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Back to Journal »International Journal of Nanomedicine» Volume 16

Author: Abd El-Halim SM, Mamdouh MA, Eid SM, Ibrahim BMM, Aly Labib DA, Soliman SM 

Published on September 17, 2021, the 2021 volume: 16 pages 6395-6412

DOI https://doi.org/10.2147/IJN.S325697

Single anonymous peer review

Editor who approved for publication: Dr. Farooq A. Shiekh

Supplementary video for "Zolmitriptan in the New SNEDDS" [ID 325697].

Shady M Abd El-Halim, 1 Mohamed A Mamdouh, 1 Sherif M Eid, 2 Bassant MM Ibrahim, 3 Dina A Aly Labib, 4 Sara M Soliman1 1 October 6, Faculty of Pharmacy, Faculty of Pharmacy and Industrial Pharmacy, University City, Giza , 12585, Egypt; 2 Analytical Chemistry, School of Pharmacy, October 6, University, October 6, City, Giza, 12585, Egypt; 3 Department of Pharmacology, Department of Medical Research, National Research Center, Giza, 12622, Egypt ; 4 Department of Medical Pharmacology, Faculty of Cairo, Cairo, Cairo, 11559, Egypt, October 6 City, 12585, Egypt Tel +20 1119994874 Email [email protected] Purpose: Current work aims to overcome Zolmitriptan (ZMT ) Has poor permeability and adverse side effects, and improves its efficacy in treating acute migraine in the following ways. Use the synergistic effect of lavender essential oil to make ZMT self-emulsified drug delivery system (ZMT-SNEDDS). Method: Based on a full factorial design (32), ZMT-SNEDDS was manufactured to statistically evaluate the influence of oil and surfactant concentration on nanoemulsion sphere size, zeta potential and drug dissolution efficiency percentage. The ATR-FTIR method was developed and verified for continuous real-time monitoring of the dissolution and penetration of ZMT. The optimal dose of ZMT-SNEDDS for efficacy studies is selected based on acute toxicity studies. The curative effect of migraine rats induced by nitroglycerin was studied, and evaluated by moving cage and heat test, electroencephalogram, electrical convulsion stimulation and brain tissue biochemical analysis. Finally, the histopathology and immunohistochemistry of the brain were examined. Results: After dilution, the optimized ZMT-SNEDDS (F5) showed 19.59±0.17 nm nano-spherical droplets, with narrow size distribution, zeta potential (-23.5± 1.17mV) and fast emulsification characteristics. The ATR-FTIR spectrum clarifies the complete time course of dissolution and penetration, confirming the excellent performance of F5. In addition, ZMT-SNEDDS (F5) has shown safety in acute toxicity studies. Compared with ZMT solution, the concentration of ZMT in the brain tissue of F5-derived rats is lower, but it has better effects on mental state, pain perception, maintaining normal brain electrical activity and delaying convulsions. It counteracts the biochemical changes in the brain caused by nitroglycerin, which is confirmed by histopathological examination. Conclusion: In short, these findings confirm the significant synergy and high performance of ZMT-SNEDDS based on lavender oil in migraine management compared with traditional Zolmitriptan solution. Keywords: ZMT-SNEDDS, acute migraine, real-time monitoring, nitroglycerin, active cage, pain sensation

Migraine is a unilateral, pulsating, recurrent headache, and a painful neurological disease. Migraine symptoms usually precede sensory or movement disorders and are called auras. Migraine patients may also experience panic, nausea, vomiting, fever, and chills, which is different from stress headaches. 1

When the vasodilator nitroglycerin (NTG) is given, migraine patients are more prone to migraine attacks. This incidence is associated with a lower total time threshold and hypersensitivity of the pain system to NTG. Systemic administration of NTG to rats induces hyperalgesia and migraine-like symptoms similar to migraine by activating the brain nucleus incorporated in nociceptive transmission and changes in the autonomic nervous system of rats. 2

Serotonin agonists triptans are currently the gold standard for abortion in the treatment of migraine. Zolmitriptan (ZMT) is a second-generation highly effective triptan drug. ZMT is a selective serotonin 1B/1D receptor agonist, which can be used as a cerebral vasoconstrictor and an inhibitor of the release of pro-inflammatory neuropeptides from the peripheral nerve terminals of the trigeminal nerve. Its prescribed dosage is 2.5 mg or 5 mg to 10 mg/day. 3

However, it is recommended to reduce the dose for patients with liver damage, because this condition may increase the incidence of hypertension due to excessive vasoconstriction due to liver microsomal enzymes' damage to ZMT metabolism. Overdose of ZMT may cause sedation or increase headache, tachycardia, tingling, pain, chest tightness, nausea or vomiting. 3 Therefore, it is worthwhile to study alternative formulation technologies, which have the same efficacy in reducing migraine symptoms, but can be administered at lower doses. Dosage to reduce its side effects.

