ZOLMITRIPTAN BRAIN TARGETING VIA INTRANASAL ROUTE USING SOLID LIPID NANOPARTICLES FOR MIGRAINE THERAPY: FORMULATION, CHARACTERIZATION, IN-VITRO AND IN-VIVO ASSESSMENT

Objective: Zolmitriptan, a class of antidepressant drugs with poor bioavailability due to its first-pass metabolism. The aim of this study was to improve systemic bioavailability and explore the brain targeting impact of nasal Zolmitriptan (Zol) solid lipid nanoparticles (SLNs) gel for migraine treatment. Methods: Stearic acid and cholesterol used as solid lipid and lecithin as a surfactant, emulsion solvent evaporation technique was used to produce Zolmitriptan SLNs. (Zol) SLNs were characterized for particle size, percent entrapment efficiency and in vitro drug release. Formula S6 showed greater percent entrapment efficiency (PEE), adequate particle size and sustained drug release behavior. Formula S6 was integrated into HPMC gel (3%) to prepare nasal gel. Zol SLN nasal gel was subjected to histopathological study to ensure brain targeting. Results: Conclusion: Intranasal administration of Solid lipid nanostructure of Zolmitriptan through the olfactory pathway in which it travels from the nasal cavity to brain tissue achieved drug targeting potential of about 90% compared with conventional Zolmitriptan tablets. The small particle size helped them to squeeze themselves through the small opening in the olfactory neurons to the brain via different endo-cystic pathways of neuronal cells in nasal tissue membranes. It was observed that all prepared Zol SLNs were in the nano-sized range with a polydispersity index of<0.5. In the cholesterol/lecithin combination, higher PEE%, better stability, and less agglomeration inclination were discovered. Results of the release profiles showed that developed Zol-SLNs were able to release Zolmitriptan in a sustained manner. Histopathological study of the brain tissues showed that Zolmitriptan SLN nasal gel can reach brain cells and localized for 24 h although the hydrophobicity of the target drug.


INTRODUCTION
Migraine is a disabling neurovascular disease with mostly unilateral throbbing headaches with a host of neurological symptoms including hypersensitivity to light, sound, smell, nausea and variety of autonomic and cognitive, emotional and motor disturbances [1]. Migraine mechanism suggests that vasoactive peptides will be released early in the attack from the primary sensory nerve terminals that innervate meningeal blood vessels. These peptides activate perivascular trigeminal nerves and cause dilatation of arteries in the meninges as well as perivascular inflammation and extravasation of plasma proteins [2]. Several triptans are 5HT1B/1D receptor agonists commonly prescribed for migraine headaches. Zolmitriptan is 4(S)-4-[3-(2-dimethyl aminoethyl)-1H5-indolyl-methyl]-1,3-oxazolan 2 one, potent and selective serotonin (5-HT1B/1D) receptor agonist which causes vasoconstriction of the blood vessels of the brain [3]. Also, reducing sterile inflammation associated with zolmitriptan antidromic neuronal transmission is another mechanism by which acute migraine attacks have been reported to be relieved [4]. It is currently available as a conventional tablet, an orally disintegrating tablet and a nasal spray (2.5 mg and 5 mg per dose) [5]. Zolmitriptan's absolute bioavailability is up to 40% for both oral and nasal dosage forms and has a very brief half-life of 1-2 h with first-pass metabolism and rapid hepatic and renal clearance. This makes the oral route unsatisfactory [6]. Zolmitriptan nasal spray administration resulted in a quicker onset of action. However, clinical evidence shows no significant improvement in other pharmacokinetic parameters, such as half-life, bioavailability and therapeutic gain, over the oral dosage forms [4]. For these reasons, our objective was to overcome the abovementioned drawbacks by formulating Zolmitriptan's nasal gel delivery system for brain targeting.
Solid Lipid Nanocarriers (SLNs) enhance the permeability of drugs owing to lipid and surfactant content and thus improve bioavailability through oral, parenteral, dermal, nasal, ocular and pulmonary routes of administration due to their tiny size [11]. Their nano-size enables them to be transcellular transported to the brain via olfactory neurons through the different endocytic pathways of neuronal cells in the olfactory membrane [12]. We aimed to improve the systemic bioavailability of Zol due to the first-pass metabolism and explore the brain targeting the impact of nasal Zol (SLNs) gel for migraine treatment.
The present research focuses on the design of Zolmitriptan solid lipid nanoparticles (SLNs) to be delivered to the brain via an intranasal route. Solid lipid nanoparticles provide an enhancement of nose-to-brain drug delivery as they enable hydrophilic molecules to pass through the lipophilic BBB. They can also safeguard the encapsulated drug against biological and/or chemical degradation [7]. The delivery of drugs to the brain is a major challenge owing to the presence of the blood-brain barrier (BBB) [8]. Various techniques have been used to enhance drug delivery to the brain. One of these techniques includes nanotechnology-based drug transport. The intranasal route used for the delivery of many drugs to the central nervous system (CNS) [9]. Intranasal delivery does not require any modification of drugs [10].

