http://informahealthcare.com/arp ISSN: 1381-3455 (print), 1744-4160 (electronic) Arch Physiol Biochem, 2014; 120(4): 131–135 ! 2014 Informa UK Ltd. DOI: 10.3109/13813455.2014.942322
ORIGINAL ARTICLE
The effects of anti-epileptic drugs on human erythrocyte carbonic anhydrase I and II isozymes Archives of Physiology and Biochemistry Downloaded from informahealthcare.com by McMaster University on 02/24/15 For personal use only.
Emine Rabia Koc¸1, Gu¨lten Erken2, Cigdem Bilen3, Zubeyde Sackes3, and Nahit Gencer3 1
Department of Neurology, 2Department of Physiology, Faculty of Medicine, Balikesir University, Balikesir, Turkey, and 3Department of Chemistry, Faculty of Art and Sciences, Balikesir University, Balikesir, Turkey Abstract
Keywords
Carbonic anhydrase (CA) is an enzyme which plays a role in various homeostatic mechanisms, such as acid-base balance and electrolyte secretion in a various tissues. This study was aimed at determining and comparing possible alterations in activity of this enzyme caused by the use of old (Carbamazepine, Phenytoin Sodium, Sodium Valproate) and new (Levetiracetam, Pregabalin, Gabapentin, Oxcarbazepine) anti-epileptic drugs. Blood samples were collected from the volunteers. The blood samples were centrifuged to separate plasma and erythrocyte package. Hemolysate was prepared from the red cells. CA I and II were purified from human erythrocytes by a simple one step procedure using Sepharose 4B-L-tyrosine-sulfonamide affinity column. CA I and II isozymes were treated with some anti-epileptic drugs, then the inhibition or activation of enzyme determined. The results of this study show that Levetiracetam is the most effective inhibitor for human erythrocytes carbonic anhydrase compared with the other anti-epileptic drugs.
Acid-base balance, anti-epileptic drugs, carbonic anhydrase, enzyme inhibition, erythrocyte, levetiracetam, valproic acid
Introduction The carbonic anhydrases (CAs, EC 4.2.1.1.) are ubiquitous zinc metallo-enzymes. The primary physiological function of these enzymes catalyse the reversible hydration of carbon dioxide to form bicarbonate and a proton and are thus involved in physiological and pathological processes (Neri & Supuran, 2011; Supuran, 2008; Supuran & Scozzafava, 2007). There are 16 different -CA isozymes or CA-related proteins in mammals. Investigation of the property of this family is very important for human health. Analyses of inhibition effects of different drugs on CA family are crucial for life. CA I and CA II are cytosolic forms of these enzymes. The isozymes CA I and CA II are involved in respiration and acid/ base homeostasis (Neri & Supuran, 2011; Supuran, 2010). Epilepsy is a common neurological disorder which is characterized by seizures. There are strong connections between epilepsy, anti-epileptic drugs and CAs. Some CA inhibitors have been used as anti-epileptic drugs. The investigations related to anti-epileptic mechanism of the drugs such as topiramate, zonisamide and sulthiame suggested that these drugs can control seizures which can be
History Received 4 February 2014 Revised 14 May 2014 Accepted 2 July 2014 Published online 7 August 2014
linked to low CO2 levels in the brain tissue by increasing CO2 levels (Aggarwal et al., 2013). Another anti-epileptic mechanism of certain CA inhibitors blocks activity of ion channels which might play a role seizure induction (Aggarwal et al., 2013; Patsalos, 2005). Active-site binding interactions of CA and its inhibitors in the crystal structures, e.g. topiramate (Maryanoff, 1998), zonisamide (Unverferth et al., 1998), lacosamide (Temperini et al., 2010) and sulthiame (Temperini et al., 2007) have been investigated by the X-ray crystallography studies (Aggarwal et al., 2013). However, as far as we know, there are no studies investigating the effects of some other used anti-epileptic drugs (Carbamazepine, Phenytoin Sodium, Sodium Valproate, Levetiracetam, Pregabalin, Gabapentin and Oxcarbazepine) on human CA enzyme activity. The current study was aimed at determining and comparing possible alterations in activity of isozymes CA I and CA II caused by use the of old (Carbamazepine, Phenytoin Sodium, Sodium Valproate) and newer (Levetiracetam, Pregabalin, Gabapentin, Oxcarbazepine) anti-epileptic drugs.
Materials and methods
Correspondence: Emine Rabia Koc¸, Department of Neurology, Faculty of Medicine, Balikesir University, Balikesir, Turkey. Tel: +90-266-6121454. Fax: +90-266-612-1459. E-mail: [email protected]
Sepharose 4B, L-tyrosine, sulfonamide, protein assay reagents, phenol red and chemicals for electrophoresis were obtained from Sigma-Aldrich Co. All other chemicals were of analytical grade and obtained from either Sigma or Merck. Medical drugs were provided by the local pharmacy.
