An Intranasal Nanoemulsion Formulation for the Management of Oxidative Stress in CNS Disorders and Neurodegeneration

Abstract

Title: A Intranasal Nanoemulsion Formulation for the Management of Oxidative Stress in CNS Disorders and Neurodegeneration The novel formulation of Tocopheryl acetate in the form of nanoemulsion for targeted drug delivery to CNS administer from nose to brain, prepared from using spontaneous emulsification method herein Tocopheryl acetate uses as oil phase or drug, Cremophore RH 40 as surfactant and PEG 400 as Co-surfactant, evaluated through various parameters herein the particle size of optimize batch was 37.66 nm, PDI was 0.308, zeta potential was -29.6 mV, in vitro drug release was 87.88±2.18%, Refractive index was 1.37±0.3, viscosity was 63.60±0.25 cp and ex vivo permeation 62.60±3.52%, behavioral study shows inhibition of oxidative stress, while the levels of GSH, CAT and SOD significantly higher and the level of MDA, AChE and β Amyloid were significantly lower with Tocopheryl acetate nanemulsion, further confirms the stability and effectively used as an antioxidant or in the management of oxidative stress in various CNS disorders along with prevention of neurodegeneration, to signify efficacy of the formulation.

Specification

Description:Title of the Invention
"A Intranasal Nanoemulsion Formulation for the Management of Oxidative Stress in CNS Disorders and Neurodegeneration"
Field of the Invention
The present invention relates to pharmaceutical preparation consist of novel nanoemulsion formulation of Tocopheryl Acetate from spontaneous emulsification method, administered through nose to brain delivery as an antioxidant for the management of oxidative stress in CNS disorders and neurodegeneration.

Background of Invention
An imbalance between free radicals and antioxidants causes oxidative stress in your body. The brain, which includes the spinal cord and central nervous system (CNS), is one of the body's most metabolically active organs and consumes 20% of all oxygen even while at rest. Free radicals are produced when oxygen is used, and the brain's increased oxygen needs result in even more reactive oxygen/nitrogen species. This free radical i. e reactive oxygen species (ROS) and reactive nitrogen species (RNS) responsible for generation of oxidative stress (Shukla, V. et al., 2011). The possibility that oxidative stress has a role in the aetiology of a number of late-onset neurodegenerative illnesses has long been acknowledged. The CNS is significantly abundant in polyunsaturated fatty acids, which harmful oxygen oxidises. Some in vitro and in vivo models suggests that oxidative stress is a crucial player in pathogenesis of neurodegenerative diseases. According to clinical and preclinical investigations, the brain and peripheral tissue of people with neurodegenerative illnesses exhibit higher levels of OS biomarkers and lower levels of antioxidant defence biomarkers. Numerous ROS damage indicators have been observed in th+e particular brain region that experiences selective neurodegeneration in PD, AD, and ALS cases (Andersen, J.K., 2004). Cumulative oxidative stress has been linked to mitochondrial dysfunction, DNA repair system degradation, and cellular damage, all of which have been linked to accelerated ageing and the emergence of neurodegenerative diseases (Niedzielska, E., et al.,2016). The blood-brain barrier, which is there and intended to protect the brain from toxins by limiting their diffusion into neurons and glia, also serves as a drawback since it blocks or lowers the uptake of some antioxidants, such as vitamin E, into the brain. Cyclin dependent kinase 5 (Cdk5) activity that is out of control has been linked to a number of neurodegenerative illnesses, including AD, ALS, HD, and PD. (Shukla, V. et al., 2011).
One of the most significant lipid-soluble antioxidants is vitamin E. Vitamin E functions as an antioxidant by having a hydroxyl group on its phenolic group on the chromanol ring, which can donate an atom of hydrogen and neutralise a wide range of free radicals, including reactive oxygen species (ROS) and act as anti-inflammatory through protein kinase C (PKC) inhibition. Vitamin E has more antioxidant potential against peroxyl radicals than other antioxidants like glutathione or β-carotene (Allan Butterfield, D., et al., 2002). Vitamin E's complicated bioavailability is affected by a number of significant variables, including the consumption of competing nutrients, variations in intestinal absorption, age, gender, smoking, obesity, and genetic polymorphisms. Nearly 30 years ago, it was published that vitamin E levels were decreased in 55 patients with AD compared to non-demented controls. So, to treat oxidative stress at CNS and maintain vitamin E level in brain we develop a novel approach of tocopherol loaded nanoemulsion for nose to brain delivery in the management of oxidative stress (Lloret, A., et al., 2019).
Oxidative stress (OS) has been proposed as one factor that plays a potential role in the pathogenesis of neurodegenerative disorders. This oxidative stress occurs in neurodegenerative disease include Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), and spinocerebellar ataxia (SCA). The current treatment for neurodegenerative diseases is based on symptoms. No any treatment targets the oxidative stress. The potential of antioxidants is limited, owing to the presence of the blood-brain barrier (BBB) by oral route. One strategy to improve the delivery of antioxidants to the brain involves the use of intranasal delivery by nose to brain pathway. Tocopheryl acetate is acts as chain-breaking antioxidant in lipoproteins and cell membranes, limiting lipid peroxidation and maintaining membrane integrity.
The present invention relates to preparation of nanoemulsion formulation of Tocopheryl Acetate using spontaneous emulsification method, administered through nose to brain delivery as an antioxidant for the management of oxidative stress in CNS disorders and neurodegeneration.