Methods
Formulations investigated in the present work are listed in [table 1]. The blank-and-drug loaded SLNs were prepared by the emulsion solvent evaporation method (o/w) followed by homogenization [13][14][15].

Formulation of SLNs of zolmitriptan
Stearic acid or Soy lecithin and cholesterol mixture were accurately weighed and dissolved in 10 ml DMSO as the internal oil phase. Then heated above the melting point of lipid at 70 °C. An aqueous surfactant solution of zolmitriptan was prepared by dissolving zolmitriptan in 5 ml methanol and adding it with tween 80 to 20 ml water. The organic phase was then poured drop by drop into a homogenizer tube containing 20 ml of the hot aqueous phase. The mixture was then homogenized (Remi Instruments Pvt. Ltd, Mumbai, India) for 30 min at 3000 rpm to form a primary emulsion (o/w) and stirred to extract the organic solvent into the continuous phase and for proper solidification of SLNs. The stirring was continued (2-3 h) at 3000 rpm to get SLN dispersion.
Zolmitriptan percentage entrapped in SLNs and the amount released was measured utilizing a UV spectroscopy (Jenway spectrophotometer, Model 6105UV/Vis, England). The maximum absorption of Zolmitriptan in phosphate buffer pH 6.8 was found to be at 283 nm [16,17]. Zolmitriptan calibration curve in pH 6.8 phosphate buffer at a range of 1-10 ug/ml at 283 nm was plotted. The specificity of the analytical method was accomplished using a blank formula (Blank SLNs) prepared from all the excipients used except drug to verify whether any element of the formulation or dissolution medium could interfere with the absorption of Zolmitriptan at a chosen wavelength.

The standard calibration curve and specificity of the analytical method used for Zolmitriptan determination
A volume of 2 ml of each Zolmitriptan loaded SLNs was centrifuged at 15000 rpm for 45 min to separate the lipid and aqueous phase. The supernatant was then diluted with methanol and filtered through 40 um filter paper. The amount of unentrapped Zolmitriptan in the supernatant was determined spectrophotometrically at λ

EE% =
283 [18]. The EE percentage was calculated using the following equation where Wi is the amount of initial drug and Wf is the amount of free drug: [19,20]. PEE values given are the averages of three estimations.

x100
Particle size estimation of the dispersions performed utilizing a Zeta-sizer 3000 PCS (Malvern Instr., England) outfitted with a 5mW helium-neon optical device. Estimations were made at 25 °C, edge 90 °, run time in any event 180 sec. The samples were appropriately dispersed in deionized water preceding the estimations. The particle size values given are the averages of 3 estimations over 5 min each.

Particle size, polydispersity index (PDI) and zeta potential analysis
It is an ultra-high-resolution scanning probe microscopy (SPM). The morphology of SLNs was examined by (AFM) (Nanosurf Easyscan 2 AFM). The experiments were conducted in the water at room temperature (20±1 °C) under atmospheric pressure operating in non-contact mode. For this analysis, triangular silicon tips have been used. This cantilever's resonant frequency was about 120 kHz.

Atomic force microscopy (AFM)
TEM (Philips, Tecrai10, Dutch) was used to examine the shape and surface morphology of the prepared zolmitriptan SLNs using a 300mesh carbon-coated copper grid and phosphotungstic acid (1%; w/v) as a negative stain. After being stained, samples were allowed to dry at room temperature for 10 min for investigation.

Transmission electron microscopy (TEM)
Scanning electron microscopy (SEM) was used to study the morphological characteristics and texture of SLNs by JSM5910 (JEOL, Japan). SEM micrographs were recorded at a magnification of 60,000x and an accelerating voltage of 20 kV.