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Purification of carbonic anhydrase from human erythrocytes by affinity chromatography The study was approved by the local ethic committee. Informed consent was obtained from the 20 healthy subjects. Erythrocytes were purified from fresh heparinised human blood obtained from the healthy volunteers. The blood samples were centrifuged at 3000 rpm for 20 min, and then the plasma and buffy coat were removed. The red cells were isolated and washed twice with 0.9% NaCl, and haemolysed with 1.5 volumes of ice-cold water. The ghost and intact cells were removed by centrifugation at 20 000 rpm for 30 min at 4 C. The pH of haemolysate was adjusted to 8.7 with solid Tris base, and applied to the prepared Sepharose 4B-Ltyrosine-sulfonamide affinity column equilibrated with 25 mM Tris-HCl/22 mM Na2SO4 (pH 8.7) (Arslan et al., 1996). The affinity gel was washed with the same buffer. The hCA I and II were eluted with 0.1 M NaCH3COO/0.5 M NaClO4 (pH 5.6). The absorbance of the protein in the column effluents was determined spectrophotometrically at 280 nm. CO2-hydratase activity in the eluates was determined, and the active fractions were collected. The purified enzymes were stored at 4 C, in order to maintain activity. The active contents of selected anti-epileptic drugs are Levetiracetam (Keppra), Pregabalin (Lyrica), Gabapentin (Neurontin), Oxcarbazepine (Trileptal) and Carbamazepine (Tegretol), Phenytoin Sodium (Epitoin), Sodium Valproate (Depakine). Total protein determination The absorbance at 280 nm was used to monitor the protein in the column effluents. Quantitative protein determination was achieved by absorbance measurements at 595 nm according to method described by Bradford (1976), with bovine serum albumin standard. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of enzymes SDS-PAGE was performed in order to verify the purified enzyme. It was carried out in 12% and 3% acrylamide concentrations for the running and the stacking gel, respectively, containing 0.1% SDS according to method described by Laemmli (1970). Sample (20 mg) was applied to the electrophoresis medium. Gel was stained for 1.5 h in 0.1% Coomassie Brilliant Blue R-250 in 50% methanol and 10% acetic acid, and then de-stained with several changes of the same solvent without the dye. CA I and CA II enzymes activities assay The CA I and CA II enzymes activities were assayed by following the hydration of CO2 according to the method described by Wilbur & Anderson (1976). CO2-hydratase activity of the enzyme was determined at room temperature in a 0.15 M Na2CO3/0.1 M NaHCO3 (pH 10.0) buffer using phenol red (pH 8.6) as an indicator, and saturated carbon dioxide concentration as substrate in a final volume of 4.2 ml. Duration (in seconds) of the colour change from red to yellow in solution was measured in a 10-ml glass tube with 1 cm diameter. The enzyme unit (EU) was calculated using the equation (t0 tc/tc), where t0 and tc are the times for pH
Arch Physiol Biochem, 2014; 120(4): 131–135
changes of the non-enzymatic and enzymatic reactions, respectively. In vitro studies for anti-epileptic drugs In this study, Carbamazepine, Phenytoin Sodium, Sodium Valproate, Levetiracetam, Pregabalin, Gabapentin, Oxcarbazepine were used as anti-epileptic drugs. Different concentrations of the anti-epileptic drugs (5.13 mM; 10.26 mM; 15.38 mM; 20.51 mM; 25.64 mM) were added to the enzyme activity determination medium in 4.2 ml of total volume. Duration (in seconds) of the colour change from red to yellow in solution was measured in a 10-ml glass tube with 1 cm diameter. Control cuvette activity in the absence of inhibitor was taken as 100%. All compounds were tested in triplicate at each concentration used. The enzyme unit (EU) was calculated using the equation (t0 tc/tc), where t0 and tc are the time periods for pH change of the non-enzymatic and enzymatic reactions, respectively. For each inhibitor, an Activity%- [Inhibitor] graph was drawn (Figure 1).
Results In this study, human erythrocyte CA I and II were purified, with a simple one step method, by using Sepharose 4BLtyrosine-sulfonamide affinity gel with the elution buffer 0.1 M NaCH3COO/0.5 M NaClO4 (pH 5.6). Purity of the enzyme was confirmed by SDS–PAGE (data not shown). Inhibition or activation effects of drugs on enzyme activity were tested under in vitro conditions. Inhibition graphs, using the drugs, are shown in Figure 1. The overall purification gave CA in a yield of 16.08% with a specific activity of 5430.06 EU/mg proteins and the overall purification was 262.57-fold (Table 1). IC50 values were calculated from Activity %-[I] graphs and are given in Table 2. CA activity in the absence of a drug was accepted as 100% activity. Levetiracetam has been shown to be the strongest inhibitor against CA I and II activities (Figure 1b).
