RESEARCH ARTICLE – Pharmaceutical Nanotechnology
Nanoparticle Formulation Improves the Anticonvulsant Effect of Clonazepam on the Pentylenetetrazole-Induced Seizures: Behavior and Electroencephalogram 1,2,10 4 4 ´ ´ ´ GERARDO LEYVA-GOMEZ, MARI´A EVA GONZALEZ-TRUJANO, EDITH LOPEZ-RUIZ, PIERRE-OLIVIER COURAUD,5,6,7 8 9 3,10 10 BABETTE WEKSLERG, IGNACIO ROMERO, FLORENCE MILLER, FLORENCE DELIE, ERIC ALLE´ MANN,10 DAVID QUINTANAR-GUERRERO1 1
´ y Posgrado en Tecnolog´ıa Farmac´eutica, Facultad de Estudios Superiores Cuautitl´an, Universidad Nacional Laboratorio de Investigacion ´ Autonoma de M´exico, Estado de M´exico 54740, M´exico 2 ´ y Atencion ´ de Quemados, Instituto Nacional de Rehabilitacion, ´ Laboratory of Connective Tissue, Centro Nacional de Investigacion M´exico, D.F., M´exico 3 Unit´e U1002 INSERM, Facult´e de M´edecine, Universit´e Paris Descartes, Sorbonne Paris Cit´e, Paris, France 4 ´ de Investigaciones en Neurociencias, Instituto Nacional de Psiquiatr´ıa Ramon ´ de la Fuente Muniz, ˜ M´exico, D.F., M´exico Direccion 5 Unit´e U1016 INSERM, Institut Cochin, Paris, France 6 Unit´e UMR8104, CNRS, Paris, France 7 UMR-S 1016, Universit´e Paris Descartes, Sorbonne Paris Cit´e, Paris, France 8 Division of Hematology and Oncology, Weill Cornell Medical College, New York 10065, New York 9 Department of Life Sciences, Faculty of Science, The Open University, Milton Keynes, United Kingdom 10 Section des Sciences Pharmaceutiques, Technologie Pharmaceutique, Universit´e de Gen`eve, Geneva 41211, Switzerland
Received 14 March 2014; revised 19 May 2014; accepted 20 May 2014 Published online 10 June 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.24044 ABSTRACT: To document the efficacy of clonazepam (CLZ) either free as a solution or loaded in solid lipid nanoparticles (CLZ-SLN) or mixed micelles (CLZ-MM), the in vitro blood–brain barrier permeability of the formulations was determined. Behavior and/or electroencephalograms (EEGs) of rodents receiving treatments were also studied. The in vitro permeability of CLZ increased when associated with SLN, but decreased in the case of MM. The occurrence of the pentylenetetrazole (PTZ)-induced seizures in mice was significantly prevented by CLZ, even when exposed a lower dose of CLZ-SLN after administration by the oral route. The behavioral severity and EEGs showing the PTZ-induced paroxystic activity in rats diminished significantly in the presence of CLZ alone (0.3 mg/kg), and were almost totally prevented in the rats treated with CLZ-SLN (equivalent to 0.3 mg/kg). The frequency, duration, and spreading of the spikes-wave of rats treated with CLZ-SLN decreased significantly as compared with CLZ alone, CLZ-MM, or the vehicle. These results show an in vitro–in vivo correlation in the enhanced blood–brain barrier permeability of SLN formulation, and a contribution of MM to the carrier effect of drugs toward the bloodstream and brain, where this pharmaceutical formulation of CLZ-SLN improves the anticonvulsant effect of this benzodiazepine, thus C 2014 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm offering additional advantages after oral administration. Sci 103:2509–2519, 2014 Keywords: absorption enhancer; anticonvulsant; clonazepam; CNS; drug transport; electroencephalogram; lipids; micelles; pentylenetetrazole; solid lipid nanoparticles
INTRODUCTION Epilepsy is a neurological disorder characterized by spontaneous and recurrent seizures.1 It occurs more frequently in children than in adults.2 Approximately 60%–70% of patients respond to conventional anticonvulsant drug treatment, but for the other 30%–40%, a single therapy is not efficient and they usually require a combination of two or more anticonvulsant drugs.3 Abbreviations used: CLZ, clonazepam; CLZ-MM, clonazepam loaded in mixed micelles; CLZ-SLN, clonazepam loaded in solid lipid nanoparticles; EEG, electroencephalogram; PTZ, pentylenetetrazole. ´ Correspondence to: Mar´ıa Eva Gonzalez-Trujano (Telephone: +525-41605084; Fax: +525-5655-9980; E-mail: evag@imp.edu.mx) Journal of Pharmaceutical Sciences, Vol. 103, 2509–2519 (2014) C 2014 Wiley Periodicals, Inc. and the American Pharmacists Association
Clonazepam (CLZ) is classified as a high-potency benzodiazepine and is sometimes used as a second-line treatment for epilepsy.4 CLZ was approved in the United States as a generic drug in 1997 and is now manufactured and marketed by several companies under various formulations. Although being first-line treatments for acute seizures, benzodiazepines are not first-line drugs for the long-term treatment of seizures because patients may develop tolerance to their anticonvulsant effects or suffer a number of adverse side effects.5 Previous research by different groups has shown that nanoparticles can enhance absorption both intestinally and across the blood–brain barrier (BBB) and deliver drugs in an area close to their site of action. Given this, the aim of the present study was to investigate whether the pharmaceutical formulation of CLZ loaded in solid lipid nanoparticles (CLZ-SLN) can improve the anticonvulsant effect of this benzodiazepine after enteral and/or
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parenteral administration, on the basis of behavioral and/or electroencephalogram (EEG) analyses of pentilenetetrazole (PTZ)-induced seizures in rodents. In addition, CLZ loaded in mixed micelles (CLZ-MM) are proposed as a degradation product of CLZ-SLN in the intestine after oral administration; this formulation was prepared to analyze, in vitro and in vivo, the possible impact on the intestinal absorption of CLZ and its bioavailability in the site of action, as an enhancer in the transportation to increase the efficacy of the anticonvulsant response.
