Lamotrigine loaded PLGA nanoparticles intended for direct nose to brain delivery in epilepsy: pharmacokinetic, pharmacodynamic and scintigraphy study

The present study aimed to investigate the brain targeting efficacy of Lamotrigine (LTG) loaded PLGA nanoparticles (LTG-PNPs) upon intranasal administration. LTG-PNPs were fabricated through the emulsification-solvent evaporation technique and evaluated for % Entrapment efficiency, particle size, in-vitro release, surface morphology, crystallinity, ex-vivo permeation & thermal behaviour. Biodistribution, gamma scintigraphy, and pharmacodynamic studies were performed in BALB/c mice, New Zealand rabbits, and Wistar rats respectively. LTG-PNPs exhibited % EE 71%; particle size 170.0 nm; Polydispersity index 0.191; zeta potential −16.60 mV. LTG-PNPs exhibited a biphasic release pattern. Biodistribution and gamma scintigraphy studies proved a greater amount of LTG in the brain following intranasal delivery of LTG-PNPs in comparison to LTG-SOL. Pharmacodynamic studies demonstrated delayed seizure onset time with LTG-PNPs in comparison to LTG-SOL. Intranasal administration of LTG-PNPs provided prolonged release, higher bioavailability, and better brain targeting bypassing the BBB. The developed formulation could be administered as a once-a-day formulation that would reduce the dosing frequency; dose; dose-related side effects; cost of the therapy and would be beneficial in the management of epilepsy as compared to the LTG-SOL. However, the proof of concept generated through these studies needs to be further validated in higher animals and human volunteers.

Keywords: Lamotrigine; nose-to-brain delivery; biodistribution; gamma scintigraphy; PLGA nanoparticles; epilepsy

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According to the World Health Organisation, epilepsy is the fourth most common neurological disorder. People with epilepsy frequently suffer from severe and life-threatening seizure-related injuries. They have 2- to 3-folds higher mortality rates than the general population. Epilepsy-related mortality can be reduced by improved seizure control. This can be managed by anti-epileptic drugs (AEDs). AEDs act by selective modification of excitability of the neurons resulting in inhibition of seizure-specific neuronal firing without affecting normal signals. LTG monotherapy has shown promise in patients suffering from the adverse effects associated with other AEDs. Moreover, LTG monotherapy reduced seizure frequency more effectively in comparison to valproate, carbamazepine, and phenytoin. Clinical studies have suggested a decrement of more than 50% in the frequency of seizure in 13–67% of patients. LTG is a broad-spectrum AED and is effective against partial as well as generalized tonic-clonic seizures when administered alone or as adjuvant therapy in combination with other AEDs. LTG inhibits sodium ion channels and thus maintains the potential of the neuronal membrane. It also suppresses the release of glutamate, an excitatory neurotransmitter [[1]].

