lacosamide on oxidative stress & pro-inflammatory cytokines in MES induced seizure in rats. Maximal electroshock seizure induction in animals resulted in generation of ROS. The brain levels of MDA were significantly elevated after MES administration in all seizure groups as compared to control animals (0h time point). Lacosamide administration (25mg kg-1) showed a significant decline in brain MDA levels in lacosamide MES group, hepatic and renal impairment groups (Table 2) at 0.5 to 8h post-dose. The effect of lacosamide on lipid peroxidation was extended up to 24h after drug administration in hepatic and renal impairment groups. Endogenous antioxidants such as GSH, SOD and catalase help in scavenging reactive oxygen species. A significant reduction was observed in the brain levels of GSH (Table 2), SOD (Supplementary Table S1) and catalase in MES induced seizures indicating oxidative stress. Lacosamide administration significantly restored the levels of endogenous antioxidants in seizure group, hepatic as well as renal impairment groups (Table 2). The effect of lacosamide on reduced glutathione levels and catalase activity was observed up to 12h and 24h post-dose in renal impairment group, respectively. -Table 2The obtained results are in agreement with previous studies which showed that lacosamide exhibited neuroprotection against pentylenetetrazol (PTZ) induced seizures. It has also been reported that lacosamide produced a significant protection against oxidative stress by ameliorating increased lipid peroxidation and restoring the levels of endogenous antioxidants


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[12]. In this study, the protective effects of lacosamide on these oxidative stress markers were observed up to 24h post-administration. Various underlying dynamic processes responsible for seizures induced neuronal death include mitochondrial dysfunction, inflammatory cascades, oxidative stress, and neuroplasticity alterations or activation of some late cell death pathways [13]. An increasing number of experimental and clinical studies indicated that activation of neuroinflammatory cascade occurs in epilepsy [14]. Lacosamide showed a significant attenuation of seizure induced elevation in brain TNF-ɑ levels in MES seizure group, hepatic impairment as well as renal impairment groups (Fig. 4). The levels of TNF-ɑ were significantly decreased up to 4h following lacosamide administration in MES seizure animals. The effect of lacosamide on inflammatory response was continued up to 24h post-dose in renal impairment group. The brain IL-1β levels were markedly stimulated by MES induced seizures and treatment with lacosamide significantly reduced the brain IL-1β concentration in seizure group as well as hepatic and renal impairment groups (Fig. 5). In case of animal with renal impairment, the effect was retained up to 8h of drug administration. Similarly, lacosamide also showed a significant decline in brain IL-6 levels at 2h post dose in MES seizure group, and up to 4h post-dose in hepatic impaired animals and up to 8h in renal impairment group (Fig. 6). This increased duration of action of lacosamide on proinflammatory cytokines might be due to extended availability of drug at site of action in renal impairment group. Extent as well as duration of drug action was varied in MES seizure group, hepatic impaired group and renal impaired group because of altered pharmacokinetics. -Figure 4-Figure 5-Figure 6-


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In previous studies, Lacosamide (interacts with CRMP-2) has also shown strong neuroprotective and anti-apoptotic effects following glutamate-induced excitotoxicity in hippocampal slices as well as in vivo animal model of status epilepticus [15]. GABA (gamma-Aminobutyric acid) is the principal inhibitory neurotransmitter in brain. In our study, lacosamide did not produce significant effect on brain GABA levels in animals (Supplementary Fig. S1) which is in agreement with previous reports showing that lacosamide did not modulate the activity of GABA neurotransmitter [15]. Apoptosis is the main process responsible for seizure induced neuronal death in epilepsy [13]. In this study, the effects of lacosamide on apoptotic markers (caspase-3 and 9) were also evaluated. Caspase-3 and 9 levels in brain tissue were measured to assess apoptosis induced by seizures in rats. Lacosamide administration did not produce any significant effect on caspase-3 levels in brain of MES induced seizure animals. While, it showed a significant reduction in brain caspases-3 levels in renal impairment group at 2h and 4h after drug administration. No significant effect of lacosamide was observed on caspase-9 concentration in brain tissue of animals (Fig. 7). -Figure 7CONCLUSION Based on the present data, following general and specific conclusive observations can be made about significant impact of experimentally induced hepatic or renal impairment on pharmacokinetic and pharmacodynamic effects of lacosamide. Overall, the findings of present study may suggest that the compromised hepatic or renal function in patients may alter the pharmacokinetic and pharmacodynamic properties of lacosamide. Hepatic as well as renal impairment resulted in prolongation of elimination t1/2 of lacosamide due to altered hepatic metabolism and decreased renal clearance. If extrapolated to humans, these results emphasize the need of monitoring the pharmacokinetic and pharmacodynamic changes of