According to reports, for conventional tablets and nasal dosage forms, the absolute bioavailability of ZMT is 40-50%. The decrease in oral bioavailability is attributed to its low permeability as a BCS class III drug and the first pass effect in the liver. 4

For a long time, the challenge of poor oral bioavailability has triggered the search for feasible solutions to this dilemma. Lipid-based formulations have gained considerable momentum as a reliable method of delivering poorly soluble or penetrating drugs. Among them, the self-nano-emulsifying drug delivery system (SNEDDS) has been established as an effective tool to improve the oral systemic bioavailability of such drugs. These systems are basically isotropic mixtures of drugs, lipids, surfactants, and one or more co-emulsifiers. They are dispersed in the gastrointestinal (GIT) aqueous medium in a fine form, resulting in a large surface area, thereby improving drug absorption. 5

SNEDDS is usually formulated with vegetable oils or semi-synthetic lipids. Recently, peppermint, lavender, and other essential oils have also been used in SNEDDS formulations. This is likely due to their potentially different treatment results, such as alleviating related migraine symptoms, such as pain and nausea, as well as easy emulsification of vegetable oils. 6,7

The evaluation of the dissolution and permeability of the drug from the dosage form involves frequent sampling of samples for analysis using UV spectrophotometry or more commonly HPLC. This type of technology involves a large number of solution replacements and re-conditioning, resulting in excessive solvent consumption, time wasting and increased energy, and if ultraviolet spectrophotometry is used, there is a risk of component interference. Therefore, the implementation of attenuated total reflection Fourier transform infrared (ATR-FTIR) real-time monitoring is beneficial to overcome these obstacles because it can provide a continuous curve of dissolved drug concentration over time. 8 FTIR spectroscopy can reveal unique information about the molecular structure of ZMT, which is called a quantitative fingerprint. 9

The purpose of this study is to enhance the penetration of ZMT into the intestinal barrier by loading ZMT into SNEDDS, compared with drug solutions, and to clarify the auxiliary effect of selected essential oils in enhancing the anti-migraine effect of ZMT. In addition, our goal is to develop an ATR-FTIR method that can be used to track real-time changes in concentration during ZMT dissolution and penetration testing. In an animal model that mimics migraine, the efficacy of SNEDDS loaded with ZMT was compared with oral ZMT solution to evaluate its effect on the serotonin precursor "tryptophan" and its correlation with the concentration of ZMT detected in the brain Changes in brain cell metabolism. In addition to histopathological and immunohistochemical examinations of brain tissue, the comparison will also include two forms of psychological and analgesic effects.

Choose the appropriate oil phase, the saturated solubility of ZMT in various oils, ie. Ginger, lavender and peppermint oils were identified. Add excess ZMT to each oil in the vial with stopper, vortex for 5 minutes, and then shake at 25±0.5°C for 72 hours until equilibrium is reached. Undissolved ZMT was removed by centrifugation at 2500 rpm for 30 minutes. Then filter the supernatant through a Millipore 0.45 µm filter. According to the method of Champaneria et al., the drug concentration in each filtrate was determined using HPLC at λ max = 222 nm. 10 The observed retention time of ZMT is 3.5 minutes.

Cremophor® EL [Polyoxyethylene 35 hydrogenated castor oil (P35HCO), HLB = 13.5], Labrasol® (HLB = 12), Brij® L4 (HLB = 9) and Brij® 93 (HLB = 4)] were tested A large amount of water is integrated into the oil/surfactant combination. The 1:1 w/w ratio of the selected oil was mixed with each of the four tested surfactants. Then the water was titrated dropwise while stirring continuously until the mixture became cloudy. The highest percentage of water that can be integrated into the oil/surfactant combination (O/S mixture) is used to determine which surfactant should be used in the SNEDDS formulation. The percentage of water incorporated is calculated as follows:

The ability of co-surfactants to improve the nanoemulsification ability of selected surfactants was tested. At the ratio of surfactant to co-surfactant (S/CoS) (1:1 w/w), the selected surfactant is compatible with three different co-surfactants (Transcutol® HP, Lauroglycol™ 90 and Propylene glycol) each of the couplings. The oil is mixed with each S/CoS mixture at a ratio of (1:1 w/w). The screening method of these combinations is the same as that of surfactants, and the CoS that allows the highest water binding is selected to construct a ternary phase diagram. 11

The purpose of creating the phase diagram is to calculate the percentage of components that will lead to the formation of a nanoemulsion. 12 Weigh an appropriate amount of each component into a vial, vortex to mix for 10 minutes, and then gently stir under heating at 37°C until a homogeneous isotropic mixture is formed. Before the visual inspection, 36 systems were created and kept at room temperature for 24 hours. 13

For all generated SNEDDS, a phase diagram was also constructed in the presence of ZMT (1% w/w) to study the effect of drug incorporation on the nanoemulsion area. In order to confirm the influence of dilution on the boundary of the nanoemulsion, all prepared systems, whether loaded with drugs or not, were diluted 100 times with deionized water, and deionized water was used as a blank at 638 nm, and the% transmittance was measured by spectrophotometry. 14

According to the clear area of ​​the established ternary phase diagram, 9 ZMT-SNEDDS were selected for further study, in which a full factorial design (32) was used to statistically evaluate the influence of oil and surfactant concentrations (X1 and X2, respectively) on the emulsion Ball size, zeta potential and percentage of drug dissolution efficiency. Three levels are assigned to each factor, as shown in (Table 1). After the statistical study, the Design Expert® software (7th edition; Stat-Ease, Inc., Minneapolis, MN) was used to calculate the willingness to select the best formula for further research. Table 1 Experimental running independent variables of 32 full factorial experimental design (recipe variables)

Table 1 Experimental running independent variables of 32 full factorial experimental design (recipe variables)

The ZMT-SNEDDS formula (0.1 mL each) was diluted to 10 mL with deionized water, and the percentage of transmittance was measured at 638 nm using deionized water as a blank. In addition, the drug precipitation and phase separation (if any) of the nanoemulsion were also examined11.