Objective of Invention
The principal objective of the invention is to develop novel formulation of Tocopheryl acetate in the form of nanoemulsion for targeted drug delivery to CNS administer from nose to brain, as an antioxidant or management of oxidative stress in various CNS disorders along with prevention of neurodegeneration.

The further objective of the invention is that Tocopheryl acetate nanoemuslsion prepared from using spontaneous emulsification method herein tocopheryl acetate uses as oil phase or drug, Cremophore RH 40 as surfactant and PEG 400 as Co-surfactant.

Another objective of the invention is that the prepared nanoemulsion preparation evaluated from particle size, PDI, zeta potential, in vitro drug release, Refractive index, viscosity, ex vivo permeation, FTIR, Behavioral Study confirms the stability and efficacy of the formulation.

Summary of the Invention

The principal objective of the invention is to develop novel formulation of Tocopheryl acetate in the form of nanoemulsion using spontaneous emulsification method herein tocopheryl acetate uses as oil phase or drug, Cremophore RH 40 as surfactant and PEG 400 as Co-surfactant effectively used as an antioxidant or management of oxidative stress in various CNS disorders along with prevention of neurodegeneration.
A Intranasal Nanoemulsion Formulation for the Management of Oxidative Stress in CNS Disorders and Neurodegeneration composed of: I) Oil Phase: Tocopheryl acetate as oil phase or drug and Smix ratio taken with effective concentration ratio from 1:9 and 9:1 respectively, II) Smix: Smix ratio prepared from effective concentration ratio of Cremophore RH 40 (Surfactant) and PEG 400 (Co-surfactant) from 2:1, 3:1 and 4:1 respectively; and III) Aqueous phase: 2 ml of distilled water as aqueous phase; converted into nanoemulsion through a process includes the following steps: I) Optimization study of Oil Phase, Smix and Aqueous Phase : Tocopheryl acetate was selected as oil phase, Distilled water as an aqueous phase and Cremophore RH 40 ( surfactant) and PEG 400 ( co surfactant) selected as Smix developed from pseudo ternary phase diagram at various ratio's herein 2:1, 3:1, 4:1 ratios of Smix were chosen in increasing concentration of surfactant with respect to co-surfactant and increasing concentration of co surfactant with respect to surfactant for a detailed study of the phase diagrams; for each phase diagram, oil phase and Smix at a predetermined ratio from 1:9 to 9:1 was uniformly mixed in different glass vial optimized from sixteen different combinations of oil and Smix were made to cover maximum ratios for the study to delineate the boundaries of phases precisely to form phase diagrams; pseudo ternary phase diagrams of oil, Smix and aqueous phase were developed using the aqueous titration method for each mass ratio of oil and Smix to get transparent, bluish and easily flowable o/w nanoemulsion, the physical state of said nanoemulsion marked on a pseudo-three-component phase diagram where one axis represents the aqueous phase, the second one represents oil phase and the third represents the Smix of surfactant and co-surfactant at a predetermined mass ratio and plotted on a triangular co-ordinates to construct the pseudo turnery phase diagram using CHEMIX turnery diagram software; II) Nanoemulsion preparation: uniformly mixing oil phase of Tocopheryl acetate with Smix (Cremophore RH 40: surfactant and PEG 400: co-surfactant) in predetermined ratio's; to said oil phase, slowly adding aqueous phase of distilled water in predetermined concentration under continuous stirring for spontaneous emulsification of oil phase and water phase to obtain final o/w nanoemulsion, evaluated through particle size, PDI, zeta potential, in vitro drug release, refractive index, viscosity, ex vivo permeation, FTIR, Behavioral Study confirms the stability and effectively used as an antioxidant or management of oxidative stress in various CNS disorders along with prevention of neurodegeneration.