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lacosamide associated with hepatic or renal co-morbidities in order to avoid drug accumulation and toxicity. METHODS The present study was conducted in Department of Pharmacology, Immunopathology and Neurology, PGIMER, Chandigarh. Drugs & Chemicals Lacosamide was received from M/s MSN Pharmachem Pvt. Ltd., India as a gift sample. Acetonitrile, Methanol and Water of HPLC grade were purchased from Merck (Merck Pvt. Ltd., Mumbai). Rat Tumor necrosis factor-α ELISA kit (Diaclone SAS, France), Interleukin1β and Rat Interleukin-6 ELISA kits (Boster Bio, USA), Rat Caspase-3 and Caspase-9 ELISA kits (Qayee Biotechnology, China) and Rat GABA ELISA Kit (Sincere Biotech, China) were used for quantitative analysis. All chemicals and reagents used for this study were of analytical grade. Experimental Animals The study protocol was approved by PGIMER Animal Ethics Committee, Chandigarh (No. 74(66)/IAEC/339R). The experiments on animals were conducted in accordance with the CPCSEA guidelines. Adult Male Wistar rats, weighing 240–280g were obtained from Advanced Small Animal Research Facility, PGIMER, Chandigarh. The animals were housed in standard laboratory conditions and 12h:12h light-dark cycle. Dose Selection Studies of Lacosamide For the selection of optimum dose, three doses of lacosamide have been tested on the basis of previous literature. Lacosamide was administered orally to animals at doses of 12.5mg kg-1, 18mg kg-1 and 25mg kg-1. After 1h of drug administration, maximal electroshock (MES) stimulation (150mA for 0.2s) was applied through bipolar ear electrodes to induce seizures in animals. Anticonvulsant activity was determined and scoring was done as: Score 1 - No


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Tonic hind limb extension (THLE) with mild jerking movements; 2 - Tonic forelimb extension followed by strong paddling movements; 3 - Complete THLE with or without paddling movements. Each dose was evaluated in five animals (n=5). The most appropriate and effective dose was selected for further pharmacokinetic and pharmacodynamic studies. Experimental Design The animals were divided into four groups: Group – 1: Control group (n=40) - All the animals were administered orally with the selected dose of Lacosamide (25mg kg-1), dissolved in distilled water. The series of blood samples (n=4 at each time point) were collected at 0h and then at 0.5, 1, 1.5, 2, 4, 8, 12, 24, 48h after administration of drug (Fig. 8). The blood collection was carried out under adequate anaesthesia via cardiac puncture and the plasma was stored at -20 °C till further analysis. Following blood collection, the animals were euthanized by cervical dislocation under deep anaesthesia and the brain tissues were isolated and preserved in phosphate buffer saline (pH 7.4) at -20 °C. Then, the levels of Lacosamide in plasma and brain were quantified (16). The levels of GABA neurotransmitter, cytokines, biochemical and apoptotic markers were estimated in brain tissue.

Group – 2: MES seizure group (n=50) - The procedure was same as mentioned in control group with slight changes. In this group, a group of 5 animals (at each time point) was administered with MES (150mA for 0.2sec) to induce seizures in rats, before preceding the blood collection. Then, the anticonvulsant effect of lacosamide was assessed by evaluating the seizure activity as per scoring system as mentioned in preliminary studies. Immediately after MES scoring, the animals were sacrificed under deep anaesthesia and the brains were isolated and preserved in phosphate buffer saline (pH 7.4) at -20°C for further analysis.

Group – 3: Hepatic impairment group (n=50) - In this group, hepatic impairment was induced in animals by administering carbon tetrachloride (1ml kg-1, i.p., CCl4 mixed with olive oil, 1:1) followed by 24h food deprivation [17]. Lacosamide was administered after the


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inducement of hepatic impairment. The further procedure was same as mentioned in Group – 2.