The rate of dispersion is a measure of the effectiveness of self-emulsification. Using the dissolution apparatus, rotating at 50 rpm and temperature at 37±0.5°C, add 1 mL of ZMT-SNEDDS to 500 mL of deionized water. Visually track the time when the scattered SNEDDS completely disappears. 14

The Malvern Zetasizer is used to analyze spherical size, polydispersity index (PDI) and zeta potential. Before the measurement, ZMT-SNEDDS was diluted 100 times in deionized water and shaken gently to form a nanoemulsion. 15

The ATR-FTIR method was implemented to determine the percentage of ZMT dissolved with respect to time. (Figure S1) shows the cycle settings of the ATR-FTIR continuous flow system (pump, dissolution device, FTIR and computer). The continuous flow starts from the dissolution apparatus container in which the formulation is placed. Use a pulse pump adjusted to 2 mL/min to pump the sample in the tube into the ATR device. The pumped solution moves towards the ZnSe crystal (ATR unit), which is tightly sealed with a metal cap. The cover plate allows the solution to move in one direction and return it to the vessel, as shown in (Figure S1 and Supplementary Video S1).

Before measuring the sample, the circulatory system conditions must be optimized, as shown below:

a) Nitrogen is used to remove air and moisture from the ATR device.

b) FTIR software parameters are carefully adjusted as follows: Measurement mode: absorption, scanning range: 750-4500 cm-1, automatic scanning for 15 minutes. SqrTriangle apodization function, the mirror speed is 2.8, and each sample is measured 45 times. The device has been adjusted for automatic atmospheric interference correction.

c) Carefully heat the solution and standard to 37°C before each measurement.

d) Pump the solution at a constant flow rate (2 mL/min) during the experiment. Stabilize the conditions by letting it flow for five minutes until all the gas that may be in the tube is removed.

Before starting the cycle, put different working concentrations of ZMT (0.2-12 µg/mL) into the dissolution vessel, and then start to flow to record different ZMT spectra.

Real-time dissolution testing is an important parameter that needs to be evaluated in the process of drug development and quality control. In order to evaluate the dissolution profile of ZMT-SNEDDS, the sample (500 mg) was put into a hard shell capsule of size 00 and placed in a dissolution vessel containing 500 mL of deionized water. The vessel was continuously stirred at 50 rpm and heated at 37°C. Then the cycle begins, pumping the solution to the FTIR prism, where the sample is continuously scanned, and the spectrum is automatically recorded. According to the equation described by Sharma et al., determine the dissolution efficiency percentage (%DE) of each system. 16

The non-eversion rat intestinal sac model was used to evaluate the penetration of ZMT from SNEDDS through the intestinal mucosa. Use phosphate buffered saline (PBS) to carefully wash the isolated small intestines of killed male Wistar rats (200-250 g) and cut them into 4 cm long sections. A certain amount of optimized SNEDDS was diluted with 1 mL of phosphate buffer (pH 6.8) and filled into the fragments. Tie both ends of the intestine firmly to avoid leakage, and then place it in a beaker containing 50 mL of phosphate buffer (pH 6.8). Use a shaking water bath to keep the receptor phase at 37 ± 0.5°C while shaking gently at 50 rpm. 5 Compare the data with the ZMT solution in deionized water. Using the ATR-FTIR method previously described in the "In vitro ATR-FTIR Dissolution" section, analyze the percentage of ZMT permeated with respect to time in the sample.

Three complete freeze-thaw cycles were performed on the selected formula, which proved its physical stability and ability to withstand thermal stress. 17

According to the method mentioned by Soliman et al., a transmission electron microscope (TEM) is used to evaluate the morphology of the best formulation. 18

Adult female Wister albino rats with a weight range of 180-200 g were obtained from the animal house of the National Research Center (Giza, Egypt). Throughout the investigation process, the animals were kept under appropriate laboratory conditions. They are fed standard pellets provided by the Animal House of the National Research Center, and they are allowed to drink freely. All experimental procedures were carried out in accordance with the "Guidelines for the Care and Use of Laboratory Animals" issued by the Institute of Laboratory Animal Resources; Committee on Care and Use of Laboratory Animals; National Institutes of Health and Research Resources, NIH publication series. 19 The animal procedure was approved by the Ethics Committee of the National Research Center of Egypt, with the license registration number (8413042021).

A single dose of ZMT-SNEDDS (0.045 mg/kg) diluted with 1 mL of distilled water was orally administered to five healthy, adult, non-pregnant female rats (18 hours fasting). This dose is equivalent to one-tenth the dose of a conventional ZMT solution; according to Paget and Barnes, the human dose is converted to an equivalent rat dose. 20 A similar group served as a negative control and was given 1 mL of distilled water once. Observe the rats closely during the first 30 minutes after administration. During the first 24 hours, report any deaths, abnormal behaviors, changes in bowel habits, or abnormal breathing. In addition, any significant changes in body weight (bwt) compared to baseline body weight (bwt) over the next 14 days are also reported. twenty one

For the induction of migraine, all rats except the negative control group were injected with NTG intraperitoneally according to the following method at a dose of 5 mg/kg every two days for 9 days (5 injections in total) Pradant et al. 22

Eighty female Wister Albino rats weighing 175-190 g were equally divided into eight groups, two control groups; a negative control group, in which the rats took 1 mL of distilled water during the experiment, and the positive control group included untreated migraines Rat. The treatment group received oral treatment, 1 hour after each injection of NTG, and the administration was as follows; the two groups received conventional ZMT solution at doses of 0.225 and 0.45 mg/kg (human doses of 2.5 and 5 mg/kg were converted to rats using Paget table) dose). According to the results of the acute toxicity study, two groups received ZMT-SNEDDS at doses (0.022 and 0.045 mg/kg), and the last two groups received normal SNEDDS at doses of 0.25 and 0.5 mL. The animals were weighed at time zero and at the end of the experiment, and the amount of food per day was fixed throughout the experiment.