CLZ-MM Preparation Method Clonazepam loaded in mixed micelles were prepared by the coprecipitation method.7 BS, soya PC (at mol fraction 0.5), and CLZ (1 mg) were dissolved in a mixture of methanol–chloroform (1:1, v/v). A film was formed after evaporation of the organic solvents at room temperature under vacuum until a constant weight was reached (48–72 h). The resulting films were dispersed in a given amount of the dispersion medium (phosphate buffer 0.067 M, pH 7.4) to give a clear micellar solution with the required concentration. These conditions allowed complete solubilization of the incorporated amount of CLZ.8
MATERIALS AND METHODS
Characterization of Formulations
Chemicals
Particle Size Analysis
Clonazepam, diazepam (DZP), PTZ, Tween 80, L,"phosphatidylcholine (L,"-PC from egg yolk, Type XVI-E, ≥99%), bile salt (BS)–sodium glycocholate hydrate, and Lucifer yellow (LY) (a marker of tight junction integrity between cells) were purchased from Sigma Chemical Company (St. Louis, Missouri). Glycerylbehenate (Compritol 888 ATO; Gattefoss´e S.A., Saint Priest Cedex, France) referred as Compritol 888 ATO was a gift from Lubrizol (State of Mexico, Mexico). Poloxamer 407 (P-407) was purchased from BASF (Mexico City, Mexico). Methanol was obtained from Sigma–Aldrich (Buchs, Switzerland), and the 2-butanone and chloroform were supplied by Productos Qu´ımicos Monterrey, S.A. de C.V. (Nuevo Leon, Mexico). Distilled water was of Milli-Q quality from Millipore (Billerica, Massachusetts). All other reagents were of at least analytical grade and were used with no additional purification. PTZ was injected intraperitoneally (i.p.), whereas CLZ was administered enterally (esophageal route, p.o.) and/or parenterally (i.p. route) at a 10 mL/kg volume. The vehicle consisted of 0.5% (w/v) in Tween 80 in saline solution (s.s.). All substances were prepared freshly on the day of the experiments.
The average size and polydispersity index were determined by the laser light scattering technique in a Zetasizer (Nano ZS-ZEN 3600; Malvern Instruments, Westborough, Massachusetts). The laser light wavelength (He/Ne, 10 mW) was 633 nm. Measurements were obtained at a 173◦ fixed angle for 60 s at a temperature of 25◦ C. The scattering intensity data were analyzed by digital correlation under a unimodal analysis mode. Dispersions were diluted with MilliQ water until the appropriate particle concentration was reached, as indicated by the particle counts per second, within the sensitivity range of the instrument. Measurements were made in triplicate for all batches prepared, and mean particle size was evaluated. CLZ-SLNs were analyzed after freeze-drying.
CLZ-SLN Preparation Method
Drug Loading and Entrapment Efficiency
The emulsification-diffusion technique with minor variations was used to prepare the CLZ-SLN.6 Typically, the organic solvent (2-butanone) and water were mutually saturated for at least 5 min at room temperature before use, to ensure the initial thermodynamic equilibrium of both liquids. All containers were of amber glass to avoid CLZ degradation because of light. In general, 200 mg of Compritol 888 ATO and 10 mg of CLZ were dissolved in 20 mL of water-saturated solvent, and this organic solution was then emulsified with 40 mL of the solventsaturated aqueous solution containing 5% (w/v) P-407 using a stirrer (Caframo RZR-1, Georgian Bluffs, Ontario; propeller: IKA 1381, IKA, Wilmington North Caroline) at 1240 rpm for 10 min. Subsequently, 160 mL of water were added to the oil-inwater emulsion to allow the diffusion of the organic solvent into the continuous phase, leading to the formation of the nanoparticles. The system was maintained at 73◦ C during the two steps of this procedure. The organic solvent was then removed from the raw SLN suspension by vacuum steam distillation at 35◦ C and 9332.5 Pa. The SLN were centrifuged (Optima LE-80K; Beckman Coulter Inc., Pasadena, California) at 183,000g for 60 min (20◦ C). Finally, they were frozen, first at −5◦ C for 30 min, then at −40◦ C for 180 min, and finally freeze-dried for 24 h at 8 Pa (Labconco , Kansas City, Missouri).
The lyophilized SLN formulation (SLN or CLZ-SLN) was digested in chloroform for 2 h and subsequently filtered. The volume was adjusted to 5 mL with chloroform. The CLZ content was analyzed spectrophotometrically at 305 nm (Varian Cary IE., Palo Alto, Australia). The non-loaded SLN samples were treated in the same way and used as the blank. The calibration curve for CLZ quantification in chloroform was linear (r2 = 0.998, range 5–70 :g/mL). The drug loading and entrapment efficiency were calculated according to Eqs. (1) and (2), respectively: Equation (1): Drug loading (DL) of CLZ in SLN
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Zeta Potential Analysis The zeta potential was determined by Electrophoretic Mobility using Laser Doppler Velocimetry in a Zetasizer (Nano ZS-ZEN 3600; Malvern Instruments, Westborough, Massachusetts) at 25◦ C in a capillary cell. Measurements were made in triplicate for all batches prepared.