Brief Description of Drawings
The following thorough explanation of the various aspects of the invention, taken in conjunction with the corresponding drawing that represents various aspects and other features of the disclosure invention.
Figure 1: Flow chart of preparation process of nanoemulsion
Figure 2: FTIR Spectra
Figure 3: Globule size and polydispersity index
Figure 4: Zeta Potential of Formulation
Figure 5: Ex vivo permeation study
Figure 6: Histopathology Study
Figure 7: A) Formulation swab collected after 30 min B) Control solution swab collected after 2 min
Figure 8: Morris water trajectory diagram A) Vehicle Control B) Scopolamine induced C) Test1 D) Test2 E) Donepzil
Detailed Description of the Invention
The following description is of exemplary embodiments only and is not intended to limit the scope, applicability or configuration of the invention in any way. Rather, the following description provides a convenient illustration through explanation and figures for implementing exemplary embodiments of the invention. Various changes to the described embodiments may be made in the function and arrangement of the ingredients described without departing from the scope of the invention.
The use of "including", "comprising" or "having" variations thereof herein is meant to encompass the items listed thereafter and equivalents thereof as well as additional items. The terms "a" and "an" herein do not denote a limitation of quantity, but rather denote the presence of at least one of the referenced items. Further, the use of terms "first", "second", and "third", and the like, herein do not denote any order, quantity, or importance, but rather are used to distinguish one element from another.
A Intranasal Nanoemulsion Formulation for the Management of Oxidative Stress in CNS Disorders and Neurodegeneration composed of: I) Oil Phase: Tocopheryl acetate as oil phase or drug and Smix ratio taken with effective concentration ratio from 1:9 and 9:1 respectively, II) Smix: Smix ratio prepared from effective concentration ratio of Cremophore RH 40 (Surfactant) and PEG 400 (Co-surfactant) from 2:1, 3:1 and 4:1 respectively; and III) Aqueous phase: 2 ml of distilled water as aqueous phase; converted into nanoemulsion through a process includes the following steps: I) Optimization study of Oil Phase, Smix and Aqueous Phase : Tocopheryl acetate was selected as oil phase, Distilled water as an aqueous phase and Cremophore RH 40 ( surfactant) and PEG 400 ( co surfactant) selected as Smix developed from pseudo ternary phase diagram at various ratio's herein 2:1, 3:1, 4:1 ratios of Smix were chosen in increasing concentration of surfactant with respect to co-surfactant and increasing concentration of co surfactant with respect to surfactant for a detailed study of the phase diagrams; for each phase diagram, oil phase and Smix at a predetermined ratio from 1:9 to 9:1 was uniformly mixed in different glass vial optimized from sixteen different combinations of oil and Smix were made to cover maximum ratios for the study to delineate the boundaries of phases precisely to form phase diagrams; pseudo ternary phase diagrams of oil, Smix and aqueous phase were developed using the aqueous titration method for each mass ratio of oil and Smix to get transparent, bluish and easily flowable o/w nanoemulsion, the physical state of said nanoemulsion marked on a pseudo-three-component phase diagram where one axis represents the aqueous phase, the second one represents oil phase and the third represents the Smix of surfactant and co-surfactant at a predetermined mass ratio and plotted on a triangular co-ordinates to construct the pseudo turnery phase diagram using CHEMIX turnery diagram software; II) Nanoemulsion preparation: uniformly mixing oil phase of Tocopheryl acetate with Smix (Cremophore RH 40: surfactant and PEG 400: co-surfactant) in predetermined ratio's; to said oil phase, slowly adding aqueous phase of distilled water in predetermined concentration under continuous stirring for spontaneous emulsification of oil phase and water phase to obtain final o/w nanoemulsion, evaluated through particle size, PDI, zeta potential, in vitro drug release, refractive index, viscosity, ex vivo permeation, FTIR, Behavioral Study confirms the stability and effectively used as an antioxidant or management of oxidative stress in various CNS disorders along with prevention of neurodegeneration.

Table No. 1: Formula of nanoemulsion

Sr. No. Name of Ingredient/ Phase Concentration Ratio Application
01 Tocopheryl acetate as Oil Phase and Smix ratio respectively 1:9 and 9:1 Key ingredient
02 Smix: Cremophore RH 40 (Surfactant) and PEG 400 ( Co-surfactant) ratio respectively 2:1, 3:1 and 4:1
Additive
03 Aqueous Phase ( Distilled Water) 2 ml Additive

General Formulation Process of nanoemulsion
I) Optimization study of Oil Phase, Smix and Aqueous Phase : Tocopheryl acetate was selected as oil phase, Distilled water as an aqueous phase and Cremophore RH 40 ( surfactant) and PEG 400 ( co surfactant) selected as Smix developed from pseudo ternary phase diagram at various ratio's herein 2:1, 3:1, 4:1 ratios of Smix were chosen in increasing concentration of surfactant with respect to co-surfactant and increasing concentration of co surfactant with respect to surfactant for a detailed study of the phase diagrams; for each phase diagram, oil phase and Smix at a predetermined ratio from 1:9 to 9:1 was uniformly mixed in different glass vial optimized from sixteen different combinations of oil and Smix were made to cover maximum ratios for the study to delineate the boundaries of phases precisely to form phase diagrams; pseudo ternary phase diagrams of oil, Smix and aqueous phase were developed using the aqueous titration method for each mass ratio of oil and Smix to get transparent, bluish and easily flowable o/w nanoemulsion, the physical state of said nanoemulsion marked on a pseudo-three-component phase diagram where one axis represents the aqueous phase, the second one represents oil phase and the third represents the Smix of surfactant and co-surfactant at a predetermined mass ratio and plotted on a triangular co-ordinates to construct the pseudo turnery phase diagram using CHEMIX turnery diagram software; and
II) Nanoemulsion preparation: uniformly mixing oil phase of Tocopheryl acetate with Smix (Cremophore RH 40: surfactant and PEG 400: co-surfactant) in predetermined ratio's; to said oil phase, slowly adding aqueous phase of distilled water in predetermined concentration under continuous stirring for spontaneous emulsification of oil phase and water phase to obtain final o/w nanoemulsion. ( Table no. 1 and Figure 1)