Twenty-four hours after the last therapeutic dose was administered, the animals were subjected to a grid floor movable cage test to evaluate the effect of the test drug on the behavior and mental state of each rat during the five-minute test period. Afterwards, a thermal test was performed to evaluate the optimal analgesic effect of SNEDDS18. The effect of treatment on the physiological activity of brain electrical activity is detected by electroencephalogram and electrical convulsion stimulation.

According to the 10-20 International Electrode Application System, all rats were subjected to electroencephalogram (EEG) under general anesthesia using standard conditions. Intermittent light stimulation is performed as a stimulation technique. twenty three

Afterwards, half of the animals in each group received electrical convulsive stimulation 24 under general anesthesia, and were stimulated with a single electrical convulsive shock (ECS) with a pulse width of 3 ms and a frequency of 60 Hz at an intensity of 10 mA. Calculate the elapsed time between the application of ECS and the occurrence of the seizure.

After electroencephalogram and electrocardiogram, all animals were sacrificed humanely by decapitation under anesthesia. Animals undergoing ECS ​​were excluded from subsequent studies involving biochemical analysis and histopathological examination.

The ZMT levels obtained in ZMT-SNEDDS and ZMT solutions in the rat brain were evaluated using high performance liquid chromatography. 10 Use FTIR to determine structural and compositional changes in the rat brain. Normalize the obtained group spectra and analyze them for the following spectral regions: (i) NH-OH region is 3700-3000/cm-1; (ii) CH stretching region is 3000-2800/cm-1; (iii) ) The fingerprint area at 1800-1000/cm-1, including the amide I band (1800-1600/cm-1). Use Origin Pro 9 software (OriginLab Corporation, Northampton, MA 01060, USA) to obtain the average value of each group of individual spectra25.

In order to evaluate the metabolic changes in the brain, a soluble brain tissue sample was diluted to 1 mL with PBS (pH: 8.2) and measured by UV spectrophotometry. Plot spectra for all groups in the 200-340 nm wavelength range. 26

Prepare brain tissue samples for histopathological examination, and then score them as follows: 0 is normal; 1, mild damage; 2, moderate damage and 3, severe damage. 27 Assess neuron-specific enolase (NSE) immunoreactivity, and score the lesion: 0" means absolutely no staining (negative); when the index is >10% for mildly immunopositive cells (weakly positive), "score 1" ; "Score 2", the percentage of strongly immunopositive neurons is between 10% and 30% (medium positive); "Score 3", where the percentage of dark brown neurons> 30% of the counted cells (strong positive).28

The higher solubility of the drug in the oil phase is essential for achieving higher drug loading and keeping the drug dissolved and avoiding precipitation during dilution. The solubility of ZMT in lavender, peppermint and ginger oil was found to be 59.375±3.72, 6.074±0.54 and 0.203±0.013 mg/mL, respectively. Lavender oil was chosen for further formulation, because the significantly higher drug solubility (p<0.001) also allows the use of a smaller amount of oil in the formulation, so the emulsification can be achieved with a smaller amount of surfactants and co-surfactants .29

The solubilizing properties of the drug may not be completely consistent with the high affinity of surfactants for oil. Therefore, the choice of S/CoS is driven by the effective emulsification of lavender oil and the ability to dissolve ZMT. Among the screened surfactants, P35HCO showed a significantly higher water binding percentage (p<0.05), as shown in (Figure S2), which may be attributed to the surfactant with the highest HLB. 30

By promoting the diffusion of oil to the hydrophobic part of the surfactant molecule, the co-surfactant can further reduce the interfacial tension, thereby improving the fluidity of the interface. 11 After screening for CoS, Transcutol® HP was able to add a significantly higher proportion of water compared with propylene glycol and lauroglycol™ (p<0.05).

Lavender oil and P35HCO/Transcutol® HP are used as the ternary phase diagram of the S/CoS system to define the self-emulsified area. The figure only shows the subtle differences in the nanoemulsion area after loading ZMT. The shaded area in the triangle, as shown in Figure S3, represents the area where SNEDDS forms nano-scale emulsion droplets with the help of gentle stirring. The large area observed reflects the high self-nano-emulsification ability. These may be attributed to the adsorption of S/CoS at the interface, which helps to improve the stability of the nanoemulsion by reducing the interface energy and providing a mechanical barrier to avoid agglomeration and phase separation. 17

The ability of SNEDDS to dilute without precipitation and/or phase separation is critical to the suitability of drug delivery. After dilution, the formed nanodispersion maintains its transparency within 24 hours, with no signs of phase separation or drug precipitation. This suggests that these systems are suitable for oral use because they can pass through the gastrointestinal tract in the ideal nanosphere form. The prepared system showed a high percentage light transmittance of more than 95%, indicating that the nanospheres have excellent emulsifying ability. 31