DL (%) =
Amount of CLZ in SLN × 100 Amount of SLN
(1)
Equation (2). Entrapment efficiency (EE) of CLZ in SLN EE(%) =
Total amount of CLZ determined in SLN Total amount of CLZ theoretically associated with SLN ×100
(2) DOI 10.1002/jps.24044
RESEARCH ARTICLE – Pharmaceutical Nanotechnology
Scanning Electron Microscopy Analysis Solid lipid nanoparticles morphology was analyzed with a field emission scanning electron microscope (JEOL-7001FA; JEOL , Peabody, Massachusets). An aqueous dispersion of the SLN was spread over a slab, dried under vacuum at room temperature, mounted on stubs, and coated with gold (21.1 nm thickness) using a film deposition system for high-vacuum conditions (EM SCD 500; LEICA, Solms, Wetzlar, Germany). After this procedure, they were observed under the microscope. R
Transmission Electron Microscopy Analysis The morphology of CLZ-MM resuspended in phosphatebuffered saline (PBS) (pH 7.4) at room temperature was studied by transmission electron microscopy (TEM; EM 410; Philips, Eindhoven, North Brabant, Netherlands) using ionized carboncoated copper grids and uranyl acetate negative staining. CLZMM was dispersed in PBS at a concentration of 1 g/L. The resulting dispersion was stirred at room temperature prior to evaluation. Micrographs were obtained at an accelerating voltage of 60 kV. Differential Scanning Calorimetry Analysis Differential scanning calorimetry (DSC) analyses were carried out on samples of the individual substances—CLZ, Compritol 888 ATO, P-407, and CLZ-SLN—at different ratios. The dried samples were weighed directly in non-hermetic aluminum pans (3–5 mg) and scanned between 0◦ C and 350◦ C at a 10◦ C/min heating rate using a DSC Q 10 differential scanning calorimeter (TA Instruments, New Castle, Delaware). A baseline value was obtained for each measurement. R
In Vitro BBB Permeability Studies Cell Culture The hCMEC/D3 cell line is the first stable human brain endothelial cell line that constitutes a unique in vitro model of human BBB.9,10 This cell line presents most of the specific properties of in vivo BBB, including the expression of tight junction proteins (claudin-5, JAM-A, and ZO-1) and the polarized expression of a variety of functional transporters.11,12 This model, already used for studying the interactions of nanocarriers with the BBB, will be the in vitro model to assess permeability of SLN and MM across the BBB.13 HCMEC/D3 cells were seeded at a density of 50,000 cells/cm2 in the upper compartment of six-well culture inserts (Millicell CM inserts; 0.4 :m porosity; Millipore) precoated with type I rat-tail collagen (provided by CELLIAL Technologies, Lens, France). They were maintained for 10–12 days in an EBM-2 basal medium (Lonza, Basel, Switzerland) containing 5% fetal bovine serum Gold (PAA; The Cell Culture Company, Pasching, Austria), 1% penicillin streptomycin (Invitrogen, Carlsbad, California), 10 mM HEPES (Invitrogen, Carlsbad, California), 1.4 :M hydrocortisone, 5 :g/mL ascorbic acid, and 1 ng/mL bFGF (Sigma Chemical Company), at 37◦ C in a 5% CO2 atmosphere. The medium was changed every 3–4 days. Twenty-four hour prior to the permeability assays, fresh medium was supplemented with Simvastatin 1 nM (Calbiochem, La Jolla, California).9–12 Permeability Assays Prior to the compound permeability studies, it is essential to find a working concentration of CLZ, and/or the nanocarriers, DOI 10.1002/jps.24044
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that will not alter the cellular complex between endothelial cells. To this end, endothelial monolayer integrity was verified by following the permeation of LY, a small hydrophilic fluorescent molecule as a paracellular diffusion marker in the presence of the formulation of interest at different concentrations. When innocuous concentrations were found, permeability assays were conducted in the same way for CLZ with and without nanocarriers. Briefly, after 10–12 days of culture, the coated culture inserts with and without endothelial cells were transferred to 6-well plates containing 2 mL of transport buffer (Hanks buffer s.s. with CaCl2 and MgCl2 , 10 mM HEPES, and 1 mM sodium pyruvate) in the abluminal chamber. At time 0, the transport buffer containing the tested compound, with or without 10 :M of LY, was placed in the luminal chambers. The transport incubations were run at 37◦ C, 95% humidity, and 5% CO2 . At different times—5, 15, 30, and 60 min—each culture insert with and without cells was transferred to the lower compartment that held fresh transport buffer. The amounts of each compound transported in the lower compartments, the upper ones at the end of the experiment, and in the working solution, were assessed by fluorometry for LY (excitation/emission peaks of 428/536 nm), or by ultra-high-performance liquid chromatography–mass spectrometry (UHPLC–MS) for CLZ and DZP. Endothelial permeability of CLZ was expressed by the ratio Pst/Psf, where Pst and Psf are the slopes of the clearance curves for the cell monolayer on coated culture inserts and for the coated culture inserts alone (i.e., without the cell monolayer), respectively, as described previously,14 allowing a concentration-independent ratio to be obtained. The increment in cleared volume between successive sampling events was calculated by dividing the amount of solute during the interval time by the donor chamber concentration (the luminal one in this study) as follows: Clearance (mL) =
X Cd
where X is the amount of LY or CLZ/DZP in the receptor (abluminal) chamber, and Cd is the donor chamber concentration at each time point. The average volume cleared is plotted versus time, and the slope estimated by linear regression analysis. Because Psf reflects the passage of the compound through the coated insert alone, it represents the maximum passage of this compound in the studied system. A higher ratio of Psf–Pst, approaching 1, indicates that the compound reaches the brain compartment easily in the in vitro BBB system. UHPLC–MS Analysis of CLZ A mobile phase consisting of a mixture of double-distilled water and acetonitrile at a flow rate of 1 mL/min and mass spectrometry detection was developed for the UHPLC (Thermo Scientific, Hemel Hempstead, UK) analysis of CLZ. The instrument consisted of: a Hypersil GOLD column (Thermo Scientific, Hemel Hempstead, UK) (50 × 2.1 mm2 ) with a mobile phase of A: H2 O + 0.1% formic acid, and B: ACN + 0.1% formic acid with a gradient of 30%–100% A in 4 min; tray temperature control: 30◦ C; column oven control: 40◦ C; detection MSQ Plus, scan mode: SIM; mass: 316 amu; span: 1 amu; time range: 0–4 min; polarity: +; cone (V): 80; ionization mode: ESI; and probe temperature: 350◦ C. ´ Leyva-gomez et al., JOURNAL OF PHARMACEUTICAL SCIENCES 103:2509–2519, 2014