Example 1
I) Optimization study of Oil Phase, Smix and Aqueous Phase : Tocopheryl acetate was selected as oil phase, Distilled water as an aqueous phase and Cremophore RH 40 ( surfactant) and PEG 400 ( co surfactant) selected as Smix developed from pseudo ternary phase diagram 2:1 ratio of Smix were chosen in increasing concentration of surfactant with respect to co-surfactant and increasing concentration of co surfactant with respect to surfactant for a detailed study of the phase diagrams; for each phase diagram, oil phase and Smix ratio of 1:9 was uniformly mixed in different glass vial optimized from sixteen different combinations of oil and Smix were made to cover maximum ratios for the study to delineate the boundaries of phases precisely to form phase diagrams; pseudo ternary phase diagrams of oil, Smix and aqueous phase were developed using the aqueous titration method for each mass ratio of oil and Smix to get transparent, bluish and easily flowable o/w nanoemulsion, the physical state of said nanoemulsion marked on a pseudo-three-component phase diagram where one axis represents the aqueous phase, the second one represents oil phase and the third represents the Smix of surfactant and co-surfactant at a predetermined mass ratio and plotted on a triangular co-ordinates to construct the pseudo turnery phase diagram using CHEMIX turnery diagram software; and
II) Nanoemulsion preparation: uniformly mixing oil phase of Tocopheryl acetate with Smix (Cremophore RH 40: surfactant and PEG 400: co-surfactant) in predetermined ratio's; to said oil phase, slowly adding aqueous phase of distilled water in predetermined concentration under continuous stirring for spontaneous emulsification of oil phase and water phase to obtain final o/w nanoemulsion. ( Table no. 1 and Figure 1)
Example 2
I) Optimization study of Oil Phase, Smix and Aqueous Phase : Tocopheryl acetate was selected as oil phase, Distilled water as an aqueous phase and Cremophore RH 40 ( surfactant) and PEG 400 ( co surfactant) selected as Smix developed from pseudo ternary phase diagram 3:1 ratio of Smix were chosen in increasing concentration of surfactant with respect to co-surfactant and increasing concentration of co surfactant with respect to surfactant for a detailed study of the phase diagrams; for each phase diagram, oil phase and Smix ratio of 5:7 was uniformly mixed in different glass vial optimized from sixteen different combinations of oil and Smix were made to cover maximum ratios for the study to delineate the boundaries of phases precisely to form phase diagrams; pseudo ternary phase diagrams of oil, Smix and aqueous phase were developed using the aqueous titration method for each mass ratio of oil and Smix to get transparent, bluish and easily flowable o/w nanoemulsion, the physical state of said nanoemulsion marked on a pseudo-three-component phase diagram where one axis represents the aqueous phase, the second one represents oil phase and the third represents the Smix of surfactant and co-surfactant at a predetermined mass ratio and plotted on a triangular co-ordinates to construct the pseudo turnery phase diagram using CHEMIX turnery diagram software; and
II) Nanoemulsion preparation: uniformly mixing oil phase of Tocopheryl acetate with Smix (Cremophore RH 40: surfactant and PEG 400: co-surfactant) in predetermined ratio's; to said oil phase, slowly adding aqueous phase of distilled water in predetermined concentration under continuous stirring for spontaneous emulsification of oil phase and water phase to obtain final o/w nanoemulsion. ( Table no. 1 and Figure 1)
Example 3