Scanning electron microscopy (SEM) studies
Using FT-IR studies (IR Prestige 21 Shimadzu, Japan) the potential interaction between the solid lipid core and the incorporated drug was investigated. The FT-IR analysis of Zolmitriptan, physical mixtures and zol loaded SLNs in a ratio of 1:1), blank-SLNs were performed with a resolution of 2 cm Fourier transfer infrared (FT-IR) spectroscopy studies −1 in the range between 4,000 and 500 cm −1 .

In vitro drug release of zolmitriptan from SLNs
The and kinetic analysis of the release data in vitro drug release of Zolmitriptan from SLNs was performed utilizing the dialysis bag technique [21]. In 12 h distilled water presoaked cellulose dialysis bag (cellulose membrane, 12,000-14000 D cut off molecular weight, Visking®, Medicell, London, UK), Zolmitriptan loaded SLNs dispersion equivalent to 10 mg of Zolmitriptan were accurately weighed and placed inside the dialysis bag and both ends of the bag were strongly closed. The bag was immersed into a beaker containing 50 ml phosphate buffer solution (PBS) of pH 6.8 which served as the receptor cell and placed in a shaker water bath at 37±0.5 °C and agitated at 100 rpm [22]. At time intervals from 0.5 to 5 h, 3 ml were removed from the receptor cell for each sample and replaced by equivalent volumes of the fresh release medium and maintained at a similar temperature. Tests were estimated spectrophotometrically at λmax 283 nm against equivalent phosphate buffer as a blank using Jenway spectrophotometer (Model 6105UV/Vis, England) [23]. The quantities of the released drug were calculated on the idea of the previously made calibration curve. Triplicate samples were analyzed and the average concentration was adopted. In vitro drug release data of Zolmitriptan from prepared SLNs were fitted to various kinetic models and analyzed to explain the mechanism of drug release.
The selected Zolmitriptan loaded SLN formula (S6) was evaluated for stability by storing and sealing in well-closed containers at 5±1, 25±1 and 45±1 °C for three weeks. The stability study was performed according to different parameters including; physical appearance, %EE, particle size (PS), and zeta potential (ZP). The changes in %EE, particle size, and zeta potential against storage time were monitored.

Formulation of zolmitriptan loaded SLNs
Formulation of Zolmitriptan loaded SLN that gave maximum entrapment, small particle size, small PDI, high zeta potential and better-sustained release effect was selected for preparation nasal gel (Tables 2 and 3). HPMC E15 polymer was used as a gelling agent for SLNs dispersion. A 3% concentration of HPMC E 15 polymer was used. HPMC E15 was added to the nanoparticle dispersion under overhead stirring at 250 rpm then, the dispersion was neutralized using 0.05% (w/w) triethanolamine. The gel was allowed to stand overnight to remove the entrapped air. The same gel formula was used to prepare a control gel containing pure drug at the same concentration.

Visual appearance, pH and drug content
Visual appearance was observed for the detection of any particular matter. The pH was measured using a calibrated digital pH meter (Model 420, ORION, USA) utilizing a pH of 4.0 and 7.0 standard buffers before use. The pH was noted by bringing the electrode close to the surface of the formulations and allowing it to equilibrate for one minute. The pH of the gel was measured at 25 °C. Gel was then diluted with methanol and filtered through 40 um filter paper. The of zolmitriptan loaded SLNs nasal gel amount of Zolmitriptan was determined spectrophotometrically at λ max 283 to determine drug content.

Viscosity study of zolmitriptan loaded SLNs nasal gel
Viscosity of the gel was measured on Brookfield viscometer (DV-II+Pro Viscometer, Middleboro, USA). The gel formula was placed in a small sample adaptor, and the viscosity was measured using spindle LV-63 at 20 rpm.

In vitro drug release study
The dialysis bag diffusion was used to study Zolmitriptan loaded SLNs gel in vitro release. Zolmitriptan loaded SLNs gel (equivalent to10 mg of the drug) were put in the dialysis bag. The method was performed as shown before at time intervals from 1 to 24 h, a placebo SLNS gel experiments were conducted. Samples were removed at predetermined time intervals from the receptor compartment and replaced by the fresh medium. The dissolved drug quantity was determined with UV spectrophotometry at λmax 283 nm against a blank. All experiments were performed in triplicate.
1-Group I: received plain gel (3% of HPMC E 15 gel without drug) to serve as a negative control group and sacrificed after 1 hour.