Discussion In the current study, the effect of various anti-epileptic drugs on human erythrocyte carbonic anhydrase enzyme activity was investigated. Epilepsy is a most common neurological disorder. Anti-epileptic drugs have been used for the treatment of epilepsy for a long time. The ideal anti-epileptic drug can be characterized by its efficacy, tolerability and ease of use in the clinical setting. Many drugs have been used for some time for the treatment of epilepsy, but recently new drugs started to be used, called new generation anti-epileptic drugs. It has been reported that the efficacy and tolerability of the new generation anti-epileptics are higher than older antiepileptic drugs (Wallace, 2001). In the current study, the following anti-epileptic drugs were used; Carbamazepine, Phenytoin Sodium, Sodium Valproate (old generation antiepileptic drugs) and Levetiracetam, Pregabalin, Gabapentin, Oxcarbazepine (new generation anti-epileptic drugs). The structures of the active substances of the used anti-epileptic drugs are shown in Figure 2. The results of the current study show that human erythrocyte CA I and II activities are inhibited by anti-epileptic drugs at different levels (Figure 1).
Effects of anti-epileptic drugs on human erythrocyte CA I and II isozymes
DOI: 10.3109/13813455.2014.942322
(b) 120
100
hCAI
100
hCAI
80
hCAII
80
hCAII
% Activity
% Activity
(a) 120
60 40
60 40
20
20
0
0 0
5
10 15 20 [Carbamazepine] µM
25
hCAI hCAII
5
10 15 20 [Levetiracetam] µM
80 60 40
100 % Activity
100 % Activity
0
20
hCAI hCAII
80 60 40 20
0
0 0
5
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15
20
25
0
5
10
15
20
25
[Gabapentin] µM
[Pregabalin] µM (f) 120
100
hCAI hCAII
80 60 40
100 % Activity
(e) 120
% Activity
25
(d) 120
(c) 120
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80 60 40 20
0
0 0
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10 15 20 [Sodium Valproate] µM
[Phenytoin Sodium] µM
25
(g) 120
% Activity
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hCAI hCAII
80 60 40 20 0 0
5
10
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[Oxcarbazepine] µM
Figure 1. Effects of Carbamazepine (a), Levetiracetam (b), Pregabalin (c), Gabapentin (d), Phenytoin Sodium (e), Sodium Valproate (f), Oxcarbazepine (g) drugs on the carbonic anhydrase enzyme activity of human erythrocyte. A purified carbonic anhydrase from human was assayed for hdyratase activity in the presence various concentrations of the above drugs. IC50 values were determined from these graphs.
Table 1. Summary of the purification of human carbonic anhydrase I and II.
Step Haemolysate Affinity chromatography
Volume (ml)
Activity (U/ml)
Total activity (U)
Protein amount (mg/ml)
Total protein (mg)
Specific activity (U/mg)
Overall yield (%)
Overall purification (fold)
25 2
41.33 83.08
1033.25 166.16
1.9980 0.0153
49.9500 0.0306
20.68 5430.06
100.00 16.08
– 262.57
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Arch Physiol Biochem, 2014; 120(4): 131–135
Levetiracetam was the most effective inhibitor for human erythrocytes carbonic anhydrase. CA is an important enzyme for homeostatic mechanisms such as pH regulation, ion transport, bone resorption and secretion of gastric, cerebrospinal fluid and pancreatic juices (Imtaiyaz Hassan et al., 2013). CA inhibitors have been used for various pathologies such as glaucoma, cerebrospinal fluid malabsorption syndrome, obesity (Newton et al., 2002; Soler et al., 1998; Supuran & Scozzafava, 2007). Because of the inhibition of CA may influence the neuronal excitation (Aggarwal et al., 2013), CA has become one of the major targets for the treatment of epilepsy. Therefore, CA inhibitors such as topiramate, zonisamide, lacosamide and sulthiame are used as anti-epileptic drugs (Supuran, 2008, 2010). One of the mechanisms for anticonvulsant activity of CA inhibitors is alterations in the ionic currents in GABAA receptors due to
the movement of ions HCO3 caused by CA inhibitors (Staley et al., 1995). In this condition membrane voltage may be affected. A further mechanism that explains reduction in neuronal excitability caused by the CA inhibitors is an increase in tonic blockade of NMDA receptors by magnesium in NMDA receptors (Lee et al., 1996). Another CA inhibitor Acetazolamide is used as an alternative to the topiramate in the treatment of increased intracranial pressure, a disease caused by cerebrospinal fluid malabsorption syndrome. It has been shown that CA inhibitors reduce the synthesis of cerebrospinal fluid in these patients (Soler et al., 1998; Newton et al., 2002). Previous studies investigated the effects of various drugs on human carbonic anhydrase enzymes. Ottlecz et al. (1999) have shown that Ampicillin sulphate inhibits human carbonic anhydrase enzyme. IC50 value of this work is 385 mm for hCA I and 774 mm for hCA II. It has been reported that the IC50 concentrations of Lamotrigine, used as an anti-epileptic and antipsychotic drug, on hCA I and II were 811.2 mM and 977.3 mM, respectively (Erzengin et al., 2014). In the current study, IC50 values of Levetiracetam were found to be 8.17 mM and 15.82 mM for hCA I and hCA II, respectively. Levetiracetame, which is a new generation anti-epileptic drug, was the most effective inhibitor for human erythrocytes carbonic anhydrase among the anti-epileptic drugs used in this study. It was reported (Kohler et al., 2007) that saccharin most likely coordinates in a deprotonated state through its
Table 2. IC50 values of the antiepileptic drugs. Drugs Carbamazepine Levetiracetam Pregabalin Gabapentin Phenytoin Sodium Sodium Valproate Oxcarbazepine
hCA I IC50 mM 18.85 8.17 16.28 10.86 15.71 19.06 11.21
hCA II
IC50 mM
22.57 15.82 24.24 20.57 22.54 24.39 18.03
Pregabalin
Carbamazepine
Figure 2. Chemical structures of antiepileptic drugs.