I) Optimization study of Oil Phase, Smix and Aqueous Phase : Tocopheryl acetate was selected as oil phase, Distilled water as an aqueous phase and Cremophore RH 40 ( surfactant) and PEG 400 ( co surfactant) selected as Smix developed from pseudo ternary phase diagram 4:1 ratio of Smix were chosen in increasing concentration of surfactant with respect to co-surfactant and increasing concentration of co surfactant with respect to surfactant for a detailed study of the phase diagrams; for each phase diagram, oil phase and Smix ratio of 9:1 was uniformly mixed in different glass vial optimized from sixteen different combinations of oil and Smix were made to cover maximum ratios for the study to delineate the boundaries of phases precisely to form phase diagrams; pseudo ternary phase diagrams of oil, Smix and aqueous phase were developed using the aqueous titration method for each mass ratio of oil and Smix to get transparent, bluish and easily flowable o/w nanoemulsion, the physical state of said nanoemulsion marked on a pseudo-three-component phase diagram where one axis represents the aqueous phase, the second one represents oil phase and the third represents the Smix of surfactant and co-surfactant at a predetermined mass ratio and plotted on a triangular co-ordinates to construct the pseudo turnery phase diagram using CHEMIX turnery diagram software; and
II) Nanoemulsion preparation: uniformly mixing oil phase of Tocopheryl acetate with Smix (Cremophore RH 40: surfactant and PEG 400: co-surfactant) in predetermined ratio's; to said oil phase, slowly adding aqueous phase of distilled water in predetermined concentration under continuous stirring for spontaneous emulsification of oil phase and water phase to obtain final o/w nanoemulsion. ( Table no. 1 and Figure 1)
Results and discussion:
FTIR analysis of drug and excipient (Pavia 2008) and (Indian pharmacopeia, 2018): FTIR spectra obtained could confirm the chemical stability of tocopheryl acetate in the nanoemulsion. FTIR of tocopheryl acetate shows characteristics C=O stretching at 1757 cm−1, aromatic C=C stretching at 1576 cm−1, strong C-O stretching at 1251 cm−11203 cm−1 and C-H stretching 2800-3000 cm−1. FTIR of nanoemulsion formulation the characteristics 1757 cm−1 tocopheryl acetate C=O stretching is seen, the aromatic C=C stretching seen around 1637 cm−1, strong C-O stretching seen at 1251 cm−1 and 1208 cm−1 and C-H stretching is seen in the range of 2800-3000 cm−1. By studying the results of the interaction studies, it is observed that all the components that are being used in the formulation which are as tocopheryl acetate as active pharmaceutical ingredient, Cremophore RH 40 as surfactant, PEG 400 as Co-surfactant as well as water are compatible with each other and shows no interaction either physical and chemical between them. (Figure 2)
Phase diagram Studies: - In combination the API tocopheryl acetate directly used as oil phase, Cremophore RH 40 used as Surfactant and PEG 400 used as Co-surfactant. Pseudoternary phase diagrams were created using aqueous phase titrations to determine the surfactant to cosurfactant ratio capable of creating the biggest isotropic NE zone. The area of nanoemulsion found in order of 4:1>3:1>2:1. It was found that the Smix ratio and its capacity to dissolve the oily phase, together with a reduction in the system's free energy, directly influence whether the region of NE is small or large. From the figures it was observed that 2:1 ratio shows less nanoemulsion region than 3:1. Again, the proportion of Surfactant was increased (Smix 4:1) the nanoemulsion region approximately same as Smix 3:1 but in case of Smix 3:1 the clarity of Smix ratto to 4:1 it was observed that area was slightly decreased it shows that further addition of surfactant does not contribute to emulsification process. Hence, 3:1 of the Smix was selected as the Nanoemulsion region. Smix both combined are practically nontoxic for human body probable lethal dose of cremophore RH 40 and PEG 400 for human body is above 40mg/kg and in rats 2000mg/kg and 30200mg/kg respectively (Stokes, A.H., et al., 2013). After the study of phase diagram, we can conclude that between those three diagrams of three different ratios the diagram which expressing the results for Ratio 3:1 has covered more area in the section in water i. e. the most essential which should be considered for formulationg a O/W Nanoemulsion. According to the above phase diagram study all the three figures shown the region of nanoemulsion which tends to form a mixture of oil in water nanoemulsion. But still after the comparision between all those figures given us the appropriate ratio of the S-mix in the formulation. The formulation that is O/W nanoemulsion it is concluded that ratio 3:1 is appropriate for the formulation.
Visual evaluation: All nanoemulsion were transparent and appeared like a homogeneous single-phase liquid, when observed for visual clarity against light. Measurement of pH: pH of the optimized NE was found to be 5.9 ± 0.7. The pH of the NE was in adequate range for nasal drug delivery system signifying its nonirritant nature. (England, R.J.A et al., 1999)
Thermodynamic stability determination: - The objective of this study is to evaluate the phase separation and effect of temperature variation on formulation. In the thermodynamic stability studies, selected formulation subjected to tests like heating cooling cycle and centrifugation. It was observed that all formulation were stable, clear liquid, no phase separation under stress conditions, this confirms the liquid formulations were stable for storage.
Table. 1 Thermal and Centrifugation stability of tocopheryl acetate nanoemulsion
Formulation Temperature Centrifugation stability at 3000 rpm
40c Room temp. 400C
1. √ √ √ √
Thermal Stability: - Formulation are subjected to various atmospheric temperature and this study shows that heating cooling cycles formulations were stable, clear liquid, no phase separation under stress conditions.
Centrifugation: - After centrifugation for 20 minutes at 3000 RPM all the formulations were still stable, clear liquid, no phase separation occurred under stress conditions. This proves that the formulation is thermodynamically stable. Refractive index determination: The refractive index, n, of a medium is defined as the ratio of the speed, c, of a wave such as light or sound in a reference medium to the phase speed, vapor pressure, of the wave in the medium presented.
Table 2. Difference in refractive indices of placebo and sample formulation
Sample Refractive index
Oil 1.44±0.5
Nano emulsion 1.37±0.3
Water 1.33±0.3
Isotropic nature of the formulation is demonstrated through the RI value of it. Percent Transmission: - This test was performed to check the transparency of the formulation which was measured in percentage. Determine by UV spectrophotometer. The percent transmission of optimized formulation was found to be 87±0.8%. The Higher transparency indicates the stability of nanoemulsion. The fact that the NEs transmittance was nearly equivalent to the aqueous phase transmittance indicates that the oil droplets were spread continuously and uniformly. The formulation is transparent because the largest dispersed phase globules were discovered to be no larger than a quarter of the wavelength of visible light.
Viscosity: - Viscosity was determined at differential RPM at same condition and same spindle number (61) of the Brookfield viscometer (DV1 viscometer). As well study of the property of the system was determined by this test.
Table. 3 Viscosity of formulation at different RPM
Speed (RPM) Viscosity(cp) Torque (%)
10 85.80±0.15 14.3
12 78±0.2 15.6
20 67.20±0.22 22.4
30 66.20±0.18 33.1
50 63.60±0.25 53
Drug Content Estimation: - 1 ml nanoemulsion was dissolved in 100ml ethanol taken in volumetric flask observed spectrometrically after triplicates reading the drug content found is given in following table.
Table. 4. Drug Content
Sr. No. Batch Drug Content (%)
1. B1 94.50%±0.8%
Drug content determination helps to find out the amount of drug entrapped in the formulation. Hence, from above study it was observed that optimized batch has maximum amount of drug content.
Globule Size and Polydispersity Index: - The Globule size in the range between 30-200nm. Optimized nanoemulsion was transparent and monophasic having mean globule size of 56.26 nm having a uniform PDI 0.715. (Figure 3)
Zeta Potential : For the dispersed system standard zeta potential range is ±30 mV. Zeta potential value for optimized batch was found to be -26.8 mV which falls within a range. (Figure 4)
In vitro drug release study: - The cumulative percentage release of tocopheryl acetate from optimized nanoemulsion was found to be 87.88±2.18% over a period of 3 hr.
Table 5. In vitro release study