Currently, immediate-release (LAMICTAL), extended-release (LAMICTAL XR), orally disintegrating (LAMICTAL ODT), and chewable (LAMICTAL CD) tablets of LTG are approved by USFDA. However, the therapeutic potential of oral LTG and its use in long-term epilepsy management has been hindered due to poor solubility (BCS Class II), non-targeted delivery, inability to cross the BBB, extensive hepatic metabolism, and food effect on the pharmacokinetics of LTG resulting in low oral bioavailability. Various LTG formulations have been tested, including solid dispersion and nanosuspension [[3]]. Although, these approaches have been successful in increasing the solubility, reducing undesirable side effects associated with high-dose, and inability to administer to unconscious patients limit the oral route as a preferred route for LTG in the management of epilepsy. Jafri et al. [[5]] developed an LTG-transdermal patch using Eudragit®RS100 (rate-controlling polymer) and DuroTak® 387-2510 (adhesive). Although this route is non-invasive and provides avoidance of hepatic first-pass effect, bioavailability could be compromised due to efflux mediated by P-glycoprotein (P-gp), which is over-expressed in BBB leading to rapid efflux of LTG. Ammar et al. [[6]] developed intravenous poly-ɛ-(d, l-lactide-co-caprolactone) LTG-nanosuspension. The developed formulation exhibited higher bioavailability with improved brain distribution of LTG in comparison to oral conventional tablets (control formulation). However, it is an invasive route and requires a skilled professional for administration, limiting its use except in emergencies. To date, none of the above-mentioned LTG formations have shown site-specific targeting with high bioavailability in the brain. Thus, the development of LTG formulations using different routes of administration that could overcome these limitations is of scientific interest. Drug delivery to the brain is always challenging due to the need to overcome the BBB. A non-invasive promising approach to deliver actives directly to the brain, bypassing BBB is the intranasal route [[7], [9]]. The nasal delivery is associated with ease of administration, faster onset of action, avoidance of first pass metabolism, large surface area for absorption, reduced non-targeted delivery with lesser systemic exposure patient accessibility, and compliance [[10], [12], [14]]. Serralheiro and his co-workers [[15]] formulated LTG-nasal gel with thermoreversible characteristics that provided release in a sustained manner in comparison to the intravenous delivery and greater LTG concentration in the brain for a very prolonged period. Nanoparticles are colloidal particles with a nano-metric size range (1–1000 nm), composed of synthetic/semi-synthetic polymers [[16]]. At present, there are some studies, wherein the combination of the intranasal delivery route and nanotechnology approaches packaging the drug to enhance brain targeting, reduce the dose and increase the bioavailability of the drug in the brain has been investigated. For instance, Bhattamisra et al. [[17]] developed rotigotine loaded chitosan nanoparticles for nose-to-brain delivery and evaluated their efficacy in human neuroblastoma cells (SH-SY5Y) and the rat model of Parkinson's disease. The results exhibited enhanced targeting efficiency and bioavailability of rotigotine in the brain. Haque et al. [[18]] formulated Venlafaxine-loaded alginate nanoparticles and administered them through the intranasal route for the treatment of depression. Brain Uptake, pharmacokinetic and pharmacodynamic studies suggested the superiority of alginate nanoparticles in comparison to Venlafaxine solution administered via the intranasal and intravenous route. Alam et al. [[19]] developed and optimized LTG nanostructured-lipid-carriers for brain delivery via the intranasal route for the management of epilepsy. The authors concluded that upon IN administration of nanostructured-lipid-carriers loaded with LTG in rats, a higher concentration in the brain could be achieved in comparison to IN and oral drug solution. Yu and his co-workers [[20]] developed an intranasal mixed micellar system based on mPEG-PLA/TPGS to improve the drug delivery in the hippocampus. The results revealed the ability of the formulation to enhance the absorption of LTG in the nasal cavity and reduced the efflux of LTG in the brain.

PLGA nanoparticles exhibit rapid uptake by the brain, sustained delivery of drug, biodegradability, biocompatibility, and less toxicity making them promising carriers for delivering drugs directly into the brain [[21], [23]]. Nigam et al. [[23]] developed LTG-loaded PLGA nanoparticles whereas Lalani et al. [[24]] formulated LTG-loaded PLGA nanoparticles functionalized with Transferrin and Lactoferrin. These results from these studies show that such nanoparticles delivered intranasally provided better biodistribution of the therapeutic drug in the brain and improved pharmacodynamics as well as provide better management of neuropathic pain.

To date, no published studies have evaluated the therapeutic effects of PLGA nanoparticles containing Poloxamer 407 via the intranasal route in an experimental model of epilepsy. The present study deals with the systematic development of CNS targeted LTG-nanoparticles proposed to be administered intranasally for the management of epilepsy. An extensive literature review reveals an absence of any study on the use of biodegradable polymer PLGA for the formulation of LTG nanoparticles. The intranasal route was chosen because of practical limitations faced in administering an antiepileptic drug to an unconscious patient. LTG was loaded in PLGA nanoparticles using the emulsification-solvent evaporation technique [[25]]. New Zealand rabbits and BALB/c mice were employed for gamma scintigraphy and biodistribution studies respectively whereas the pharmacodynamic studies were performed in Wistar rats. It was speculated that nasal administration of LTG-PNPs along with Poloxamer 407 would overcome P-gp efflux resulting in brain targeting and improved antiepileptic efficacy.