After dilution, SNEDDS is expected to achieve rapid and complete dispersion under gentle stirring. The dispersion time depends on the composition, as shown in (Table 1). Under different oil levels, increasing the surfactant concentration has a significant effect on increasing the emulsification time. This is most likely due to the high viscosity provided by P35HCO, which delays the penetration of water into the colloid or gel phase formed on the surface of the formulation. 32 A similar effect was observed in increasing oil concentration, which may be due to the shortage of surfactant systems at larger oil volumes. Other researchers have described similar trends. 33,34

The small droplet size is an indicator of emulsion stability and improved drug bioavailability. Smaller droplets dispersed in the GIT will result in faster drug diffusion and improved drug dissolution. 17 The average spherical size of the dilution system ranges from 13.97±0.06 nm to 35.05±0.44 nm. The low PDI values ​​of all formulations ranging from 0.101±0.011 to 0.351±0.001 resulted in a uniform ball size distribution.

The Zeta potential determines the charged surface of the beads, which is usually an indicator of the physical stability of the emulsion, because the electrostatic repulsion between the beads prevents coalescence. After dilution, SNEDDS containing lavender oil and P35HCO produces a negatively charged emulsion with a zeta potential ranging from -17.23±1.32 mV to -32.2±3.76 mV. The fatty acids and/or surfactants in the oil phase are most likely to generate negative charges. Linoleic acid and linoleic acid account for more than 80% of the total fatty acids in lavender oil. The main component of P35HCO is glycerol polyethylene glycol hydroxystearate, which forms the hydrophobic part together with fatty acid glycerol polyethylene glycol ester. 35,36

(Figure 1) shows the 3D response surface plot and the quantitative linear relationship between the expected values ​​of all dependent variables and the synthetic ball size, Zeta potential, and RE response percentage. Figure 1 3D response surface plot and the resulting quantitative linear relationship between (A) pellet size, (B)% zolmitriptan dissolution efficiency, and (C) Zeta potential and the predicted values ​​of all dependent variables.

Figure 1 3D response surface plot and the resulting quantitative linear relationship between (A) pellet size, (B)% zolmitriptan dissolution efficiency, and (C) Zeta potential and the predicted values ​​of all dependent variables.

ANOVA analysis showed a significant reduction in the size of the spheres (p<0.0001), while the surfactant concentration increased from 10% to 40%. The observed decrease may be due to a further decrease in interfacial tension, as the increase in the amount of surfactant and the accompanying decrease in free energy may cause the droplets to deform or break. In addition, the surfactant film formed at the interface is more densely packed, thereby hindering the coalescence of droplets and enhancing the stability of the emulsion. 37 On the other hand, increasing the oil concentration from 10% to 50% resulted in a significant increase in the size of the pellets (p <0.0001). This may be due to insufficient emulsifiers at the interface at higher oil levels. In addition, a higher oil content may cause the emulsion to have a higher viscosity, thereby reducing the emulsification efficiency. 38

Statistical analysis of Zeta potential revealed the non-significant effect of oil concentration (p=0.176). In contrast, higher surfactant levels significantly reduced the zeta potential value (p<0.0001). Since the hydroxyl group is discharged from the o/w interface, which will reduce the surface potential, there are similar observations about the effect of non-ionic surfactants on lowering the zeta potential value above the critical concentration. 37

Zolmitriptan is an anti-migraine drug, so rapid dissolution is a basic requirement. Conventional analytical methods (such as HPLC) require frequent sampling and continuous replacement of the extracted solutions, so continuous monitoring is advantageous because it can provide a complete real-time profile of the dissolution pattern of the active drug. Since FTIR spectra are regarded as molecular fingerprints because they display characteristic functional groups in the form of structurally confirmed bands, real-time monitoring of drug concentration using FTIR improves method selectivity39 (Figure 2A). ZMT is (4S)-4-({3-[2-(dimethylamino)ethyl]-1H-indol-5-yl}methyl)-1,3-oxazolidin-2-one, and its FTIR spectrum shows The structure of the following characteristic peaks is that NH stretches at 3380 cm-1, CH stretches at 2926 cm-1, and C=O stretches at 1738 cm-1. Figure 2 FTIR spectrum: (A) Zolmitriptan structure and its characteristic peaks and (B) Zolmitriptan calibration concentration (0.2–12 µg/mL), the illustration shows the selected calibration peaks.

Figure 2 FTIR spectrum: (A) Zolmitriptan structure and its characteristic peaks and (B) Zolmitriptan calibration concentration (0.2–12 µg/mL), the illustration shows the selected calibration peaks.