Sr. No. %CDR (Q) Time (Min) T Square Root T Log (%) release Log T Cumulative % drug remain Log % remain
1 0.33±0.02 0 0 -0.48 99.67 1.99
2 3.368±0.3 20 4.47 0.53 1.30 96.63 1.98
3 26.38±1.2 40 6.32 0.80 1.60 73.62 1.86
4 50.53±1.8 60 7.74 0.89 1.77 49.47 1.69
5 67.89±2.1 80 8.94 0.95 1.90 32.11 1.50
6 76.57±1.75 100 10 1 2 23.43 1.36
7 81.47±2.33 120 10.95 1.04 2.07 18.53 1.26
8 82.60±2.01 140 11.83 1.07 2.14 17.4 1.24
9 83.74±2.2 160 12.64 1.10 2.20 16.26 1.21
10 87.89±2.18 180 13.41 1.13 2.25 12.11 1.08
11 66.38±1.88 200 14.14 1.15 2.30 33.62 1.52

Ex vivo permeation Study: - Diffusion kinetic were carried out using goat nasal mucosa for the optimized batch in phosphate buffer pH 6.4 for a period of 3h using diffusion cell apparatus. The drug content was measured in PB using UV spectrophotometric method at λmax 284nm. To study the significance as well as side effects of the prepared nanoemulsion on nasal route. (Figure 5)
Table 6. Ex vivo permeation Study
Sr. No. Time (Min) % CDR
1. 0 2.226415±0.39
2. 20 5.245283±0.52
3. 40 13.16981±0.48
4. 60 18.83019±1.11
5. 80 27.13208±1
6. 100 35.81132±0.98
7. 120 38.45283±1.20
8. 140 45.24528±1.17
9. 160 47.88679±1.15
10. 180 50.90566±2.15
11. 200 62.60377±3.52
Nasal Ciliotoxicity Study: - Goat nasal mucosa was used to assess the formulation's excipients' potential harmful effects. The mucosa treated with the negative control (PBS pH 6.4) indicates no nasociliary damage and has an intact epithelial layer, whereas the mucosa treated with the positive control (isopropyl alcohol) shows severe damage. On the other hand, optimized formulation also showed an intact epithelial layer with no traces of toxicity. (Figure 6 and 7)
Stability Study: - Physical stability of nanoemulsion was confirmed with insignificant changes observed in zeta potential and particle size. Tocopheryl Acetate nanoemulsion found chemically stable as drug content of the formulation was observed unchanged during accelerated stability study. Table 7.13 gives results obtained after stability.
Table 7. Stability Study
Sr. No. Parameter 0 Month 3 Month
1. Drug Content 94.50%±0.8% 92%±0.78%
2. Particle Size (nm) 56.26 32.57
3. Zeta Potential (mV) -26.8 -29.6
Shelf-life Estimation
Shelf Life of Tocopheryl acetate Nanoemulsion at 50° C Temperature
a) Determination of rate constant (k0 )
A0 = 94.50% (initial concentration)
At = 89.88% (concentration after time 't') and
t= 30 days (time period between initial concentration and concentration after storage)


Rate Constant= 0.154/day

b) Determination of shelf life (t90 )
a= 94.50% (initial concentration)
k0 = 0.154/day (rate constant)


t90= 61 days
Shelf Life of Tocopheryl acetate Nanoemulsion at Room Temperature (26-28 0C):
a) Determination of Rate Constant (k0 )
A0= 94.50% (initial concentration)
At= 91.86% (concentration after time't') and
t= 30 days (time period between initial concentration and concentration after storage)


Rate Constant= 0.088/day
b) Determination of shelf life (t90 )
a= 94.50% (initial concentration)
k0 = 0.088/day (rate constant)


t90= 107 days
Shelf Life of Tocopheryl acetate Nanoemulsion at 10°C
a) Determination of Rate Constant (k 0 )
A0= 94.50% (initial concentration)
At= 91% (concentration after time 't') and
t= 30 days (time period between initial concentration and concentration after storage)