LTG and PLGA (50:50) were received as gratis samples from IPCA Laboratories (Mumbai, India) and Evonik Healthcare (Mumbai, India) respectively. Poloxamer 407 was supplied by Sigma-Aldrich (Mumbai, India) whereas acetone, potassium dihydrogen phosphate, sodium chloride, methanol, and acetonitrile were purchased from Merck Scientific (Mumbai, India). Dialysis-membrane (MWCO 12–14 KDa) and polycarbonate syringe filters (0.22 µm) were sourced from Himedia Laboratories Pvt. Ltd. (Mumbai, India).

The emulsification-solvent evaporation technique was employed in the fabrication of LTG-PNPs [[7], [27]]. Organic and aqueous phases were prepared by addition of 25 mg LTG and PLGA in 10 ml of acetone and Poloxamer 407 in 10 ml of double-distilled water respectively. The organic phase was added in a drop-wise manner to the aqueous phase, which was further processed in a high-speed Probe sonicator (BioLogics Inc., Virginia, USA). The resultant mixture was gently stirred on a magnetic stirrer at 25 ± 2 °C to evaporate acetone. Critical material attributes namely the amount of PLGA and surfactant concentration was optimized using a 32 factorial design (data not shown).

The nanoparticle suspension was subjected to centrifugation (Remi Instruments Pvt. Ltd., Mumbai, India) at 20,000 rpm for 20 min at 4 ± 1 °C. The supernatant was collected and analyzed for free LTG by HPLC [[28]] after suitable dilution. % EE was calculated as in Equation (1) [[29]].

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$$\text{Entrapment efficiency}\left(\mathrm{\%}\right)=\left[\frac{(\text{Total amount of drug}-\text{Amount of free drug})}{\text{Total amount of drug}}\right]\mathrm{}\times \mathrm{}100$$ (1)

LTG was estimated by HPLC (Shimadzu LC-2010-CHT, Shimadzu, Tokyo, Japan). LTG separation was performed using a Grace Smart C18 column (Thermo Fisher Scientific, Mumbai, India); (250 × 4.6 mm; 5 μm) maintained at 25 °C. The mobile phase consisting of 25:25:50v/v/v mixture of Methanol:Acetonitrile:Phosphate buffer (pH 7.0) was isocratically pumped at 1 ml/min. LTG retention time was 5.9 min and its estimation wavelength was 225 nm. The calibration range was 15–50 μg/ml (R2 =.9997).

The mean particle size, ζ-potential, and PDI of LTG-PNPs were evaluated by Zetasizer Nano ZS (Malvern Instrument Ltd; UK) based on the dynamic light scattering principle. All the measurements were performed at 25 °C with a fixed angle of 137°. PDI was also determined to evaluate the homogeneity or heterogeneity of the nanoparticle dispersion, wherein PDI <0.5 suggests a monodisperse and homogenous population [[27], [30]].

A modified dialysis bag technique was employed to monitor LTG release. 2 ml LTG-SOL/LTG-PNPs (equivalent to 5 mg LTG) was added to the dialysis bag, which was placed in a 10 ml beaker containing 50 ml of release medium, maintained at 37 ± 2 °C, with continuous stirring. At 1, 2, 3, 4, 6, 8, 12 & 24 h, aliquots (5 ml) were taken from the receptor compartment and replaced with a release medium. The samples were estimated using HPLC after suitable dilutions [[31]].

The data of in-vitro release from LTG-PNPs was fit into release models like zero order, first order, Higuchi, Korsmeyer–Peppas, and Hixon–Crowell [[2], [16]].

For lyophilization [[33]], 2 ml LTG-PNPs and 2 ml 10% w/v mannitol were introduced into vials. The freezing (Labconco, Missouri, USA) was carried out at −50 °C for 48 h followed by primary drying at −30 °C and 150 mTorr for 24 h. The last step consisted of secondary drying at 22 °C and 50 mTorr for 6 h.

Thermograms of LTG and LTG-PNPs were recorded using a differential scanning calorimeter (Shimadzu Corporation, Tokyo, Japan). Empty aluminium pans were taken and samples (10 mg) were crimped followed by scanning at 25–300 °C. Heating rate and nitrogen purging were done at 10 °C/min and 20 ml/min respectively [[16], [34]].