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PHARMACOLOGICAL EVALUATION Animals Male Swiss Webster mice (25–30 g) and male Wistar rats (280– 300 g) were used. All animals were housed under standard laboratory conditions (22 ± 1◦ C, 12–12-h light/dark cycle) and maintained on a standard pellet diet (Lab diet ) with water ad libitum. The experiments were carried out in compliance with the guidelines laid down by the local ethics committee of the Instituto Nacional de Psiquiatr´ıa “Ram´on de la ˜ Fuente Muniz” (“New Alternatives for Epilepsy,” project number INPNC3280.1/SEP08, approved March 29, 2005), and with international norms for the care and use of laboratory animals in experimental procedures. During handling, an isotonic s.s. injection (0.9% NaCl) was administered daily for 3 days before testing. R
PTZ-Induced Seizures in Mice Groups of mice received p.o. or i.p. administration of the vehicles (s.s. or unloaded SLN); CLZ (0.01–3 mg/kg i.p., and 0.01–1 mg/kg p.o.), or CLZ-SLN (0.01–1 mg/kg, i.p., and 0.003–1 mg/kg, p.o.). The mice were then placed individually in an acrylic glass box for 30 min before PTZ administration (80 mg/kg, i.p.). Immediately after receiving a CLZ injection, the animals were returned individually to the acrylic glass box and observed for 30 min. Latency to the onset of the first focal (myoclonic), generalized (tonic–clonic), or tonic behavioral seizure, or the time of death, was recorded. A dose-response curve was constructed using the parameters of the seizure response according to each administration route. At least five doses and six mice per dose were used to build the curve to determine the median effective dose (ED50 ). PTZ-Induced Seizure Phenotype in Mice Myoclonus, tonic–clonic, and tonic seizures were taken as measures of the progressive nature of the PTZ-induced seizure phenotype on the severity scale, where generalized clonus is deemed a more significant event than partial myoclonus, and tonic hind limb extension is regarded as the most severe component of the phenotype.15 Latencies to the onset of each type of seizure were recorded by analyzing the behavioral responses after the i.p. injection of PTZ, as described below. Myoclonus (myoclonic body jerks): characterized by a progressive decrease in motor activity and partial clonic seizure activity affecting the face, head, and/or forelimbs. Partial or focal seizures were brief, typically lasting 1 or 2 s, occasionally accompanied by vocalizations. Partial seizures occurred either individually or in multiple discrete episodes before generalization. Generalized (tonic–clonic seizure): characterized by the sudden loss of upright posture, whole body clonus involving all four limbs and tail, rearing, and autonomic signs. Some mice exhibited multiple generalized seizures, irrespective of their subsequent status for tonic hind limb extension. Tonic seizure: a generalized seizure characterized by tonic hind limb extension; associated with death. Behavioral and EEG Analyses of PTZ-Induced Seizures in Rats Surgical Procedure Rats were anaesthetized with an i.p. injection of a mixture TM TM of ketamine (Anesket ; 100 mg/kg) and xylazine (Rompun ; ´ Leyva-gomez et al., JOURNAL OF PHARMACEUTICAL SCIENCES 103:2509–2519, 2014
20 mg/kg). Indwelling bipolar electrodes oriented stereotaxically (2.4 mm posterior to bregma, 4.3 mm from the midsagittal plane, and 8.0 mm from the skull surface), following Paxinos and Watson,16 were placed in the central nucleus of the left and right amygdala; two stainless steel electrodes were implanted epidurally into the frontal cortex to record the electrocorticograms. The electrode assembly was then fixed to the skull using two screws and dental acrylic glue. After allowing 7 days for recovery, groups of rats were processed for behavioral and EEG analyses 30 min before and after the PTZ-induced seizures (40 mg/kg, i.p.). Behavioral and EEG Analyses For each experiment, rats were individually placed in a soundinsulated observation box for constant, synchronous recordings of behavior, and EEG. During the procedure, a concurrent EEG of the prefrontal cortex and left and right central amygdale nucleus was recorded. EEG signals were amplified and filtered at 3 and 100 Hz for low and high pass, respectively, with a Grass Model 8-18D. Electrical activity was also recorded and stored in a Grass Telefactor Model 15 polygraph (Astro-Med., Warwick, Rhode Island) at a sampling rate of 100 Hz. EEG traces were analyzed using Windaq analysis software. After a basal register of 30 min, rats were administered i.p. with vehicle (n = 7), CLZ in dispersion (0.3 mg/kg; n = 6), CLZ-SLN (0.3 mg/kg of CLZ; n = 6), or CLZ-MM (0.3 mg/kg of CLZ; n = 6). After 30 min of treatment, an injection of a 40-mg/kg i.p. dosage of PTZ was applied to induce seizures, which were observed as spike-wake (S-W) discharges. S-W is defined as a depolarizing and hyperpolarizing discharge described as a burst of greater amplitude (voltage, 3–7 Hz) than those observed in the basal EEG. Behavioral seizure severity (BSS) was scored according to Racine’s scale17 : (1) sudden behavioral arrest and/or motionless staring; (2) facial jerking with muzzle or muzzle and eye; (3) neck jerks; (4) seizure in a sitting position; (5) convulsions including clonic and/or tonic–clonic seizures while lying on the belly, and/or pure tonic seizures; and, (6) convulsions including clonic and/or tonic–clonic seizures while lying on the side and/or jumping wildly.17 S-W duration and frequency, and behavioral seizure severity (BSS) were analyzed after the PTZ-induced seizures for at least 30 min. After the experiments, all animals were anesthetized with sodium pentobarbital (Cheminova de Mexico, S.A. de C.V., Mexico City, Mexico) and perfused with a 4% formaldehyde solution (J.T. Baker, Phillipsburg, New Jersey). Their brains were excised and sliced into 30-:m thick coronal sections with a microtome (Cryostate Microm, Pittsburgh, Pennsylvania), stained by Nissl’s technique, and scanned to establish the precise location of the electrodes.16
STATISTICAL ANALYSIS In vivo data are shown as mean ± standard error of the mean. A one-way analysis of variance (ANOVA) on ranks followed by a Student–Newman–Keuls test was performed to compare groups. A statistical difference was determined for p < 0.05 values using SIGMA STAT software, version 2.3. In vitro data are also presented as mean ± standard error of the mean. A oneway ANOVA Dunnett test was performed to compare groups. A statistical difference was determined for p < 0.05 values using the Analyse it software. R
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Figure 2. In vitro hCMEC/D3 cells assay. Data represented as Pst/Psf ratios. DZP was used as the performance reference. Each point represents the average (mean ± SEM) of one assay performed in triplicate. Mean scores proved significant using a one-way ANOVA Dunnett test. * p < 0.05.