NH CH3 2H H3C
N O
OH
NH2
Leveracetam
Phenytoin Sodium
CH3 O H
O− NH2
O
O
Na+
N
N
NH O
Gabapenn
Oxcarbazepine O
O
OH NH2
N O
Sodium Valproate O H3C
O−
CH3
Na+
NH2
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DOI: 10.3109/13813455.2014.942322
Effects of anti-epileptic drugs on human erythrocyte CA I and II isozymes
nitrogen atom to the catalytically active zinc ion. Additionally, ureido-substituted benzenesulfonamide moieties were evidenced when the inhibitor was bound within the enzyme active site (Pacchiano et al., 2010, 2011). The inhibition mechanism of the drugs used in our study may be the drug derivatives containing carboxyl and amide groups inhibited hCA I and II in the same way. Many drug side effects may result from CA isozyme inhibition. For example, respiratory acidosis is probably the cause of some side effects observed during acetazolamide therapy, such as fatigue, headache, altered taste sensations, and respiratory distress (Wistrand & Lindqvist, 1991). Given the physiological importance of CA, the metabolic impact of medically important drugs should receive greater study, not only erythrocyte hCA I and II but also whole CA isozymes. For example, in two recent studies, total hepatic CA (I, II, III, IV) activity was shown to be diminished in the streptozotocininduced diabetic rat (Dodgson & Watford, 1990; Moynihan & Ennis, 1990). As mentioned above, drug-enzyme interaction studies have gained a great interest over the recent years. Our study is thus a contribution to the literature data on the inhibitors or carbonic anhydrase enzymes. Results of our study provide some useful information.
Conclusions In conclusion, in the current study, for the first time to the authors’ knowledge, the inhibitory effects of some older and newer anti-epileptic drugs on activity of hCA I and II enzymes was compared. In this study, the strongest inhibitors against hCA I and II activities were found to be Valproic acid among the old generation anti-epileptic drugs and Levetiracetam among the new generation anti-epileptic drugs. The results of the current study show that clinicians should be careful when prescribing anti-epileptic drugs as possible adverse effects can be produced as a result of inhibition of carbonic anhydrase as well as the use of these drugs in the treatment of different clinical conditions.
Declaration of interest The authors report no declarations of interest.
References Aggarwal M, Kondeti B, McKenna R. (2013). Anticonvulsant/antiepileptic carbonic anhydrase inhibitors: A patent review. Expert Opin Ther Patents, 23:717–24. Arslan O, Nalbantoglu B, Demir N, et al. (1996). A new method for the purification of carbonic anhydrase isozymes by affinity chromatography. Turk J Med Sci, 26:163–6. Bradford MM. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of proteindye binding. Analyt Biochem, 72:248–51. Dodgson SJ, Watford M. (1990). Differential regulation of hepatic carbonic anhydrase isozymes in the streptozotocin-diabetic rat. Arch Biochem Biophys, 277:410–14. Erzengin M, Bilen C, Ergun A, Gencer N. (2014). Antipsychotic agents screened as human carbonic anhydrase I and II inhibitors. Arch Physiol Biochem, 120:29–33.