The diffractograms of LTG, PLGA, Poloxamer 407, and LTG-PNPs were taken on a Rigaku Miniflex Diffractometer (Applied Rigaku Technologies, Inc., Texas, USA). The samples were mounted on a sample holder and diffractograms were recorded in an angular range of 0–80° 2θ at a step size of 0.01° and a scan rate of 1 s/step [[35]].

TEM of LTG-PNPs was performed using Jeol JEM 2100 (Jeol Limited, Tokyo, Japan), operating at an acceleration voltage of 120 kV. LTG-PNPs were fixed on carbon-coated copper microgrids. The sample was dried at room temperature (25 ± 2 °C) and treated with a phosphotungstic acid solution (2% w/v) and imaged in the microscope [[36]].

The spectra for LTG , and LTG-PNPs were recorded in ATR mode FTIR spectrophotometer (Bruker Optics, Germany). Scanning was done in the range of 500–4000 cm−1 at a resolution of 2 cm−1 [[2], [16]].

Ex-vivo permeation was performed using goat nasal mucosa, which was mounted on a Franz diffusion cell (0.58 cm2). 1 ml LTG-SOL/LTG-PNPs (equivalent to 2.5 mg LTG) was kept in the donor chamber. The receptor chamber comprised 20 ml of 30% methanolic phosphate buffer (pH 6.4), maintained at 37 ± 2 °C, under constant stirring. At fixed time intervals, samples were withdrawn from the receptor chamber and analyzed.

Histopathological studies of LTG-PNPs were performed and compared with PBS (negative control), IPA (positive control), and LTG-SOL. Freshly excised goat nasal mucosa was incubated with sample followed by rinsing of the mucosa with PBS (pH 6.4), 2 h post-treatment. The mucosa was fixed in 10% buffered formalin and embedded in paraffin. Staining was performed with haematoxylin and eosin. Pathologists reviewed coded slides to eliminate bias [[9], [37]].

The radiolabelling of LTG was done with 99mTc by direct labelling method with stannous chloride under reductive conditions [[38]]. 99mTc-LTG-SOL was further employed in gamma scintigraphy and biodistribution studies [[39]].

The radiolabelling of LTG-PNPs was performed similarly to the radiolabeling of LTG-SOL. 99mTc-LTG-PNPs were employed in gamma scintigraphy and biodistribution and studies [[39]].

Biodistribution studies were as per protocol approved by the Institutional Animal Ethics Committee vide INM/IAEC/16/03 and CPCSEA guidelines. Healthy BALB/c mice (20–25 g) were anaesthetized with Xylazine and Ketamine cocktail anaesthesia. The animals were distributed into the following groups:

Gr.A: 99mTc- LTG-SOL (20 µCi/10 µl) (0.001 mg LTG; equivalent to 0.833 mg/kg body weight) injected by i.v. route through tail vein.

Gr.B: 99mTc- LTG-SOL (20 µCi/10 µl) (0.001 mg LTG) administered by the intranasal route.

Gr.C: 99mTc- LTG-PNPs (20 µCi/10 µl) (0.001 mg LTG) administered via intranasal route.

Each group was further subdivided, wherein each subgroup consisted of 3 animals. After formulation administration (t = 0 h), sacrifice done at the following time points:

Subgroup 1:15 min.

Subgroup 2:30 min.

Subgroup 3:60 min.

Subgroup 4:120 min.

Subgroup 5:240 min.

Subgroup 6:480 min.

At different time points, blood collection was done by cardiac puncture. Brain dissection was carried out followed by washing it with normal saline. Radioactivity in blood and brain was determined using a gamma scintillation counter (Capintec, Inc. New Jersey, USA). % radioactivity/g in brain and blood was calculated and plotted vs. time. Cmax, Tmax, and AUC0–480 were calculated using WinNonlin software (Pharsight Corporation NC, USA).