Histopathological study of brain tissue
In vivo histopathological study of brain tissues carried out of the previously chosen S6 zolmitipitan SLNs gel and plain gel (3% of HPMC E 15 gel without drug) on male albino rats (adult/weighing 200-250 g). The protocol of the study was approved by the October University of Modern Science and Arts (MSA) Ethics Committee (PT8/ec8/2018f). The animals were kept under standard laboratory conditions, received a balanced diet of commercially available pellet rat feed, water and libitum, i.e. temperature of 22±3 _C and relative humidity of 30-70% under a 24-hour light/dark cycle. The animals were housed in polypropylene cages, four animals per cage with free access to standard laboratory diet and water. Rats were sacrificed using thiopental (50 mg/kg) and brains were removed, cleaned with cold saline (0.9%). Brain tissues fixed in 10% formalin and embedded in paraffin wax were sonicated at 6 µm thickness on a rotatory microtome, then stained with eosin and hematoxylin [24]. The stained sections were observed using the digital microscope and digital photomicrographs by using the attached camera.
Statistical analysis was performed using the GraphPad Prism version 8.1.2 software utilizing analysis of variance (ANOVA) or the paired ttest, where appropriate, and statistical significance was set at <0.05.

RESULTS AND DISCUSSION
Zolmitriptan exhibits its maximum absorption at 283 nm and obeyed Beer's law in the range of 1-10 µg/ml. There was a good linear relationship between the Zolmitriptan concentration and its absorption (R The standard calibration curve and specificity of the analytical method used for Zolmitriptan determination 2 = 0.996).  [25] also, the lipid types highly affected the PEE%. The mixture of cholesterol and lecithin showed significantly greater levels of PEE than stearic acid (S4, S5 and S6) at p<0.001. It could be explained by the length of cholesterol is about 13.8A˚, which is much shorter than the length of stearic acid. A shorter length could generate more random molecular packing, allowing more space to drug accommodation [26]. The increase in drug entrapment efficiency might be also, due to lecithin effect: high Tc (phase transition temperature), decrease in membrane permeability, therefore, preventing drug leakage, hence the increase in Zolmitriptan content within the prepared SLN [27]

Percent entrapment efficiency (PEE %) of zolmitriptan in SLNs
. Vijayan et al. showed that the entrapment efficiency (%) was directly proportional to lecithin concentration and obviated that low entrapment efficiency was due to low affinity between drugs at different solvent (organic and aqueous) [13]. Manjunath and his colleague revealed that the entrapment efficiency of the drug in the lipids depended upon the variable characters such as miscibility, the solubility of the drug in the lipid melt, physical and chemical structure of the lipid matrix and crystalline state of lipid components [28]. Varshosaz et al. showed that by increasing the amount of cholesterol in SLN, the PEE is much increased [29]. Particle size measurement was done to ensure that particles of the Zol-SLNs in the nanometer range. We observed that all prepared SLNs were in the nano-sized range (average particle size values ranged from 144.9±40.3 nm to 581.2±29.6 nm), with a polydispersity index of<0.5 as shown in [table 2]. The particle size increased significantly with the increase of lipid concentration (Stearic acid and cholesterol) at p<0.0001. The results indicate that the amount of stearic acid has a positive influence on the mean size of the SLN. It is logical that increased particle size was observed with higher amounts of stearic acid. Lack of sufficient surfactant to cover the particle surface is the likely reason for increased particle size [30]. The particle size of SLNs prepared from the combination of cholesterol and lecithin was significantly greater than those prepared from stearic acid at p<0.0001, but by increasing the concentration of lecithin to 75 mg it showed a greater reduction in particle size of cholesterol SLNS to 248.8±9.1 at p<0.0001, which could be attributed to the excessive surfactant molecules present rapidly cover the new surfaces reducing the surface tension and thus facilitating the particle partition during emulsification [31]. As a result, an increase in the concentration of surfactant (Lecithin) results in a reduction in the SLN particle size. The interaction between cholesterol and surface-active agent might increase in surface curvature of oil droplets and leading to the small size of SLN after cooling down [32]. Jain et al. observed that lecithin addition prevents the agglomeration of particles [33]. Measuring zeta potential allows for predictions of colloidal dispersion storage stability [33]. Particle aggregation is less likely to occur for charged particles with high zeta potential due to electric repulsion. Compared to SLNs formulations, formulations containing cholesterol and lecithin combination showed a significant greater zeta potential values at p<0.01 as shown in table 2. Severino et al. showed that stearic acid nanoparticles were negatively charged, but the zeta potential was rather low [34].