RESULTS
a lower temperature of the two signals caused by the solubilization of CLZ by Compritol 888 ATO and P-407. For this reason, the CLZ peak disappeared completely. No chemical interaction with this sample can be ascribed and each compound exhibits its characteristic temperature (Fig. 1c-IV). SLN was subsequently identified by two peaks: the first for P-407 and the second broadly for Compritol 888 ATO. The same slight solubilization effect was observed for the second peak because of a prior rubber-like state (Fig. 1c-V). Finally, the CLZ-SLN thermogram (Fig. 1c-VI) showed peaks for P-407 and Compritol 888 ATO, whereas no signal of CLZ was recorded under these conditions, probably because of the molecular dispersion of CLZ and/or prior solubilization in the other materials (Fig. 1c).
Characterization of Formulation
In Vitro BBB Permeability Studies
Scanning electron microscopy analysis showed that CLZ-SLN had a solid spherical matrix with no drug crystal formation (Fig. 1a). The size was very similar to the data obtained by dynamic light scattering (Table 1). The zeta potential value was −20.8 mV and was associated with the mechanism of steric stabilization conferred by the P-407. CLZ-SLN drug loading was 0.75%, and this was used for the in vivo evaluations. The entrapment efficiency corresponded to 0.11%. Figure 1b shows the micrograph of CLZ-MM through TEM. The micelles observed are of a size close to that measured by dynamic light scattering. DSC thermograms are presented in Figure 1c. An endotherm at 72.43◦ C associated with the melting point was shown for pure Compritol 888 ATO (Fig. 1c-I), whereas an endotherm at 55.63◦ C was related to the glass transition temperature for the pure P-407 (Fig. 1c-II). An endotherm at 240.38◦ C and an exotherm at 290.61◦ C were because of the melting point and the decomposition of CLZ (Fig. 1c-III). The physical mixture of Compritol 888 ATO, P-407, and CLZ showed a shift toward
In vitro LY permeability was measured using hCMEC/D3 cells in presence of CLZ or DZP at a concentration of 10 :M, of CLZSLN at a concentration up to 0.05 :g/mL, and of the CLZ-MM preparation diluted from 1/100 to 1/25: values were similar to LY permeability value measured with LY alone (data not shown). These results demonstrate that these compounds can be used at 10 :M without disturbing the cellular complexes between endothelial cells. In vitro CLZ permeability studies were performed using appropriate concentrations with hCMEC/D3 cells. In these experiments, CLZ permeability was evaluated, either alone or associated with SLN or MM; DZP was used as a control for a compound that presented the ability to reach the brain easily.11 To facilitate comparisons of the compounds and formulations, these results are expressed as a Pst–Psf ratio, as described in the Materials and Methods section. The Pst–Psf ratio for CLZ alone is 0.94 ± 0.01 (Fig. 2), which is similar to the one for DZP (0.86 ± 0.01), meaning that CLZ presents a high permeability across the in vitro BBB. However, when CLZ is associated with nanocarriers, the ratios are quite different. Indeed, on the one hand, when incorporated into SLN, the Pst–Psf ratio of CLZ is higher than that for CLZ alone (1.09 ± 0.06) giving a statistical difference (p < 0.05); on the other hand, CLZ incorporated into MM shows a lower Pst–Psf ratio: 0.67 ± 0.01 than CLZ alone (p < 0.0001). Therefore, even though CLZ permeability is already high across the in vitro BBB, this permeability improves with the SLN formulation.
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Figure 1. Scanning (a) and transmission (b) electron micrograph of CLZ-SLN (a; bar = 100 nm) and CLZ-MM (b; bar = 500 nm), respectively. DSC thermograms (c) of Compritol R 888 ATO (I); P-407 (II); CLZ (III); physical mixture of Compritol R 888 ATO, P-407, and CLZ (IV); SLN (V); and CLZ-SLN (VI).
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Table 1.
Mean Particle Size and Z-Potential of the Formulations
Formulation SLN CLZ-SLN MM CLZ-MM
DOI 10.1002/jps.24044
Particle Size (nm)
Z-Potential (mV)
± ± ± ±
− 23.5 ± 0.52 − 20.8 ± 1.7 – –
300 332 12.4 16.5
5.2 9.8 1.8 1.4
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Figure 3. Dose-response curves of the anticonvulsant effect of CLZ ( ) or CLZ-SLN ( ) on myoclonus (a or d), generalized (tonic–clonic) (b or e), and tonic seizures (c or f), induced by PTZ, respectively, and evaluated at different doses administered parenterally (via i.p.) or enterally (via p.o.) in mice. Each point represents the average (mean ± SEM) of at least six repetitions. Mean scores proved significant using a one-way ANOVA Dunnett test. * p < 0.05.