Brain targeting indices-DTE & DTI [[2], [23], [41]] were calculated as below:

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$$\text{DTE \%}=\mathrm{}\frac{{\mathrm{AUC}}_{\text{brain i}.\mathrm{n}.}\mathrm{}}{{\mathrm{AUC}}_{\text{blood i}.\mathrm{n}}}\mathrm{}\times \mathrm{}100$$ (2)

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$$\text{DTI \%}=\mathrm{}\frac{\raisebox{1ex}{${\mathrm{AUC}}_{\text{brain i}.\mathrm{n}}$}\!\left/ \!\raisebox{-1ex}{${\mathrm{AUC}}_{\text{blood i}.\mathrm{n}}$}\right.}{\raisebox{1ex}{${\mathrm{AUC}}_{\text{brain i}.\mathrm{v}}$}\!\left/ \!\raisebox{-1ex}{${\mathrm{AUC}}_{\text{blood i}.\mathrm{v}}$}\right.}\mathrm{}\times \mathrm{}100$$ (3)

Gamma scintigraphy imaging was performed on NewZealand rabbits as per IAEC protocol INM/IAEC/16/03. Healthy rabbits (2.0–2.5 kg) were anaesthetized by Xylazine (5 mg/kg) and Ketamine (40 mg/kg). 99mTc-LTG-SOL and 99mTc-LTG-PNPs (100 µCi/100 µl; containing 0.071 mg LTG) were administered intranasally (50 µl per nostril). The rabbits were placed on an imaging board and imaging was done (SPECT LC75–005, Diacam Siemens AG, Germany) [[41]].

The anticonvulsant activity of LTG-formulations was performed as per protocol MPC/IAEC/06/2017 in Albino Wistar rats (male; 200–350 gm) [[2]]. Random distribution of the animals was done into four different groups as given below:

Group A: Normal saline (intranasal administration).

Group B: LTG-SOL (intranasal administration; 0.833 mg/kg BW)

Group C: LTG-SOL (intravenous administration; 0.833 mg/kg BW)

Group D: LTG-PNPs (intranasal administration; 0.833 mg/kg BW)

Each group consisted of 18 animals and was subdivided as follows; each subgroup containing 6 animals. After administering the formulation, PTZ (100 mg/kg BW) was given at the following time points to induce convulsions.

Subgroup 1: After 15 min.

Subgroup 2: After 30 min.

Subgroup 3: After 60 min.

The ability of the formulation to delay the onset of seizure was recorded.

A stability study of LTG-PNPs was done at 4 ± 2 °C and 25 ± 2 °C/60 ± 5% RH (Hicon Instruments, New Delhi, India). The samples were withdrawn at 3 M and 6 M intervals and monitored for % EE, particle size, and % CDR at 24 h [[42]].

The results are expressed as Mean ± SD. Statistical analyses were performed using an independent t-test and one-way ANOVA by employing SPSS®. A value of p <.05 was considered significant.

The emulsification-solvent evaporation method [[2], [16]] was employed in the manufacturing of LTG-PNPs. The optimization was performed using 32 factorial design (data not shown). The optimized formulation parameters along with the results are depicted in Table 1. LTG-PNPs had an entrapment efficiency of around 71%, a mean particle size of 170 nm, PDI 0.191, and zeta potential of 16.60 mV. A size <200 nm has higher chances of getting internalized into brain cells, making intranasal LTG-PNPs a suitable option for brain targeting.

Table 1. Optimized formulation parameters for LTG-PNPs.

LTG-SOL exhibited incomplete release of only 42% (Figure 1) after 24 h due to poor aqueous LTG solubility [0.17 mg/ml; BCS Class II]. Complete release (94.5%) from LTG-PNPs could be explained by nanometric size range and the presence of Poloxamer 407, which improves permeation, wetting, solubilization, and dissolution by forming pores in the matrix and facilitates LTG release.

PHOTO (COLOR): Figure 1. In-vitro release profiles of LTG-SOL & LTG-PNPs.

LTG-PNPs exhibited a biphasic release pattern, wherein rapid release (19.83%) occurred within the first 2 h and the remaining 75–80% LTG released in a prolonged fashion in the remaining 22 h. The results are in line with the findings of other investigators [[2], [43]]. Initial burst release could be due to adsorption of LTG on the surface of polymeric nanoparticles, the occurrence of the free LTG in the external phase, and a large surface:volume ratio. Prolonged-release was due to LTG's complete and homogeneous encapsulation within the matrix of PLGA causing slow diffusion towards the aqueous media [[44]]. Thus, with LTG-PNPs, clinicians can achieve loading and maintenance doses due to initial burst release and prolonged release respectively. This dual release pattern is advantageous in preventing the fluctuations in the LTG plasma level, thereby providing favourable pharmacokinetics.