The developed real-time ATR-FTIR method was verified according to the ICH guidelines by evaluating the following parameters:

a- Linearity and sensitivity: Perform baseline correction on the obtained ZMT spectrum, and then select the characteristic ZMT peak at 1745 cm-1 for calibration (Figure 2A). The relationship between ZMT concentration and absorption was found to be linear, as shown in the Beer curve (Figure 2B), with a small intercept (0.0005) and slope (0.0001) in the concentration range of (0.2-12 µg/mL) And the coefficient of correlation (0.9891).

b- Accuracy and precision: It is found that the accuracy of the developed method is 98.7%, which is ensured by applying the Beer-Lambert law to quantify different blind ZMT concentrations. Method repeatability (intra-day precision) was determined by analyzing three different ZMT concentrations three times in the same day, and the calculated RSD was 0.345. Using the same procedure to determine the ZMT concentration on three different days, the RSD was 0.946.

c- Robustness: The developed FTIR method was found to be robust and not affected by slight changes in method parameters, such as flow rate fluctuations. It was studied by deliberately changing the flow rate by ±0.1 mL/min. In addition, the influence of temperature fluctuations is studied by the ±0.5 change of the dissolution temperature. The method was found to be stable and not significantly affected by these changes, thus ensuring the robustness of the method.

d-Selectivity: FTIR spectrum is considered to be the fingerprint of each molecule, indicating its characteristic active functional groups, thereby confirming its identity. Therefore, by monitoring the obtained peaks, the identity of ZMT can be confirmed. In addition, the overlay of ordinary SNEDDS (formula without ZMT) and pure ZMT confirms the selectivity of this method to ZMT, as shown in (Figure S4).

(Figure 3A) describes the ZMT dissolution results of different formulations in deionized water. All prepared SNEDDS showed rapid and almost complete drug dissolution within the first 10 minutes. The rapid dissolution can be attributed to the rapid emulsification caused by the lower surface free energy of the system and the larger surface area caused by the small sphere size. 40 Analysis of variance showed that both oil and surfactant concentrations significantly affected% DE (p<0.0001), ranging from 68.67±1.4% to 82.37±1.2%. %DE is directly affected by the size of the spheres, and the size of the spheres is affected by changes in the oil concentration, as described above. Figure 3 The dissolution rate of Zolmitriptan in vitro: (A) The percentage of Zolmitriptan dissolved in different preparations in deionized water and (B) The FTIR spectrum shows the change of the concentration of Zolmitriptan dissolved in F5 over time .

Figure 3 The dissolution rate of Zolmitriptan in vitro: (A) The percentage of Zolmitriptan dissolved in different preparations in deionized water and (B) The FTIR spectrum shows the change of the concentration of Zolmitriptan dissolved in F5 over time .

In addition, due to the significant increase in system viscosity, the increase in surfactant concentration will slow down the emulsification process, thereby delaying the dissolution of the drug. 32 The dissolution results obtained are consistent with the emulsification time, and the system with the faster emulsification time shows the highest dissolution efficiency. According to the Design-Expert® software, the optimized formula is analyzed based on the following set of criteria; oil concentration is 30%, ball size <20 nm, ZP is within the range, and the maximum %DE. Therefore, F5 was selected as the optimized SNEDDS with a desirability value of 0.632. Figure 3B details the gradual increase in ZMT concentration from F5 to completely dissolved.

Relying on in vitro dissolution data alone may not be sufficient to successfully predict oral absorption and in vivo behavior. Penetration studies through excised living tissue can provide valuable data on the rate and extent of drug absorption. 33

Compared with ZMT solution, SNEDDS F5 shows a significant improvement in ZMT penetration, as shown in (Figure S5). The significant difference between the two drug forms can be attributed to the formation of the nanoemulsion as a colloidal carrier, which has been considered to enhance the penetration of the drug formulated in SNEDDS, and the inherent penetration of the excipients used by changing the fluidity of the membrane The enhancement allows for increased diffusion. 41

Exposing the selected formula (F5) to three freeze-thaw cycles did not show any signs of instability, indicating that the system is stable. There was no phase separation or drug precipitation by visual inspection, and statistical analysis showed that there was no significant change in the size of the pellet, zeta potential and %DE (P>0.05).

As shown in (Figure S6), the TEM of the reconstructed F5 SNEDDS shows spherical discrete oil balls with a diameter in the range of 19 nm, which is consistent with the results obtained using Zetasizer.

The results of the acute toxicity study showed that within 14 days after the administration of ZMT-SNEDDS, the rats did not die, and there was no change in the bowel habits or behavior of the rats. In addition, the weight of rats given ZMT-SNEDDS was within the normal range, showing an increase of 3.35% from the baseline bwt before ZMT-SNEDDS administration, which was relatively consistent with the amount of food consumed, and was considered normal for age and duration experiment of. However, it was significantly lower than the negative control (Table 2). Table 2 Effects of treatment on body weight (Bwt) in acute toxicity and effectiveness studies

Table 2 Effects of treatment on body weight (Bwt) in acute toxicity and effectiveness studies

Histopathological examination of kidney tissue showed normal glomerular structure and intact renal tubular epithelial lining (Figure S7A). The liver showed normal histological structure of liver lobules (Figure S7B). All these findings indicate that ZMT-SNEDDS can be used in preclinical studies within the safety range of the conventional selective serotonin agonist ZMT.

Therefore, acute toxicity studies have confirmed that ZMT-SNEDDS is non-toxic at a dose of 0.045 mg/kg. Therefore, the selected doses for conducting ZMT-SNEDDS efficacy studies are 0.0225 and 0.045 mg/kg.

Regarding the efficacy study in the current work, the weight of the negative control rats that received only distilled water during the entire experiment increased slightly by 3.87%, while the weight of the positive control rats significantly decreased by 3.7% of the baseline weight and positive Compared with the control group, the baseline weight percentages of all treatment groups increased significantly, but were still significantly lower than the negative control group. This was probably due to digestive system side effects such as indigestion and the use of triptan drugs including ZMT42 Nausea that occurred as part of the serotonin syndrome suffered by the treated patients (Table 2).