Anticonvulsant Response of CLZ Alone and CLZ-SLN in Mice Via the i.p. administration route, latency to the onset of the first myoclonus in mice from the vehicle groups (s.s. or unloaded SLN) was observed at 0.87 ± 0.15 and 0.92 ± 0.18 min, respectively. This latency increased significantly (p < 0.05) when the mice were treated with CLZ or CLZ-SLN at a dose of 0.01 mg/kg (3.25 ± 0.44 or 3.68 ± 0.52 min, respectively). At a dose of 3 mg/kg, mice showed almost total protection against myoclonus in both cases (23.89 ± 3.97 and 25.24 ± 4.75 min, respectively) (Fig. 3a). Regarding the generalized seizure (tonic– clonic), latency to onset in the vehicle and SLN groups was observed at 1.04 ± 0.18 and 1.32 ± 0.34 min, respectively. Administration of CLZ or CLZ-SLN produced a delay in these convulsions from 7.76 ± 4.64 and 6.72 ± 4.74 min at 0.03 mg/kg to 25.29 ± 4.72 and 30 ± 00 min at 0.3 mg/kg (Fig. 3b). Finally, tonic seizure and mortality were deferred by 26.64 ± 3.36 and 23.54 ± 4.11 min, respectively, at 0.03 mg/kg, with total prevention at 0.1 mg/kg in both cases (Fig. 3c). ´ Leyva-gomez et al., JOURNAL OF PHARMACEUTICAL SCIENCES 103:2509–2519, 2014
Under the oral administration, latency to the onset of the first myoclonus in mice receiving s.s. or unloaded SLN was observed at 0.72 ± 0.03 and 0.91 ± 0.04 min, respectively. Using this route, myoclonus was significantly delayed by 1.55 ± 0.18 min in the presence of CLZ at 0.01 mg/kg up to total inhibition at a dose of 0.3 mg/kg, whereas CLZ-SLN significantly delayed the occurrence of myoclonus at a dose of 0.001 mg/kg (13.57 ± 6.13 min). This anticonvulsant response was observed in a dose-dependent manner up to total inhibition at 0.1 mg/kg (Fig. 3d). Generalized seizures were also significantly delayed in the presence of CLZ at 7.24 ± 5.20 min from 0.01 mg/kg until total inhibition was achieved at 0.3 mg/kg (Fig. 3e). This type of seizure was significantly delayed in the presence of CLZ-SLN compared with the anticonvulsant response observed with a solution of CLZ, which began from 0.003 mg/kg (18.44 ± 6.46 min) up to total inhibition, achieved at 0.01 mg/kg (30 ± 00 min) (Fig. 3e). Tonic seizures and mortality were completely avoided in both cases of CLZ tested at the doses mentioned and using p.o. administration (Fig. 3f). DOI 10.1002/jps.24044
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Table 2.
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Pentylenetetrazole (40 mg/kg, i.p.)-Induced Paroxistic Activity in Rats Receiving Vehicle, CLZ-MM, or CLZ-SLN
Group Vehicle CLZ-MM CLZ CLZ-SLN
Latency to the Onset of First DSW (min) 0.72 0.93 6.73 15.14
± ± ± ±
Seizure Score Reached (Median)
0.12 0.16 4.68* 4.95#
6 (5–6) 3 (1–5) 1 (0–6) 0 (0–3)
n=7 n=6 n=6 n=6
Duration of the Seizure (Score Reached 4–6) (s) 70.16 55 60 0
± ± ± ±
16.6 25 0 0
n=5 n=2 n=l n=0
*p < 0.001 versus vehicle; # versus CLZ, ANOVA on ranks followed SNK’s test. DSW: discharge spike-wave.
Behavioral and EEG Analysis in Rats Latency to the onset of paroxystic activity was significantly delayed in rats receiving a solution of CLZ or CLZ-SLN, a response that was also significantly different between the two groups (Table 2). The following behavioral scores for seizures after PTZ administration, following Racine,17 were observed: five out of seven rats receiving the vehicle showed a seizure score of 6, whereas two out of seven had a seizure score of 5 (Table 2). Rats receiving CLZ-MM and then PTZ showed no scores of 6; only one of the rats receiving CLZ alone reached a seizure score of 6; and no rat in the CLZ-SLN group had this score (Table 2). These results indicate a reduction in the severity of behavioral scores in rats dosed with CLZ-MM, although not significant. CLZ alone inhibited the severity of the seizures, but the most important inhibition of convulsive behavior was observed when the rats were treated with CLZ-SLN, where only two out of six showed motionless staring, indicating a score below 4–6, whereas four of six showed no convulsive behavior. After PTZ, the rats in the vehicle group showed an accumulative S-W bursting of 273 ± 59 s and paroxystic activity was propagated to the left and right amygdales (Fig. 4). This activity resembles that observed in the rats injected with CLZ-MM (375 ± 77 s) and CLZ alone (255 ± 76 s); however, less S-W bursting was observed in the two rats that showed paroxystic activity (147 ± 66 s) and in those pretreated with SLN. It is important to note that S-W bursting in the vehicle (a), CLZ-MM (c), and some rats in the CLZ alone (b), groups was observed in the cortex and both amygdales, demonstrating the spread of paroxystic activity (Fig. 4). In contrast, where convulsive activity was observed in the CLZ-SLN group, the S-W bursting appeared in the EEG from the cortex, and no activity was propagated in the amygdales (Fig. 4). The S-W bursting observed in the cortex of the rats that received CLZ-SLN plus PTZ was probably induced by the presence of sleep spindles (Fig. 4), an activity between 7 and 16 Hz that occurs during early stage of sleep seen in the brain as a burst of activity.18 The coronal sections of the brain stained by Nissl’s technique and then scanned, allowed the identification of the precise location of the electrodes (Fig. 5).