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Imtaiyaz Hassan M, Shajee B, Waheed A, et al. (2013). Structure, function and applications of carbonic anhydrase isozymes. Bioorg Med Chem, 21:1570–82. Kohler K, Hillebrecht A, Wischeler JS, et al. (2007). Saccharin inhibits carbonic anhydrases: Possible explanation for its unpleasant metallic aftertaste. Angew Chem Int Ed Engl, 46:7697–9. Laemmli DK. (1970). Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature, 227:680–83. Lee J, Taira T, Pihlaja P, et al. (1996). Effects of CO, on excitatory transmission apparently caused by changes in intracellular pH in the rat hippocampal slice. Brain Res, 706:210–16. Maryanoff BE, Costanzo MJ, Nortey SO, et al. (1998). Structure-activity studies on anticonvulsant sugar sulfamates related to topiramate. Enhanced potency with cyclic sulfate derivatives. J Med Chem, 41: 1315–43. Moynihan JB, Ennis S. (1990). Acetazolamide-insensitive carbonic anhydrase activities in liver and tonic skeletal muscle of adult male rats with streptozotocin-induced diabetes mellitus. Biochem J, 272: 553–6. Neri D, Supuran CT. (2011). Interfering with pH regulation in tumours as a therapeutic strategy. Nat Rev Drug Discov, 10:767–77. Newton PN, Thai LH, Tip NQ, et al. (2002). A randomized, doubleblind, placebo-controlled trial of acetazolamide for the treatment of elevated intracranial pressure in cryptococcal meningitis. Clin Infect Dis, 35:769–72. Ottlecz A, Romero JJ, Lichtenberger LM. (1999). Effect of ranitidine bismuth citrate on the phospholipase A2 activity of Naja naja venom and Helicobacter pylori: A biochemical analysis. Aliment Pharmacol Ther, 13:875–81. Pacchiano F, Aggarwal M, Avvaru BS, et al. (2010). Selective hydrophobic pocket binding observed within the carbonic anhydrase II active site accommodate different 4-substituted-ureido-benzenesulfonamides and correlate to inhibitor potency. Chem Commun (Camb), 46:8371–3. Pacchiano F, Carta F, McDonald PC, et al. (2011). Ureido-Substituted benzenesulfonamides potently inhibit carbonic anhydrase IX and show antimetastatic activity in a model of breast cancer metastasis. J Med Chem, 54:1896–902. Patsalos PN. (2005). Properties of anti-epileptic drugs in the treatment of idiopathic generalized epilepsies. Epilepsia, 46:140–8. Soler D, Cox T, Bullock P, et al. (1998). Diagnosis and management of benign intracranial hypertension. Arch Dis Child, 78:89–94. Staley KJ, Soldo BL, Proctor WR. (1995). Ionic mechanisms of neuronal excitation by inhibitory GABA, receptors. Science, 269: 977–81. Supuran CT, Scozzafava A. (2007). Carbonic anhydrases as targets for medicinal chemistry. Bioorg Med Chem, 15:4336–50. Supuran CT. (2008). Carbonic anhydrases: Novel therapeutic applications for inhibitors and activators. Nat Rev Drug Discov, 7:168–81. Supuran CT. (2010). Carbonic anhydrase inhibitors. Bioorg Med Chem Lett, 20:3467–74. Temperini C, Innocenti A, Mastrolorenzo A, et al. (2007). Carbonic anhydrase inhibitors. Interaction of the anti-epileptic drug sulthiame with twelve mammalian isoforms: Kinetic and X-ray crystallographic studies. Bioorg Med Chem Lett, 17:4866–72. Temperini C, Innocenti A, Scozzafava A, et al. (2010). The Coumarinbinding site in carbonic anhydrase accommodates structurally diverse inhibitors: The anti-epileptic lacosamide as an example and lead molecule for novel classes of carbonic anhydrase inhibitors. J Med Chem, 53:850–4. Unverferth K, Engel J, Hofgen N, et al. (1998). Synthesis, anticonvulsant activity, and structure-activity relationships of sodium channel blocking 3-aminopyrroles. J Med Chem, 41:63–73. Wallace SJ. (2001). Newer anti-epileptic drugs: Advantages and disadvantages. Brain & Develop, 23:277–83. Wilbur KM, Anderson NG. (1976). Electrometric and colorometric determination of carbonic anhydrase. J Biolog Chem, 176:147–51. Wistrand PJ, Lindqvist A. (1991). Design of carbonic anhydrase inhibitors and the relationship between the pharmacodynamics and pharmaco-kinetics of acetazolamide. In: Botre` F, Gros G, Storey BT, eds. Carbonic Anhydrase. VCH Verlagsgesellschaft, Weinheim: Vch Publishers, 352–74.
ORIGINAL ARTICLE
The effects of anti-epileptic drugs on human erythrocyte carbonic anhydrase I and II isozymes Archives of Physiology and Biochemistry Downloaded from informahealthcare.com by McMaster University on 02/24/15 For personal use only.
Emine Rabia Koc¸1, Gu¨lten Erken2, Cigdem Bilen3, Zubeyde Sackes3, and Nahit Gencer3 1
Department of Neurology, 2Department of Physiology, Faculty of Medicine, Balikesir University, Balikesir, Turkey, and 3Department of Chemistry, Faculty of Art and Sciences, Balikesir University, Balikesir, Turkey Abstract
Keywords
Carbonic anhydrase (CA) is an enzyme which plays a role in various homeostatic mechanisms, such as acid-base balance and electrolyte secretion in a various tissues. This study was aimed at determining and comparing possible alterations in activity of this enzyme caused by the use of old (Carbamazepine, Phenytoin Sodium, Sodium Valproate) and new (Levetiracetam, Pregabalin, Gabapentin, Oxcarbazepine) anti-epileptic drugs. Blood samples were collected from the volunteers. The blood samples were centrifuged to separate plasma and erythrocyte package. Hemolysate was prepared from the red cells. CA I and II were purified from human erythrocytes by a simple one step procedure using Sepharose 4B-L-tyrosine-sulfonamide affinity column. CA I and II isozymes were treated with some anti-epileptic drugs, then the inhibition or activation of enzyme determined. The results of this study show that Levetiracetam is the most effective inhibitor for human erythrocytes carbonic anhydrase compared with the other anti-epileptic drugs.