Rate Constant= 0.116/day

b) Determination of shelf life (t90 )
a= 99.3% (initial concentration)
k0 = 0.116/day (rate constant)


t90= 81.46 days
k0 = 0.22/day (rate constant)
The result is mentioned in the table 8
Table.8. Shelf Life of formulation
Evaluation Temperature
150C Room Temp. (26-28 0C): 500C
Rate Constant 0.116 0.088 0.154
Shelf life 81.46/days 107/days 61/days
Drug Content 91% 91.86% 89.88%
In vivo Study
Nasal Mucociliary Transport Time: Nasal mucociliary transport time was observed between 27 to 32 min for the test formulation and for saline solution time was observed between 2 to 6 min. Test formulation showed better residence of formulation in the nasal cavity. Figure 20 showed images of swab after nasal administration of sample.
Acute intranasal toxicity (AINT) study
The acute trial at a dose of 100 mg/rat/day revealed no mortality or aberrant clinical symptoms in rats. During the 14-day post-dosing observation period, no substantial body weight gain was detected in rats.
Behavioral Study
a) Morris Water Maze: - The Escape Latency Time (ELT) was measured to assess spatial memory and results are displayed in Figure no. 22 during the pre-treatment session the test was conducted on day 10 and we found that there was no significant difference in the ELT of all groups. On the 14th-daytocopheryl acetate treatment was found to improve spatial memory in a dose-dependent manner with a significant reduction in the ELT at 25mg/kg and 42mg/kg as compared to the negative control. Additionally, donepezil (5mg/kg), an AChE inhibitor showed significant improvement in spatial memory on both the days during treatment regimen (P<0.001) in comparison with only the negative control group. The trajectory diagrams are also captured with the help of the Logitech webcam. (Hosseini, M.J. et al., 2022) (Figure 8)
Locomotor activity in open field
Immediately after the Morris water maze test, the rat were evaluated for their locomotor activity in the open field due to the administration of Scopolamine. The crossing number of the Scopolamine group significantly decreased compared to the control. It is the same for the rearing and Self-rearing which sees its activity increase in the animals receiving the both doses of 25 mg/kg and 42 mg/kg tocopheryl acetate nanoemulsion contrary to the Scopolamine group that decreases. (Augustsson, H., 2004).
Biochemical Estimation:
Estimation of Lipid peroxidation: -
Elevated MDA levels have been found to be a decisive marker of in vivo lipid peroxidation which occurs due to oxidative stress induced cell wall impairment. As depicted in figure 27 and 28 scopolamine administration significantly increased MDA levels as an indicator of oxidative stress in both whole brain and hippocampus + cortex compared to control. Both, whole brain and hippocampus + cortex samples were estimated for the level of malondialdehyde. Significantly scopolamine + 25 mg/kg nanoemulsion and Scopolamine 42mg/kg nanoemulsion decreases malondialdehyde level in whole Brain and hippocampus + cortex as compared to control. (Yadang, F.S.A., et al., 2020)
Estimation of Catalase Activity: -
CAT activity was enhanced significantly after Tocopheryl acetate nanoemulsion administration at 25, 42 mg/kg (***P<0.001) as compared with negative control. Donepezil which was used as a positive control significantly increased CAT activity at 5mg/kg dose (***p<0.001) in homogenate of both, whole brain and hippocampus + cortex. (Yadang, F.S.A., et al., 2020)
Estimation of Reduced glutathione (GSH): -
As shown in figure 31 & 32, Our data revealed that the scopolamine treated rats showed significant decline in GSH levels which related to the imbalance between ROS and antioxidant levels, as well as induction of oxidative stress condition in Brain. The data analysis indicated a significant rise in GSH levels in the whole brain and Hippocampus + cortex of rats treated with 25, 42 mg/kg tocopheryl acetate nanoemulsion as compared to negative control. Donepezil which was used as a positive control significantly increased GSH activity at 5mg/kg dose in homogenate of both, whole brain and hippocampus + cortex. (Yadang, F.S.A., et al., 2020)
Estimation of superoxide Dismutase (SOD): -
SOD activity was enhanced significantly after administration of tocopheryl acetate nanoemulsion with doses 25, 42 mg/kg as compared with negative control. Donepezil which was used as positive control significantly increased SOD activity at 5mg//kg dose in homogenate of whole brain and hippocampus + cortex. (Yadang, F.S.A., et al., 2020).
Estimation of β Amyloid: -
β Amyloid protein was decreased significantly after administration of 25 mg/kg and 42 mg/kg doses of toocopheryl acetate nanomeuulsion in comparision with negative control. Donepezil which was used as positive control significantly decreased β Amyloid protein level at 5 mg/kg dose in homogeneate of both whole brain and Hippocampus. (Esparza, T.J., et al., 2018)
Estimation of β Amyloid: -
β Amyloid protein was decreased significantly after administration of 25 mg/kg and 42 mg/kg doses of toocopheryl acetate nanomeuulsion in comparision with negative control. Donepezil which was used as positive control significantly decreased β Amyloid protein level at 5 mg/kg dose in homogeneate of both whole brain and Hippocampus. (Esparza, T.J., et al., 2018)
Estimation of Acetylcholinesterase activity: -
Scopolamine administration resulted in significant increase in the brain AchE activity. Tocopheryl acetate (25 mg/kg and 42 mg/kg) treated groups significantly decreased the elevated AchE activity respectively in comparison of negative control rat in both brain and hippocampus + cortex. The standard treatment also has less AchE level when compared to negative control rat in homogenate of both, brain and hippocampus + cortex. (Kumar A et al., 2010.)
Conclusion: -
Additionally, it was discovered that the drug's concentration in the brain was higher, supporting the effectiveness of NE's targeting via the i.n. route. Behavioral Study shows that Scopolamine induced oxidative stress in rats was successfully reversed when administered with Tocopheryl acetate nanoemulsionintranasally along with the donepezil. While the levels of GSH, CAT, and SOD were significantly higher, the level of MDA was significantly lower in the group treated with Tocopheryl acetate NE via the intranasal route along with Donepezil. Also, the AchE and β amyloid levels decreased in NE-treated animals as compared to Scopolamine induced group which indicated that tocopheryl acetate nanoemulsion may help to reduce Alzheimer's biomarkers.
Scope of the Invention
The present invention relates to novel The novel formulation of Tocopheryl acetate in the form of nanoemulsion for targeted drug delivery to CNS administer from nose to brain, prepared from using spontaneous emulsification method herein Tocopheryl acetate uses as oil phase or drug, Cremophore RH 40 as surfactant and PEG 400 as Co-surfactant, evaluated through various parameters herein the particle size of optimize batch was 37.66 nm, PDI was 0.308, zeta potential was -29.6 mV, in vitro drug release was 87.88±2.18%, Refractive index was 1.37±0.3, viscosity was 63.60±0.25 cp and ex vivo permeation 62.60±3.52%, behavioral study shows that scopolamine induced oxidative stress in rats were successfully reversed when administered with Tocopheryl acetate nanoemulsion intranasally along with the donepezil. While the levels of GSH, CAT and SOD significantly higher and the level of MDA, AChE and β Amyloid were significantly lower in the group treated with Tocopheryl acetate NE via intranasal route and Donepezil by oral route. The formulated nanoemulsion confirms the stability and effectively used as an antioxidant or in the management of oxidative stress in various CNS disorders along with prevention of neurodegeneration, to signify efficacy of the formulation, could be a promising treatment in CNS compare to existing formulations, in future.