DISCUSSION Clonazepam has proven particularly effective against certain types of myoclonic seizures, such as juvenile myoclonic epilepsy and progressive myoclonic epilepsy, as well as against seizures caused by flashing lights (photosensitivity).19 Children with Lennox–Gastaut syndrome also frequently benefit from its use, though not all studies have reported its utility for long-term treatment. The factors influencing the decision on whether DOI 10.1002/jps.24044
to add CLZ to antiseizure treatment for patients with uncontrolled episodes may include potential interactions, side effects, and the mechanisms of action of the various medications. These facts evidenced the need to look for new antiepileptic drugs, or better pharmaceutical formulations to improve the efficacy of those currently in use. Like other benzodiazepines, CLZ is a GABA neurotransmitter enhancer via modulation of the GABAA receptor,20 whereas PTZ-induced seizures are initiated by a nonselective antagonism and blockade of brain GABAA receptors after systemic administration.21,22 PTZ is one of the first convulsing agents selected to induce convulsions in rodents and thus allow studies of new alternatives for anticonvulsant or antiepileptic drug treatment.23–26 It is also considered a reliable predictor of a drug’s ability to raise the seizure threshold and of its potential activity against human myoclonic jerks and S-W seizures.27 In our study, tonic seizures and mortality induced by PTZ were completely prevented in the presence of CLZ under different administration conditions. Also, CLZ produced a significant and dose-dependent delay in the onset of myoclonic and tonic–clonic seizures, but this anticonvulsant response was obtained at lower dose levels with the CLZ-SLN formulation (from 0.2 mg/kg with CLZ alone down to 0.003 mg/kg with CLZ-SLN), suggesting an improvement in the bioavailability of this benzodiazepine after p.o. as compared with i.p. administration. Our results reinforce the recent study by Nair et al.,28 describing higher activity of carbamacepine when associated to SLN in the treatment of seizures when compared with the free drug. No difference was observed in the convulsive response in mice receiving a solution of CLZ or CLZ-SLN via i.p.; however, the paroxystic activity in rats evaluated on the basis of seizure behavior and EEG activity in the cortex and the right and left amygdales showed a higher efficacy of CLZ in the SLN formulation, compared with CLZ in solution. In these experiments, CLZ-SLN prevented convulsive behavior completely in four out of six rats, whereas in the two rats that showed seizures, a significant delay in onset, a reduction of the severity score, and prevention of propagation were all observed. The mechanism of action through which SLN increases the efficacy of CLZ is still unknown, but when administered orally, there are two possible simultaneous mechanisms at the intestinal level29 that may explain SLN absorption30 : (1) a fraction of SLN might adhere to the intestinal wall and undergo a slight degradation by lipase, after which it is absorbed into the systemic circulation with no important structural modification31 ; and (2) another fraction is degraded almost completely by the lipases32–34 and the metabolism of triglycerides and diglycerides to monoglycerides, fatty acids, and glycerol takes place. It has been reported that two interactions affect the presence of lipids at the gut: first, the degradation of tri-, di-, and ´ Leyva-gomez et al., JOURNAL OF PHARMACEUTICAL SCIENCES 103:2509–2519, 2014
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Figure 4. Representative EEG recordings of the paroxystic activity on the prefrontal cortex (CxPf) and left and right central amygdale (Am-L and Am-R) of rats pretreated with vehicle (a), CLZ (b), CLZ-MM (c), and CLZ-SLN (d), on PTZ-induced seizures (40 mg/kg, i.p.). Latency to the onset of the seizure is expressed in minutes; the stage of convulsive behavior associated with paroxystic activity is also indicated below the EEG register. Note the presence of seizures and S-W bursts propagated in all sites (a–c) as compared with the epileptic activity inhibition in (d) that only shows spikes with sleep spindles.
monoacylglycerols by lipase and colipase in positions 1 and 3 of the glycerol32 ; and second, the combination with BSs to form micelles to enhance drug absorption.32,35 It is important to mention that if the effect of promoting absorption is required, a molecular dispersion of the drug in the lipid is needed. In our case, this fact was demonstrated by DSC experiments. Given that SLN possess adhesive properties to the gut wall, the release is carried out in the absorption site leading to an effective concentration gradient between gut wall and blood.36–38 Lipids encourage absorption of drugs functioning as a kind of Trojan horse.30 These mechanisms may also occur between blood and brain. Later, another fraction of the remaining SLN may be emulsified by the biliary salts and alkalis39–42 and subse´ Leyva-gomez et al., JOURNAL OF PHARMACEUTICAL SCIENCES 103:2509–2519, 2014
quently absorbed as mixed micelles.43 The degradation rate occurs faster in shorter fatty acids. In our sample, there is a significant percentage of larger molecules because Compritol is a mixture of approximately 15% mono-, 50% di-, and 35% triglycerides of behenic acid, whereas other fatty acids account for less than 20%. In addition to the lipid degradation, the stabilizer located at the surface of the SLN shows an impediment that controls the degradation rate. The stabilizing layer controls the anchoring of the lipase–colipase complex; for example, in case of the use of lecithin or sodium cholate as stabilizers, the anchoring of the complex leads to fast degradation, but in case sterically stabilizing polymers such as poloxamero 407 are used, there is a steric hindrance of the anchoring of the R
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Figure 5. Schemes showing the precise location of the electrodes in the left (O) and right (•) amygdala of the experimental rats. CeL: central amygdaloid nucleus, lateral division; CeM: central amygdaloid nucleus, medial division; CeC: central amygdaloid nucleus, capsular part.