LTG-PNPs followed the Higuchi model kinetics (R2 =.99) and Korsmeyer–Peppas model (R2 =.9987). The n (release exponent) value was 0.6298, suggesting anomalous transport which is considered to be a combination of polymer erosion/degradation and drug diffusion mechanisms [[1], [46], [48]].

LTG (Figure 2(A)) exhibited a sharp endothermic peak at 217.02 °C referring to its melting temperature and suggesting its crystalline nature. The sharp endothermic peak of LTG disappeared in the LTG-PNPs (Figure 2(B)), suggesting that LTG is present inside the lipid matrix provided by PLGA and its transition has occurred from the crystalline to amorphous state. These observations are in agreement with the results reported by other authors [[19], [24]].

PHOTO (COLOR): Figure 2. DSC Thermogram of (A) Pure LTG (B) LTG-PNPs.

LTG shows sharp and intense peaks at 8.2°, 10.2°, 13.1°, 25.9°, and 27.9° 2θ, suggesting the crystalline nature of LTG (Figure 3(A)). The XRD pattern of PLGA exhibited a dome-shaped region (Figure 3(B)) and the absence of sharp intense peaks, which indicated that it is amorphous. Figure 3(C) shows a sharp and intense peak of Poloxamer 407. The sharp and intense peaks of LTG were found to disappear in the XRD scan of LTG-PNPs (Figure 3(D)) indicating the amorphous nature of LTG, which is molecularly dispersed in the PLGA matrix. Similar observations were reported by Seju et al. [[12]].

Graph: Figure 3. X-ray diffractogram of (A) pure LTG (B) PLGA (C) Poloxamer 407 (D) LTG-PNPs.

TEM of LTG-PNPs (Figure 4) exhibited spherical, discrete particles with nanometre size range homogeneously distributed throughout the formulation and possessing narrow, uniform size distribution. The findings of this study are in line with particle size and PDI measurements.

PHOTO (COLOR): Figure 4. TEM image of LTG-PNPs.

FTIR chromatograms of pure LTG (Figure 5(A)) exhibited characteristic peaks at wavelengths 716, 791, 1140, 1617, 3446 cm−1, which were found corresponding to C = H, C–Cl, C–N, C = C, and N–H stretching respectively. The presence of LTG characteristic peaks and the absence of extra peaks in LTG-PNPs (Figure 5(B)) indicated compatibility between LTG and excipients [[49]].

PHOTO (COLOR): Figure 5. FTIR spectra of (A) Pure LTG (B) LTG-PNPs.

The permeability coefficients for LTG-SOL & LTG-PNPs were found to be 1.316 (±0.27) × 10−3cm/h and 3.264 (±0.33) × 10−3cm/h, respectively. LTG-PNPs exhibited 2.5-fold higher drug permeation when values were compared with LTG-SOL (Figure 6). This could be justified by the nanometric size of LTG-PNPs and the presence of Poloxamer 407 on the surface of nanoparticles, which is a well-known permeation enhancer. Poloxamer 407 adherence to the nasal mucosa occurs via hydrogen bond formation. Additionally, Poloxamer 407 also acts as an inhibitor of P-gp (efflux pump) and thereby facilitates the permeation of LTG (P-gp substrate) [[50]].

PHOTO (COLOR): Figure 6. Ex-vivo permeation of LTG-SOL & LTG-PNPs across goat nasal mucosa.

No significant signs of damage/harmful effects (such as erosion, inflammation, or cell destruction) were observed in the histopathology of nasal mucosa treated with LTG-PNPs (Figure 7(D)). The images were similar to that of PBS (negative control; Figure 7(A)), indicating the safety of LTG-PNPs for intranasal administration. Heavy mucosal damage (indicated by the detachment of cilia, cell necrosis, and widening of the epithelial cell) was visualized in the mucosa treated with IPA (Figure 7(B)) & LTG-SOL (Figure 7(C)). Similar observations were reported by Seju et al. [[12]].