Particle size, polydispersity index (PDI) and zeta potential analysis
AFM images of Zolmitriptan loaded SLN showed that lipid composition did not influence nanoparticle morphology. Nanoparticles appeared in [ fig. 1] to be round-shaped and SLN's mean particle diameter appeared to be smaller (around 150 nm) compared to the particle size estimate. Additionally, no roughness is observed on the particle surface.

Transmission electron microscopy (TEM)
TEM micrographs revealed the formation of nanoparticles of narrow particle size distribution [ fig. 2]. Micrographs showed that the SLN were smooth, spherical and homogenous nanovesicles. Fig. 3 showed a smooth and spherical shape of the particles. Also, particles generally agglomerated owing to carriers ' lipid nature, surfactant presence, and sample preparation before SEM analysis.

Scanning electron microscopy (SEM) studies
Zolmitriptan and lipid compatibility studies were carried out by FTIR, and the results were given in [ fig. 4]. The pure zolmitriptan exhibits characteristic peaks at 3352 cm

Fourier transfer infrared (FT-IR) spectroscopy studies
-1 corresponding to the aromatic secondary amine N-H stretching, 2974.23 cm-1 corresponding to aromatic C-H stretching, 1735.93 cm-1 corresponding to C=O stretching and 1257.59 cm-1corresponding to C-N aliphatic amine stretching) as appeared in fig. 4. The FTIR spectra of Zolmitriptan and lipid physical mixture (cholesterol, Lecithin and stearic acid) exhibit the same characteristic peaks due the aromatic secondary amine N-H stretching at 3348.42 cm-1, C = O stretching at 1735.93 cm-1, and C-N aliphatic amine stretching at 1257.59, However they showed a slight shift in aromatic C-H stretching at 2931.8, 2978.09 and 2920.52 cm-1 respectively. Thus, it is evident that all the characteristic peaks that were present in the spectra of pure drugs replicated almost in the same region in the spectra of Zolmitriptan and lipid physical mixture (cholesterol, Lecithin, and stearic acid) indicating that there is no significant interaction between the drugs and the lipids.

In vitro
Cholesterol SLNs exhibited significantly higher initial burst release than Stearic acid at p<0.01 and both followed by sustained release. The initial burst effect was ranged from (22.01±1.23 to 41.34±1.56) within one hour. The initial drug release from the Zolmitriptan loaded SLNs was performed in PBS (pH 6.8 at 37 °C) using the dialysis bag technique over 5 h and the cumulative release percentages are illustrated in [ fig. 5]. The release profiles of the investigated SLN formulations shows a biphasic behavior, an initial fast release that lasted for 0-1 h, followed by a sustained slow release behavior. Burst release can be useful to improve the penetration of the drug, while, sustainedrelease supplied the drug over a prolonged period [33].
in vitro burst release was probably caused by the drug adsorbed on the nanoparticle surface or precipitated from the superficial lipid matrix [35]. Muntimadugu E et al. reported that initial burst release was mainly due to weakly bound drug on the surface of nanoparticles [36]. Baig et al. reported that the initial burst release rate was affected by the change of concentration of the lipid (Stearic acid and cholesterol) and surfactant (Lecithin) in the external phase. When the lipid concentration increased, the initial burst release rate decreased. Whereas surfactant concentration increases, the initial burst release rate increases due to the increased solubility of the drug in the external phase [37]. SLNs showed prolonged cumulative drug release percent and it decreased with increasing lipid concentration as shown in fig. 5. Due to the high concentration of drug in the inner phase [38]. The drug release of Zolmitriptan loaded SLNs was ranged from 50.24±5.6to 75.75±5.6 at 5 h SLNs suggests homogeneous entrapment of the drug throughout the systems. Furthermore, in vitro release profiles for the selected prepared SLNs were applied on various kinetic models (zero-order, first-order, and Higuchi equations), to fig. out the mechanism of drug release. The highest r value for drug release was obtained for Higuchi's equation at 0.975, 0.885, 0.916, 0.9655, 0.976 and 0.965 for S1, S2, S3, S4, S5 and S6 respectively. Release pattern of the drug from almost all SLNs formulations was best fitted into the Higuchi equation that describes the diffusion of the drug from homogenous and granular matrix systems [35]. Considering these observations, it was concluded that all the batches of SLNs had the potential for sustained drug delivery. This finding is in good agreement with the previous studies [39,13] Formulation of Zolmitriptan loaded SLNs that gave maximum entrapment efficiency percent, small particle size, small polydispersity index, high zeta potential, and better-sustained release effect were selected for physical stability studies, preparation of gel for nasal administration and brain histopathological studies. On that basis, we have chosen formula S6 for further investigations. During the storage period, there was no change in color or creaming and phase separation in the S6 formulation. There was a nonsignificant increase in particle size of SLN stored at 5 °C, 25 °C and 45 °C after 3 w at. At greater temperature 45 °C, the entrapment efficiency percent was none significantly decreased compared to other storage circumstances. The non-significant change in size and %EE due to the partial leakage of zolmitriptan from SLN. SLN stability also showed a non-significant decrease by raising the storage temperature as shown in the zeta potential outcomes. The results were presented in [table 3].