Acid-base balance, anti-epileptic drugs, carbonic anhydrase, enzyme inhibition, erythrocyte, levetiracetam, valproic acid
Introduction The carbonic anhydrases (CAs, EC 4.2.1.1.) are ubiquitous zinc metallo-enzymes. The primary physiological function of these enzymes catalyse the reversible hydration of carbon dioxide to form bicarbonate and a proton and are thus involved in physiological and pathological processes (Neri & Supuran, 2011; Supuran, 2008; Supuran & Scozzafava, 2007). There are 16 different -CA isozymes or CA-related proteins in mammals. Investigation of the property of this family is very important for human health. Analyses of inhibition effects of different drugs on CA family are crucial for life. CA I and CA II are cytosolic forms of these enzymes. The isozymes CA I and CA II are involved in respiration and acid/ base homeostasis (Neri & Supuran, 2011; Supuran, 2010). Epilepsy is a common neurological disorder which is characterized by seizures. There are strong connections between epilepsy, anti-epileptic drugs and CAs. Some CA inhibitors have been used as anti-epileptic drugs. The investigations related to anti-epileptic mechanism of the drugs such as topiramate, zonisamide and sulthiame suggested that these drugs can control seizures which can be
History Received 4 February 2014 Revised 14 May 2014 Accepted 2 July 2014 Published online 7 August 2014
linked to low CO2 levels in the brain tissue by increasing CO2 levels (Aggarwal et al., 2013). Another anti-epileptic mechanism of certain CA inhibitors blocks activity of ion channels which might play a role seizure induction (Aggarwal et al., 2013; Patsalos, 2005). Active-site binding interactions of CA and its inhibitors in the crystal structures, e.g. topiramate (Maryanoff, 1998), zonisamide (Unverferth et al., 1998), lacosamide (Temperini et al., 2010) and sulthiame (Temperini et al., 2007) have been investigated by the X-ray crystallography studies (Aggarwal et al., 2013). However, as far as we know, there are no studies investigating the effects of some other used anti-epileptic drugs (Carbamazepine, Phenytoin Sodium, Sodium Valproate, Levetiracetam, Pregabalin, Gabapentin and Oxcarbazepine) on human CA enzyme activity. The current study was aimed at determining and comparing possible alterations in activity of isozymes CA I and CA II caused by use the of old (Carbamazepine, Phenytoin Sodium, Sodium Valproate) and newer (Levetiracetam, Pregabalin, Gabapentin, Oxcarbazepine) anti-epileptic drugs.
Materials and methods
Correspondence: Emine Rabia Koc¸, Department of Neurology, Faculty of Medicine, Balikesir University, Balikesir, Turkey. Tel: +90-266-6121454. Fax: +90-266-612-1459. E-mail: [email protected]
Sepharose 4B, L-tyrosine, sulfonamide, protein assay reagents, phenol red and chemicals for electrophoresis were obtained from Sigma-Aldrich Co. All other chemicals were of analytical grade and obtained from either Sigma or Merck. Medical drugs were provided by the local pharmacy.
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Purification of carbonic anhydrase from human erythrocytes by affinity chromatography The study was approved by the local ethic committee. Informed consent was obtained from the 20 healthy subjects. Erythrocytes were purified from fresh heparinised human blood obtained from the healthy volunteers. The blood samples were centrifuged at 3000 rpm for 20 min, and then the plasma and buffy coat were removed. The red cells were isolated and washed twice with 0.9% NaCl, and haemolysed with 1.5 volumes of ice-cold water. The ghost and intact cells were removed by centrifugation at 20 000 rpm for 30 min at 4 C. The pH of haemolysate was adjusted to 8.7 with solid Tris base, and applied to the prepared Sepharose 4B-Ltyrosine-sulfonamide affinity column equilibrated with 25 mM Tris-HCl/22 mM Na2SO4 (pH 8.7) (Arslan et al., 1996). The affinity gel was washed with the same buffer. The hCA I and II were eluted with 0.1 M NaCH3COO/0.5 M NaClO4 (pH 5.6). The absorbance of the protein in the column effluents was determined spectrophotometrically at 280 nm. CO2-hydratase activity in the eluates was determined, and the active fractions were collected. The purified enzymes were stored at 4 C, in order to maintain activity. The active contents of selected anti-epileptic drugs are Levetiracetam (Keppra), Pregabalin (Lyrica), Gabapentin (Neurontin), Oxcarbazepine (Trileptal) and Carbamazepine (Tegretol), Phenytoin Sodium (Epitoin), Sodium Valproate (Depakine). Total protein determination The absorbance at 280 nm was used to monitor the protein in the column effluents. Quantitative protein determination was achieved by absorbance measurements at 595 nm according to method described by Bradford (1976), with bovine serum albumin standard. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of enzymes SDS-PAGE was performed in order to verify the purified enzyme. It was carried out in 12% and 3% acrylamide concentrations for the running and the stacking gel, respectively, containing 0.1% SDS according to method described by Laemmli (1970). Sample (20 mg) was applied to the electrophoresis medium. Gel was stained for 1.5 h in 0.1% Coomassie Brilliant Blue R-250 in 50% methanol and 10% acetic acid, and then de-stained with several changes of the same solvent without the dye. CA I and CA II enzymes activities assay The CA I and CA II enzymes activities were assayed by following the hydration of CO2 according to the method described by Wilbur & Anderson (1976). CO2-hydratase activity of the enzyme was determined at room temperature in a 0.15 M Na2CO3/0.1 M NaHCO3 (pH 10.0) buffer using phenol red (pH 8.6) as an indicator, and saturated carbon dioxide concentration as substrate in a final volume of 4.2 ml. Duration (in seconds) of the colour change from red to yellow in solution was measured in a 10-ml glass tube with 1 cm diameter. The enzyme unit (EU) was calculated using the equation (t0 tc/tc), where t0 and tc are the times for pH
Arch Physiol Biochem, 2014; 120(4): 131–135
changes of the non-enzymatic and enzymatic reactions, respectively. In vitro studies for anti-epileptic drugs In this study, Carbamazepine, Phenytoin Sodium, Sodium Valproate, Levetiracetam, Pregabalin, Gabapentin, Oxcarbazepine were used as anti-epileptic drugs. Different concentrations of the anti-epileptic drugs (5.13 mM; 10.26 mM; 15.38 mM; 20.51 mM; 25.64 mM) were added to the enzyme activity determination medium in 4.2 ml of total volume. Duration (in seconds) of the colour change from red to yellow in solution was measured in a 10-ml glass tube with 1 cm diameter. Control cuvette activity in the absence of inhibitor was taken as 100%. All compounds were tested in triplicate at each concentration used. The enzyme unit (EU) was calculated using the equation (t0 tc/tc), where t0 and tc are the time periods for pH change of the non-enzymatic and enzymatic reactions, respectively. For each inhibitor, an Activity%- [Inhibitor] graph was drawn (Figure 1).
Results In this study, human erythrocyte CA I and II were purified, with a simple one step method, by using Sepharose 4BLtyrosine-sulfonamide affinity gel with the elution buffer 0.1 M NaCH3COO/0.5 M NaClO4 (pH 5.6). Purity of the enzyme was confirmed by SDS–PAGE (data not shown). Inhibition or activation effects of drugs on enzyme activity were tested under in vitro conditions. Inhibition graphs, using the drugs, are shown in Figure 1. The overall purification gave CA in a yield of 16.08% with a specific activity of 5430.06 EU/mg proteins and the overall purification was 262.57-fold (Table 1). IC50 values were calculated from Activity %-[I] graphs and are given in Table 2. CA activity in the absence of a drug was accepted as 100% activity. Levetiracetam has been shown to be the strongest inhibitor against CA I and II activities (Figure 1b).
Discussion In the current study, the effect of various anti-epileptic drugs on human erythrocyte carbonic anhydrase enzyme activity was investigated. Epilepsy is a most common neurological disorder. Anti-epileptic drugs have been used for the treatment of epilepsy for a long time. The ideal anti-epileptic drug can be characterized by its efficacy, tolerability and ease of use in the clinical setting. Many drugs have been used for some time for the treatment of epilepsy, but recently new drugs started to be used, called new generation anti-epileptic drugs. It has been reported that the efficacy and tolerability of the new generation anti-epileptics are higher than older antiepileptic drugs (Wallace, 2001). In the current study, the following anti-epileptic drugs were used; Carbamazepine, Phenytoin Sodium, Sodium Valproate (old generation antiepileptic drugs) and Levetiracetam, Pregabalin, Gabapentin, Oxcarbazepine (new generation anti-epileptic drugs). The structures of the active substances of the used anti-epileptic drugs are shown in Figure 2. The results of the current study show that human erythrocyte CA I and II activities are inhibited by anti-epileptic drugs at different levels (Figure 1).
Effects of anti-epileptic drugs on human erythrocyte CA I and II isozymes
DOI: 10.3109/13813455.2014.942322
(b) 120
100
hCAI
100
hCAI
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hCAII
80
hCAII
% Activity
% Activity
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60 40
60 40
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20
0
0 0
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[Gabapentin] µM
[Pregabalin] µM (f) 120
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hCAI hCAII
80 60 40
100 % Activity
(e) 120
% Activity
25
(d) 120
(c) 120
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(g) 120
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[Oxcarbazepine] µM
Figure 1. Effects of Carbamazepine (a), Levetiracetam (b), Pregabalin (c), Gabapentin (d), Phenytoin Sodium (e), Sodium Valproate (f), Oxcarbazepine (g) drugs on the carbonic anhydrase enzyme activity of human erythrocyte. A purified carbonic anhydrase from human was assayed for hdyratase activity in the presence various concentrations of the above drugs. IC50 values were determined from these graphs.
Table 1. Summary of the purification of human carbonic anhydrase I and II.