PHOTO (COLOR): Figure 7. Histopathological condition of goat nasal mucosa after treatment with (A) Phosphate buffer saline (PBS) pH 6.4 (B) Isopropyl alcohol (IPA) (C) LTG-SOL (D) LTG-PNPs.

PHOTO (COLOR): Figure 8. Drug concentration versus time plot upon administration of 99mTc-LTG-SOL (intranasal and intravenous). and 99mTc-LTG-PNPs (intranasal) in BALB/c mice in (A) blood (B) brain.

The radioactivity in the brain and blood are recorded in Table 2. % radioactivity/g vs. time in blood and brain was represented in Figure 8(A,B) respectively. AUC0–480, Cmax, and Tmax are represented in Table 3. LTG-PNPsi.n. showed higher brain/blood ratios as compared to LTG-SOLi.v. & LTG-SOLi.n. indicating better brain targeting. Brain AUC0–480 for LTG-PNPsi.n. was 1.8 & 15.8 folds higher than the values for LTG-SOLi.v. and LTG-SOLi.n. indicating the better ability of PLGA based LTG nanoparticles to enhance the drug availability at the target site. The relative superiority of LTG-PNPs for brain delivery in comparison to LTG-SOL could be explained by the nanoparticulate nature; avoidance of enzymatic degradation; enhancement in permeation and P-gp inhibition by Poloxamer 407 [[13], [52]]. Upon intravenous administration of LTG-SOL, AUC0–480 blood values were 11 folds higher than AUC0–480 brain values suggesting major accumulation of the formulation in systemic circulation and only minor amounts in the brain, which is due to the inability of LTG present in LTG-SOL to cross BBB.

Table 2. Biodistribution of 99mTc-LTG-SOLi.v., 99mTc-LTG-SOLi.n., 99mTc-LTG-PNPsi.n. in blood and brain at different time intervals in BALB/c Mice*.

1 *Each value is mean ± SD of three estimations.

Table 3. Pharmacokinetic Parameters of 99mTc-LTG-SOLi.v., 99mTc-LTG-SOLi.n. and 99mTc-LTG-PNPi.n. in BALB/c Mice.

AUC0–480 blood and brain values for LTG-SOL administered intranasally were substantially lower than corresponding counterpart AUC values for intravenous LTG-SOL & LTG-PNPs. This could be due to the brief contact time of the nasal solution which is due to the absence of any viscosity modifier. DTE and DTI for 99mTc-LTG-SOL & 99mTc-LTG-PNP are shown in Table 4, indicating 1.8-folds higher values of DTE and DTI for nanoparticles than solution indicating a significantly (p <.05) higher amount of LTG accumulation in the brain exhibiting promising outcomes via intranasal administration. The brain elimination T1/2 of 99mTc-LTG-PNPsi.n., 99mTc-LTG-SOLi.n. & 99mTc-LTG-SOLi.v. were 963.33 min, 336.32 min & 305.21 min, respectively. 3-folds increase in T1/2 signifies that nanoparticles slow the decline in brain concentration in the elimination phase.

Table 4. Drug targeting efficiency [DTE (%)] and direct target organ transport [DTI (%)] following administration of 99mTc-LTG-SOLi.n. and 99mTc-LTG-PNPsi.n.

After administering 99mTc-LTG-SOL/99mTc-LTG-PNPs, images were recorded at 15, 30 and 60 min post-administration (Figure 9(A,B)). Only a trace quantity of LTG-SOL could reach the brain whereas most of LTG reached the buccal cavity. LTG reaching the buccal cavity gets transported to the stomach area through the oesophagus [[53]]. Conversely, administrations of LTG-PNPs resulted in greater amounts of LTG getting accumulated in the brain, suggesting targeted drug delivery. Similar findings were reported by other researchers [[42]].

PHOTO (COLOR): Figure 9. Gamma scintigraphy images in rabbits following intranasal administration of (A) 99mTc-LTG-SOL. and (B) 99mTc-LTG-PNPs.