Group – 4: Renal impairment group (n=50) - For the evaluation of lacosamide pharmacokinetics in this group, renal impairment was produced by the validated method of diclofenac sodium (25mg kg-1, i.p.) induced renal injury model [18]. Then, the further procedure was same as given in Group – 2. - Figure 8 Lacosamide bioanalysis The levels of lacosamide in plasma and brain samples were predicted by liquid chromatographic (LC) method as previously described by Martinez et al. [19]. The RP-HPLC system (Hitachi) was consisted of two L-2130 pumps, an L-2200 auto sampler and an L-2455 photodiode array detector. Data collection and integration were achieved using Hitachi Model D-2000 Elite Chromatography data station software (version 3.0). The analytical column, Waters C18 (5µm, 250 x 4.60mm), was used for separation. The mobile phase was comprised of a mixture of water (65%), acetonitrile (26.2%) and methanol (8.8%). Detection was achieved with ultraviolet detection at 210nm. Biochemical Estimation The whole brain tissue was rinsed with 0.9% sodium chloride solution and homogenized (10%) in cooled phosphate buffer saline (pH 7.4). The brain tissue homogenates were centrifuged at 10,500 g for 20 min at 4°C to get the post mitochondrial supernatant, which were used for further drug bioanalysis and biochemical assays.

Estimation of Lipid Peroxidation Brain lipid peroxidation was estimated by measuring the levels of thiobarbituric acid-reactive substances by the method of Wills [20] as described previously [21]. The level of LPO was


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expressed as nanomoles of MDA/mg of protein (nmol mg-1 protein). Tissue protein content was measured by Biuret method.

Estimation of Reduced Glutathione Reduced glutathione (GSH) was estimated by the method of Jollow et al. [22] as described previously by Kumar et al. [21]. GSH levels were calculated and expressed as µmol mg protein-1.

Estimation of Superoxide Dismutase (SOD) Activity Superoxide dismutase activity was estimated by the method of Kono [23]. The method was described previously [24]. The SOD activity was calculated and expressed as units per mg protein.

Estimation of Catalase Activity Catalase activity was assayed by the method of Claiborne [25]. The procedure was followed as described earlier by Kumar et al [24]. Catalase activity was calculated and expressed as micromoles of H2O2 decomposed per minute per mg protein (µmol mg-1 pr). Proinflammatory Cytokines Estimation

Brain TNF-α level The levels of brain TNF-α were estimated by commercially available ELISA kit (Diaclone SAS, France). All materials and methodology were provided in the kit by the manufacturer and performed as per manufacturer’s instructions.

Brain IL-1β and IL-6 levels Brain Interleukin-1β & Interleukin-6 levels were estimated by ELISA as per the manufacturer’s instructions provided in the kit (BosterBio, USA). Required materials and methodology were provided in the kit.


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Estimation of brain GABA levels The levels of gamma-Aminobutyric acid (GABA) neurotransmitter were estimated using ELISA as per manufacturer’s instructions (Sincere Biotech, China). Assessment of Caspase-3 & Caspase-9 levels Caspase-3 and Caspase-9 concentrations in brain tissue were quantified using commercially available ELISA kits as per instructions provided by manufacturer (QayeeBio, China). Statistical Data Analysis Data is represented as mean ± S.E.M. The plasma and brain tissue-concentration data was fitted to pharmacokinetic modeling using pharmacokinetic modeling software. After fitting the data to compartmental modeling, all pharmacokinetic parameters were determined as follows: peak concentration (Cmax), time to reach peak drug concentration (Tmax), Area under concentration-time curve (AUC), elimination half-life (t1/2), and clearance (CL). In view of a kinetic compartmental analysis, the concentration-time data set was submitted to a weighted nonlinear least squares regression analysis: considering both the absorption and disposition phases, the mean drug plasma or brain tissue levels were fitted either with one-compartment or two compartment first-order models. The model that best fitted the data was determined with the sum of squares of the residuals, with the Akaike’s information criterion and by visual inspection of the observed and predicted values. Pharmacodynamic data was statistically analyzed using the One Way Analysis of Variance (ANOVA) followed by Tukey’s post-hoc analysis and p

Evaluation of Brain Pharmacokinetic and Neuropharmacodynamic

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