Viscosity study of zolmitriptan loaded SLN nasal gel
The viscosity of Zolmitriptan loaded SLN nasal gel (gelled with HPMC E15) was recorded to be 3000 cp as shown in [table 4]. It has to be noted that the viscosity of the gels is due to the polymer (HPMC E15) used.
The gel showed an initial burst release of drug during the first 1 hour as discussed before. Following that, the drug entrapped into the gel was released gradually as shown in [ fig. 6]. About 73.21±1.44%, 75.32±2.54% were released after 12 and 24 h respectively.

In vitro release study
In vitro release profiles for Zolmitriptan loaded SLN nasal gel was applied on various kinetic models (zero-order, firstorder, and Higuchi equations), to fig. out the mechanism of drug release. The highest r value for drug release was obtained for Higuchi's equation at 0.967 compared to that of zero-order at 0.838 and first order at 0.912. The mechanism of drug release was found to be Higuchi model. We concluded that Zolmitriptan loaded SLN nasal gel has a sustained drug release effect. This finding is in good agreement with the previous studies [39,13].  Histological examination of brain tissue

Parameter
Histopathological analysis was conducted on sections of brain tissue treated with formula S6 Zolmitriptan SLNs gel and control plain gel (3% of HPMC E 15 gel) free from drug at a different time interval [ fig. 7]. Histopathological examination of hippocampus normal pyramidal cells of brain tissue, (a) show no appearance of the drug after 1hour while, (b) showed a slight difference in cells condensation in CA2 layer after 1 hour. (C) Showed CA2 region with the intact pyramidal cell after four hours. While; (D) show a slight difference in cells structure in CA2 layer after four hours. (e) Showed normal neurons (arrowhead) and white matter (arrow) after 24 h. (f) Showed spots of the drug appear around the cell after 24 h. These promising results ensure that zolmitriptan drugs achieve higher brain targeting with 24 h which helps us to enhance efficacy in the treatment of migraine headaches specially that many patients suffer from nausea and/or vomiting that can make the oral treatment ineffective. Zolmitriptan (Zol) solid lipid nanoparticles (SLNs) gel success to reach the brain cells via permeation through nasal mucosa through the olfactory region thus; passing hepatic metabolism providing anti-migraine activity. The prepared Zol SLNs formulations were characterized for various parameters, and maximum entrapment efficiency was obtained with small particle size. Sustained release of ZMT SLNs was best fitted into the Higuchi equation that describes the diffusion of the drug from homogenous and granular matrix systems. Histological examination of brain tissue showed that Zolmitriptan achieved a higher brain targeting with 24 h, which help us to enhance efficacy in the treatment of migraine headaches.

CONCLUSION
The brain targeting via nasal drug delivery system circumvent the pre-systemic metabolism thus increasing the bioavailability of zolmitriptan. Further, future clinical studies should be done on that technique to show the significance of an increase in bioavailability compared to commercial dosage forms.