The onset time of seizures of LTG-SOL & LTG-PNPs was recorded (Table 5) and represented in Figure 10. All treatment groups exhibited a delay in onset of seizures as compared to the control group, confirming the antiepileptic effect of LTG. In subgroup 1, onset times of seizures were 131.36 s (control group); 187.08 s (intranasally administered LTG-SOL), 160.68 s (intravenously administered LTG-SOL), and 241.55 s (intranasally administered LTG-PNPs). Intravenous administration of LTG-SOL exhibited only a slight delay in the onset of seizures. This could be explained by only a marginal amount of LTG crossing the BBB and the majority being present in the systemic circulation. Intranasally administered LTG-SOL & LTG-PNPs exhibited a delay in the onset of seizures as compared to i.v. administration of LTG-SOL, confirming the ability of the nasal route to provide direct access to the brain. However, the delay in the onset of seizures was not very significant since the nasal formulations could not have reached the brain within 15 min. This was confirmed by the 30 min and 120 min Tmax of LTG-SOL and LTG-PNPs respectively.

PHOTO (COLOR): Figure 10. Onset time of seizures in Pentylenetetrazole (PTZ)-induced convulsions in Albino Wistar rats.

Table 5. Time (s) for the development of seizures in male Albino Wistar rats (n = 6).

• 2 *p <.05; Significant as compared to control group, One-way ANOVA followed by Tukey's test.
• 3 *$<.05; Very significant as compared to LTG-SOL i.n. & LTG-PNPs i.v., One-way ANOVA followed by Tukey's test.

In subgroup 2, onset times of seizures were 135.25 s (control group); 255.78 s (intranasally administered LTG-SOL), 165.26 s (intravenously administered LTG-SOL), and 440.84 s (intranasally administered LTG-PNPs). In subgroup 3, onset times of seizures were 128.45 s (control group); 210.06 s (intranasally administered LTG-SOL), 160.35 s (intravenously administered LTG-SOL), and 355.85 s (intranasally administered LTG-PNPs). In both the subgroups, delay in onset of seizures upon administering LTG-PNPs is very significant (p <.05) as compared to intranasal and intravenous administration of LTG-SOL.

The improved efficacy exhibited by the nasally administered nanoparticles indicates a higher amount of LTG reaching the brain and exhibiting greater therapeutic action. The higher amount can be attributed to nanosize, increased surface area, and Poloxamer 407's ability to inhibit P-gp mediated efflux. Our findings of pharmacodynamic studies are in agreement with the results of our gamma scintigraphy and biodistribution studies.

The results of stability studies are shown in Table 6. LTG-PNPs were stable at 4 °C for 6 M and 25 °C/60% RH for 3 M as there was no significant change in % EE, particle size, and % CDR at 24 h. At 25 °C/60% RH, changes in critical quality attributes beyond 3 M could be due to aggregation of particles and degradation of the polymer. Based on this study, it was concluded that LTG-PNPs should be stored at 4 °C where they would remain stable.

Table 6. Stability study of LTG-PNPs (P10).

In the present investigation, Lamotrigine could be successfully entrapped in PLGA based nanoparticles using the Emulsification-solvent evaporation technique. The optimization was carried out by 32 full factorial design to achieve desired properties using different amounts of PLGA and concentrations of Poloxamer 407. The optimized formulation showed satisfactory entrapment efficiency and amorphization of the LTG by getting entrapped within the PLGA matrix. DLS measurements suggested nanometric size and narrow size distribution, which was confirmed by TEM imaging. Dissolution profiles of LTG-PNPs suggested a biphasic release pattern, providing loading and maintenance dose. Cytotoxicity was evaluated using excised goat nasal mucosa, which suggested PLGA nanoparticles be a safe carrier. Gamma scintigraphy imaging in rabbits where suggested localization of LTG in the brain upon administration of intranasal LTG-PNPs which were validated by biodistribution studies done in BALB/c mice. Pharmacodynamic studies also confirmed the delay in the onset of convulsions by LTG-PNPs in comparison to LTG-SOL administered by the intranasal and intravenous route. Intranasal administration of LTG-PNPs provided prolonged release, higher bioavailability, and better brain targeting bypassing the BBB. The developed formulation could be administered as a once-a-day formulation in epilepsy that would reduce the dosing frequency; dose; dose-related side effects; cost of the therapy and would be beneficial in the management of epilepsy as compared to LTG-SOL. However, the proof of concept generated through these studies needs to be further validated in higher animals and human volunteers.

No potential conflict of interest was reported by the author(s).