Step Haemolysate Affinity chromatography
Volume (ml)
Activity (U/ml)
Total activity (U)
Protein amount (mg/ml)
Total protein (mg)
Specific activity (U/mg)
Overall yield (%)
Overall purification (fold)
25 2
41.33 83.08
1033.25 166.16
1.9980 0.0153
49.9500 0.0306
20.68 5430.06
100.00 16.08
– 262.57
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Levetiracetam was the most effective inhibitor for human erythrocytes carbonic anhydrase. CA is an important enzyme for homeostatic mechanisms such as pH regulation, ion transport, bone resorption and secretion of gastric, cerebrospinal fluid and pancreatic juices (Imtaiyaz Hassan et al., 2013). CA inhibitors have been used for various pathologies such as glaucoma, cerebrospinal fluid malabsorption syndrome, obesity (Newton et al., 2002; Soler et al., 1998; Supuran & Scozzafava, 2007). Because of the inhibition of CA may influence the neuronal excitation (Aggarwal et al., 2013), CA has become one of the major targets for the treatment of epilepsy. Therefore, CA inhibitors such as topiramate, zonisamide, lacosamide and sulthiame are used as anti-epileptic drugs (Supuran, 2008, 2010). One of the mechanisms for anticonvulsant activity of CA inhibitors is alterations in the ionic currents in GABAA receptors due to
the movement of ions HCO3 caused by CA inhibitors (Staley et al., 1995). In this condition membrane voltage may be affected. A further mechanism that explains reduction in neuronal excitability caused by the CA inhibitors is an increase in tonic blockade of NMDA receptors by magnesium in NMDA receptors (Lee et al., 1996). Another CA inhibitor Acetazolamide is used as an alternative to the topiramate in the treatment of increased intracranial pressure, a disease caused by cerebrospinal fluid malabsorption syndrome. It has been shown that CA inhibitors reduce the synthesis of cerebrospinal fluid in these patients (Soler et al., 1998; Newton et al., 2002). Previous studies investigated the effects of various drugs on human carbonic anhydrase enzymes. Ottlecz et al. (1999) have shown that Ampicillin sulphate inhibits human carbonic anhydrase enzyme. IC50 value of this work is 385 mm for hCA I and 774 mm for hCA II. It has been reported that the IC50 concentrations of Lamotrigine, used as an anti-epileptic and antipsychotic drug, on hCA I and II were 811.2 mM and 977.3 mM, respectively (Erzengin et al., 2014). In the current study, IC50 values of Levetiracetam were found to be 8.17 mM and 15.82 mM for hCA I and hCA II, respectively. Levetiracetame, which is a new generation anti-epileptic drug, was the most effective inhibitor for human erythrocytes carbonic anhydrase among the anti-epileptic drugs used in this study. It was reported (Kohler et al., 2007) that saccharin most likely coordinates in a deprotonated state through its
Table 2. IC50 values of the antiepileptic drugs. Drugs Carbamazepine Levetiracetam Pregabalin Gabapentin Phenytoin Sodium Sodium Valproate Oxcarbazepine
hCA I IC50 mM 18.85 8.17 16.28 10.86 15.71 19.06 11.21
hCA II
IC50 mM
22.57 15.82 24.24 20.57 22.54 24.39 18.03
Pregabalin
Carbamazepine
Figure 2. Chemical structures of antiepileptic drugs.
NH CH3 2H H3C
N O
OH
NH2
Leveracetam
Phenytoin Sodium
CH3 O H
O− NH2
O
O
Na+
N
N
NH O
Gabapenn
Oxcarbazepine O
O
OH NH2
N O
Sodium Valproate O H3C
O−
CH3
Na+
NH2
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DOI: 10.3109/13813455.2014.942322
Effects of anti-epileptic drugs on human erythrocyte CA I and II isozymes
nitrogen atom to the catalytically active zinc ion. Additionally, ureido-substituted benzenesulfonamide moieties were evidenced when the inhibitor was bound within the enzyme active site (Pacchiano et al., 2010, 2011). The inhibition mechanism of the drugs used in our study may be the drug derivatives containing carboxyl and amide groups inhibited hCA I and II in the same way. Many drug side effects may result from CA isozyme inhibition. For example, respiratory acidosis is probably the cause of some side effects observed during acetazolamide therapy, such as fatigue, headache, altered taste sensations, and respiratory distress (Wistrand & Lindqvist, 1991). Given the physiological importance of CA, the metabolic impact of medically important drugs should receive greater study, not only erythrocyte hCA I and II but also whole CA isozymes. For example, in two recent studies, total hepatic CA (I, II, III, IV) activity was shown to be diminished in the streptozotocininduced diabetic rat (Dodgson & Watford, 1990; Moynihan & Ennis, 1990). As mentioned above, drug-enzyme interaction studies have gained a great interest over the recent years. Our study is thus a contribution to the literature data on the inhibitors or carbonic anhydrase enzymes. Results of our study provide some useful information.
Conclusions In conclusion, in the current study, for the first time to the authors’ knowledge, the inhibitory effects of some older and newer anti-epileptic drugs on activity of hCA I and II enzymes was compared. In this study, the strongest inhibitors against hCA I and II activities were found to be Valproic acid among the old generation anti-epileptic drugs and Levetiracetam among the new generation anti-epileptic drugs. The results of the current study show that clinicians should be careful when prescribing anti-epileptic drugs as possible adverse effects can be produced as a result of inhibition of carbonic anhydrase as well as the use of these drugs in the treatment of different clinical conditions.
Declaration of interest The authors report no declarations of interest.
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