complex leading to slower degradation.36 It is an important point to consider the stabilizing layer that controls the particle degradation velocity because there is a certain limitation in exchanging the lipid particle matrix.30 It reported 60% degradation of Dynasan 116 SLN in 120 min after lipase– colipase incubation using total surfactant content 0.5% (P-407). Our preparation of Compritol SLN has 5% of P-407, which would mean less degradation. Although in the case of Dynasan 118, SLN showed about 20% degradation after lipase–colipase incubation.36 These percentages allow the estimate that what could happen with SLN degradation using high concentration and steric hindrance of poloxamero 407; degradation is possibly very slow and the effect of CLZ in the brain is enhanced because of CLZ-SLN as demonstrated in our in vitro experiments and supported by a recent reported study.44 On the contrary, the increase of the therapeutic effect exclusively by passage through the BBB45,46 could be explained by the following mechanisms: (1) adsorption of SLN into the capillary walls followed by a release from this site to the brain47,48 ; and (2) adhesion to the endothelial cell membrane followed by endocytosis by the endothelial cells and the release (passive diffusion) of the drug within these cells for delivery to the brain.49–53 Our results suggest that these mechanisms are likely to be involved in the increase in anticonvulsant efficacy observed with CLZ-SLN compared with CLZ in solution. It is important to note that SLN itself may induce a delay in latency to PTZ-induced myoclonus and generalized seizures, suggesting that the lipid proportion in the formulation plays an important role in the anticonvulsant activity of CLZ by producing a possible synergism. This effect observed in vivo could be attributed to an additive mechanism of CLZ-SLN and CLZ-SLN degradation products (by mixed micelles) that increases passage across the intestinal wall54 and the BBB. Recent advances have demonstrated that lipids have broad information-carrying functions in the central nervous system as both ligands and substrates for proteins. Lipids alter the geometric properties of membranes, control protein traffic, and provide messenger molecules that mediate communication between cells. The phospholipid bilayer and associated lipids provide not only a permeability barrier but also a structured environment essential for the proper role of membrane-bound proteins.55 Lipidomic analyses may provide a powerful tool for elucidating the specific roles of lipid intermediates in cell signaling.56 Therefore, a deeper knowledge of the complexity of lipid signaling will increase our understanding of the role of lipid metabolism in various central nervous system disorders, and open new opportunities to develop drugs and therapies for neurological diseases.57,58 The enhancement of the anticonvulsant response obtained with CLZ R
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contained in a lipid vehicle such as SLN indicates that the proportion of lipid content plays an important role in modulating the excitability of the brain. “Excitability” is commonly used in literature, especially for medical and biological field, to characterize the state or activity of the nerve centers such as the brain and spinal cord. Normal and abnormal patterns of behavior require a certain degree of synchronization of firing in a population of neurons that are influenced by synaptic and nonsynaptic interconnections. Neurochemical transmission between neurons involves a number of steps that can be selectively altered to affect neuronal excitability such as in epilepsy. The efficacy of CLZ-SLN was observed not only in the reduction of the severity of convulsive behavior, but also in the inhibition of the propagation of paroxystic activity, as observed in the EEG recordings. Previous studies have shown that the ketogenic diet is a successful therapy for controlling seizures in children59 and, in some cases, also in adolescents60 and adults.61 The mechanisms of action by which this therapy induces anticonvulsant effects are not entirely clear, but it has been suggested that fatty acids may be used to synthesize the three ketone bodies—$-hydroxybutyrate, acetoacetate, and acetone—such that they enter the brain and replace glucose for energy, whereas in the developing brain, they constitute essential building blocks for the biosynthesis of cell membranes and lipids. Moreover, chronic ketosis is also thought to modify the tricarboxylic acid cycle, which would increase glutamate and, subsequently, GABA synthesis in the brain.62 The ketogenic diet generates adaptive changes to brain energy metabolism that increase energy reserves. Ketone bodies are a more efficient fuel than glucose as they induce an increase in the number of mitochondria that could help neurons remain stable when under the increased energy demands occasioned by a seizure, thus perhaps conferring neuroprotection. It is hypothesized that some of these mechanisms of action might be involved in the synergistic anticonvulsant response observed with CLZ-SLN in this study, though further research is needed to corroborate this hypothesis.
CONCLUSION The results of this investigation show an in vitro--in vivo correlation in the enhanced brain permeability of CLZ in the CLZSLN formulation, and a contribution of CLZ-MM (proposed as a degradation product of CLZ-SLN in the intestine) to the carrier effect of drugs into the bloodstream and brain. In this regard, the p.o. route of administration becomes strategic to assist the action of SLN. This research demonstrates the potential of the SLN formulation for transporting drugs to the brain as an alternative anticonvulsant drug in the therapeutic treatment of epilepsy, which produced diminution in the excitatory activity by itself.
ACKNOWLEDGMENTS ´ Cardoso, Jos´e We wish to thank Bernardo Contreras, Raul Luis Calderon, and the students Antonio Escobar and Daniela ´ Silem Chavez for their technical assistance. The authors thank Pierre Olivier Couraud, Ph.D., for donating the cells. This study was partially supported by INP3280, PAPIIT project IN 222411-3 entitled “Evaluation of solid nanoparticles as vectors of drugs,” CONACyT 80811 and 226454. Gerardo Leyva-G´omez ´ Leyva-gomez et al., JOURNAL OF PHARMACEUTICAL SCIENCES 103:2509–2519, 2014
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acknowledges the support provided by CONACyT, Mexico (177426) and the Postgraduate Program in Chemical Sciences at the UNAM, Mexico. The authors declare no conflict of interest. This work is consistent with the Journal’s guidelines for ethical publication.
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