Behavioral and sensory evaluations are carried out through both; a walking test is performed using a grid floor cage, a hot plate test is used to evaluate the mentality of the rat and to evaluate the integrity of the surrounding sensation. The negative control group recorded 99.5 exercises during the 5-minute duration of the grid floor activity test, and the pain assessment using the hot plate showed complete sensation, because the rats responded quickly to the temperature adjusted to 52°C , Because they have paws after 9 seconds of licking, indicating a normal withdrawal reflex to heat.

On the other hand, the positive control group had migraine-like symptoms, manifested as a significant reduction in movement through the grid floor cage, 59.79% less than the negative group (Table 3), indicating loss of interest or mental apathy, if accompanied by Depression with severe pain. 43 In addition, the incubation period of rat paw licking in the hot plate test was 27.77% less than that of the negative control group (Table 3) 44 Table 3 Selective serotonin agonists Zolmitriptan (ZMT) and ZMT-SNEDDS and their products containing lavender oil Comparison of the effects of common preparations on the psychological state of the use of grid floor cages and their analgesic effects. Hot plate heat test

Table 3 The effect of selective serotonin agonists Zolmitriptan (ZMT) and ZMT-SNEDDS and their lavender oil-containing preparations on the psychological state of the grid floor movable cage test and the use of the hot plate heat test test Comparison of analgesic effects

The occurrence of pain associated with depression can be explained by the correlation between pro-inflammatory cytokines and reduced serotonin in the brain. 45 This explains the relationship between the results of the hot plate test and the active cage test in the migraine rat model. our research. It also explains the subsequent improvement results of these tests on rats treated with ZMT solution or ZMT-SNEDDS serotonin agonist; as compared with the positive control group, the rats in the treatment group have significantly increased exercise, ZMT solution (0.225 and 0.45 mg/kg) increased 113.75, 145, 116.25, 147.5, 58.95 and 109.17%, respectively. ZMT-SNEDDS (0.022 and 0.045 mg/kg) and pure SNEDDS contained lavender oil (0.25 and 0.5 ml), respectively. It is worth noting that the highest increase in walking was observed in the group treated with high-dose ZMT solution and ZMT-SNEDDS (Table 3).

In addition, compared with the positive control group, compared with the positive control group, ZMT solution (0.225 and 0.45 mg/kg), ZMT-SNEDDS (0.025) and the positive control group, the hot plate paw licking latency significantly increased by 87.38, 89.69, 51.69, 115.07, 67.69 and 63.07%. mg/kg) and pure SNEDDS contain lavender oil (0.25 and 0.5 mL) respectively. Obviously, high doses of ZMT-SNEDDS showed the highest analgesic effect (Table 3). Sanna et al. explained the analgesic effect of lavender. He pointed out that giving mice lavender oil at a single dose of 100 mg/kg can relieve neuropathic pain, and its efficacy is equivalent to pregabalin. They attributed the effect of lavender oil to a significant decrease in the level of inducible nitric oxide synthase in the nervous system and a significant decrease in phosphorylation of ERK1, ERK2 and JNK1, and also inhibited the endocannabinoid degrading enzyme "fatty acid" Amidohydrolase" .46

When the electrical signals of the brain were evaluated by EEG to further study the therapeutic effect of ZMT-SNEDDS and ordinary SNEDDS containing lavender compared with ZMT solution, the background activities of all groups were within the normal α range. There is no clear focal or paroxysmal discharge, and no evidence of slowing or rapid activity of normal EEG signals (Table 4). Table 4 The effects of selective serotonin agonists Zolmitriptan (ZMT) and ZMT-SNEDDS and their common preparations containing lavender oil on electroencephalogram (EEG) and electroconvulsive shock stimulation test (ECS) research on electroencephalogram and epilepsy Comparison of the effects of seizures)

Table 4 The effects of selective serotonin agonists Zolmitriptan (ZMT) and ZMT-SNEDDS and their common preparations containing lavender oil on electroencephalogram (EEG) and electroconvulsive shock stimulation test (ECS) research on electroencephalogram and epilepsy Comparison of the effects of seizures)

ECS was also performed to confirm the aforementioned in vivo findings, because a delayed seizure in response to ECS indicates normal activity. The negative control group showed the longest incubation period among all the test groups, because the rats began to have mild seizures 12 seconds after exposure to a single ECS shock, but the results were not significantly different from the other groups (Table 4).

When administered in high and low doses, HPLC assays are used to detect the concentration of ZMT reaching the brain from the solution and SNEDDS to correlate the concentration in the brain with the pharmacological effects achieved and histopathological and histochemical parameters. The ZMT concentration obtained from the SNEDDS low-dose solution is 85% lower than the concentration obtained from the ZMT low-dose solution. The concentration of SNEDDS high-dose ZMT is 68.61% lower than that of ZMT high-dose solution. However, the effects of low-dose and high-dose ZMT and ZMT solutions from ZMT-SNEDDS on in vivo parameters were not significantly different (Table 5). It is expected that there will be no evidence of ZMT in the brain tissue homogenates of the negative control, positive control, or general treatment group because they did not receive the drug (Table 5). Table 5 The levels of zolmitriptan (ZMT) (μg/mL) in brain homogenate and ZMT-SNEDDS of rats receiving conventional treatment

Table 5 The levels of zolmitriptan (ZMT) (μg/mL) in brain homogenate and ZMT-SNEDDS of rats receiving conventional treatment

FTIR spectroscopy is a very reliable technique for studying the structure, conformation and functional state of biomolecules. The 47 FTIR spectrum (Figure 4) was completed as a qualitative verification test based on the established hypothesis of ZMT serotonin agonism, and compared ZMT-SNEDDS on the detection of tryptophan (serotonin precursor) in the brain of rats treated with solution and SNEDDS And the effects of SNEDDS in rats treated with ordinary SNEDDS containing lavender oil. Figure 4 FTIR measures changes in brain structure and composition.