It is to be understood that the present invention is not limited to the embodiments described above, it should be clear that various modifications and alterations can be made along with various features of one embodiment included in other embodiments, within the scope of the present invention.

, Claims:We claim,
1- A intranasal nanoemulsion formulation for the management of oxidative stress in CNS disorders and neurodegeneration comprises of:

a Tocopheryl acetate as an oil phase or drug;

a Smix ratio of Cremophore RH 40 (Surfactant) and PEG 400 (Co-surfactant);

and a distilled water as aqueous phase characterized in that an effective concentration ratio of said oil phase to Smix from 1:9 to 9:1 respectively and said Smix of Cremophore RH 40 (Surfactant) and PEG 400 (Co-surfactant) in effective concentration from 2:1, 3:1 and 4:1 respectively, converted into nanoemulsion using spontaneous emulsification method effectively used as an antioxidant or management of oxidative stress in various CNS disorders along with prevention of neurodegeneration.

2- The process to prepare intranasal nanoemulsion formulation as claimed in claim 1 includes the following steps: wherein,

I) Optimization study of Oil Phase, Smix and Aqueous Phase : Tocopheryl acetate was selected as oil phase, Distilled water as an aqueous phase and Cremophore RH 40 ( surfactant) and PEG 400 ( co surfactant) selected as Smix developed from pseudo ternary phase diagram at various ratio's herein 2:1, 3:1, 4:1 ratios of Smix were chosen in increasing concentration of surfactant with respect to co-surfactant and increasing concentration of co surfactant with respect to surfactant for a detailed study of the phase diagrams;

for each phase diagram, oil phase and Smix at a predetermined ratio from 1:9 to 9:1 was uniformly mixed in different glass vial optimized from sixteen different combinations of oil and Smix were made to cover maximum ratios for the study to delineate the boundaries of phases precisely to form phase diagrams;

pseudo ternary phase diagrams of oil, Smix and aqueous phase were developed using the aqueous titration method for each mass ratio of oil and Smix to get transparent, bluish and easily flowable o/w nanoemulsion, the physical state of said nanoemulsion marked on a pseudo-three-component phase diagram where one axis represents the aqueous phase, the second one represents oil phase and the third represents the Smix of surfactant and co-surfactant at a predetermined mass ratio and plotted on a triangular co-ordinates to construct the pseudo turnery phase diagram using CHEMIX turnery diagram software;

II) Nanoemulsion preparation: uniformly mixing oil phase of Tocopheryl acetate with Smix (Cremophore RH 40: surfactant and PEG 400: co-surfactant) in predetermined ratio's;

to said oil phase, slowly adding aqueous phase of distilled water in predetermined concentration under continuous stirring for spontaneous emulsification of oil phase and water phase to obtain final o/w nanoemulsion.

Documents