Figure 4 FTIR measures changes in brain structure and composition.

The average spectra of the negative control, positive control and treated rat brain cell homogenates in the 3000-4000 cm-1 and 1000-2000 cm-1 spectral regions are shown in Figure 4. There were significant differences in the spectral parameters of the negative and positive controls between the groups, indicating that NTG-induced migraine caused significant changes in the structure and composition of the brain homogenate. The spectrum of ZMT-SNEDDS high-dose and normal SNEDDS treatment group is close to that of the negative control group. No significant changes were observed between the negative control and the ZMT-SNEDDS high-dose treatment group. These results indicate that SNEDDS has a protective effect on NTG-induced changes. Since oleamide analogs allow selective serotonin receptor subtype modulation, the 48 amide bands detected at 1000-2000 cm-1 indicate that the protein in the brain points to tryptophan, which is a pre-serotonin body.

Ultraviolet spectrophotometry (Figure 5) was performed to clarify the mechanism of structural changes between groups. The spectrum of the negative control group showed that the light absorption of the protein was at 200 nm. All groups have the same light absorption wavelength but different peaks. There were no significant structural changes between all groups. Figure 5 Ultraviolet spectrophotometry to determine changes in brain metabolism.

Figure 5 Ultraviolet spectrophotometry to determine changes in brain metabolism.

Histopathology and immunohistochemistry studies confirmed the results of in vivo efficacy experiments. Histopathological examination showed that high-dose ZMT-SNEDDS had the best effect. The cerebral cortex structure was normal and the cell arrangement was normal. As a negative control rat, the score was (0), followed by high-dose ZMT solution and low-dose ZMT-SNEDDS. The score is 1 for rats, and the score for rats treated with low-dose ZMT solution is 2. On the other hand, animals given low and high doses of ordinary SNEDDS had the same histological images as the untreated group, with a score of 3.

The results of immunohistochemical study of the cerebral cortex of the negative control large mouse with NSE staining showed that there was no detectable staining score (0), as shown in Figure S16. The positive control group showed a score (3), in which most of the cortex was stained and was characterized by increased NSE reactivity (Figure S17). Low-dose ZMT solution scores (2), where the percentage of strong immunopositive neurons is between 10% and 30% (Figure S18). High-dose ZMT solution and low-dose ZMT-SNEDDS score (1), index>10% mild immunopositive cells (Figure S19 and S20). On the other hand, high ZMT-SNEDDS showed no detectable staining score (0) (Figure S21). Low-dose and high-dose ordinary SNEDDS showed the same induction group score (3) picture, in which most of the cortex was stained (Figure S22 and S23).

The pain encountered in the migraine model in our study may be due to the dilation of cerebral blood vessels caused by NTG, which is an effective precursor of nitric oxide (NO), 49 may also be due to inflammation of the pain-sensitive meninges due to nerves Pannexin-1 stimulates the trigeminal afferent nerve, then Caspase-1 is activated, then pro-inflammatory mediators are released and the nuclear factor kappa-B is activated, and finally the inflammatory signal spreads around the pia mater blood vessels. 49 In addition, NO can be significantly produced by cerebral vascular endothelial cells, and plays a key role in the inflammatory process of brain cells. 50 However, these effects and related depressive symptoms, as well as histopathological and immunohistochemical changes, were offset and improved by the administration of ZMT solution and ZMT-two doses of SNEDDS. In addition, ordinary SNEDDS containing lavender oil shows promising effects on these symptoms, which may strengthen the hypothesis of the possibility of synergy with ZMT in the ZMT-SNEDDS formulation.

ZMT is a selective serotonin agonist "triptans" used to treat migraine headaches, but its reported side effects may reduce patient compliance. Therefore, it is worth considering another formulation method, which has the same efficacy in alleviating migraine symptoms, but can be administered at a lower dose to reduce its side effects, and ZMT-SNEDDS can achieve this. Compared with the ZMT solution, the optimized ZMT-SNEDDS F5 shows nanospheres with better penetration. By integrating the ATR-FTIR technology in the continuous flow cycle, the rapid dissolution was successfully monitored in real time. For each dose level, the concentration of ZMT in brain tissue obtained from F5 is much lower than that of conventional ZMT solution, and their effects are equivalent, confirming the superiority and effectiveness of F5. Lavender oil has shown promising effects in relieving pain, demonstrating its value as a supplement for migraine treatment. Therefore, the synergy achieved by lavender oil in ZMT-SNEDDS has a high potential for migraine relief. However, all these promising effects of the new formulation ZMT-SNEDDS containing lavender oil should be further clinically tested before being administered to patients.

The authors report no conflicts of interest in this work.

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