Description:1
FORM-2
THE PATENTS ACT, 1970
(39 of 1970)
&
THE PATENTS RULES, 2003
COMPLETE
SPECIFICATION
(See section 10 and rule 13)
Quercetin loaded Bio-SNEDDS for the Breast Cancer
G D Goenka University, an Indian university of Sohna Gurugram Road, Sohna, Haryana, India, 122103
THE FOLLOWING SPECIFICATION PARTICULARLY DESCRIBES THE INVENTION AND THE MANNER IN WHICH IT IS TO BE PERFORMED
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FIELD OF INVENTION:
The present invention relates to pharmaceutical science field, which aims at QbD Approach for the Development of Quercetin loaded Bio-SNEDDS for the Breast Cancer Management.
BACK GROUND:
Breast cancer is the most common malignant tumor, and being the first cause of death in the world. Current studies have shown that quercetin has the great potential to become an alternative or complementary drug to prevent and treat breast cancer through its estrogenic and antiestrogen effects. But its poor water solubility, inadequate bioavailability and intense biotransformation confinement limit the clinical application of quercetin. so, various approaches have been used to enhance the solubility, dissolution rate, and hence, bioavailability of quercetin including solid dispersions, nanosuspensions,microemulsions, and solid lipid nanoparticles.
Many researchers has reported the different quercetin loaded nanoformulations. For instance, one study has developed the quercetin loaded solid dispersion and nanosuspension but even after the successful preparation the maximal solubility of quercetin loaded nanosuspension was limited to 0.4 mg/ml (Lei Gao et al.,2011) and similary, a study has reported the SLNs of quercetin which enhanced the bioavailability of quercetin by five times but these nanoparticles frequently had stability issues that they precipitated out during storage. Surplus preparation of these formulations requires use of hazardous solvents such as chloroform, acetone and ethanol etc (Houli li et al., 2009).
Gaber et al., have demonstrated that Quercetin is the major polyphenolic flavonoid found in food products, including berries, apples, cauliflower, tea, cabbage, nuts, and onions that have traditionally been treated as anticancer and antiviral, and used for the treatment of allergic, metabolic, and inflammatory
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disorders, eye and cardiovascular diseases, and arthritis. Pharmacologically, quercetin has been examined against various microorganisms and parasites, including pathogenic bacteria, viruses, and Plasmodium, Babesia, and Theileria parasites. Additionally, it has shown beneficial effects against Alzheimer’s disease (AD), and this activity is due to its inhibitory effect against acetylcholinesterase. It has also been documented to possess antioxidant, antifungal, anti-carcinogenic, hepatoprotective, and cytotoxic activity. Quercetin has been documented to accumulate in the lungs, liver, kidneys, and small intestines, with lower levels seen in the brain, heart, and spleen, and it is extracted through the renal, fecal, and respiratory systems. The current review examines the pharmacokinetics, as well as the toxic and biological activities of quercetin.
In light of potential data on quercetin for cancer treatment, we propose the use of bio-SNEDDS with natural bioactive oils (i.e., pomegranate seed oil (PSO)). This is an unmet need as there is currently very little or no research on quercetin SNEDDS with bioactive oils (PSO) for breast cancer; only the combination of quercetin SNEDDS alone or in combination with other bioactive compounds such as curcumin and genistein is under clinical study. And development of quercetin loaded bio-SNEDDS is an efficient and convenient method and has the great patient compliance. There are many techniques which are used to develop SNEDDS such as high pressure homogenization, ultrasonication method etc. and maintains the drug in dissolved state, in small droplets of oil all over its transit through GIT, prevents dissolution related problems which is rate-limiting factor and hence facilitates faster onset of action.
OBJECTIVE OF THE INVENTION:
1. It is an object of the invention to provide a method for preparing quercetin loaded bioactive-self nanoemulsifying drug delivery system (SNEDDS) having anticancer effect against breast cancer cells.
2. It is another object of the invention to provide quercetin loaded bioactive-self nanoemulsifying formulation of Quercetin loaded bio-SNEDDS.
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3. It is another object of the invention to provide quercetin loaded bioactive-self nanoemulsifying drug delivery system (SNEDDS).
4. It is another object of the invention to provide use of bio-SNEDDS with natural bioactive oils (i.e., pomegranate seed oil (PSO)).
SUMMARY
The present study aims to develop quercetin loaded bioactive-self nanoemulsifying drug delivery system (SNEEDS) that offers anticancer effect against breast cancer cells. Solubility study of quercetin in different oils, surfactants and co-surfactants were performed.Pseudo ternary phase diagram was constructed for the selected mixture components to set the suitable range for each component.Then the experimental design approach was used to optimize the bio-SNEDDS that possess small globule size with enhanced emulsification and dissolution rates. Hence, we have successfully developed quercetin loaded bioactive-SNEDDS which enhance the bioavailability, increase the permeability through the membrane and reduced the first pass metabolism along with the synergistic effect with bioactive oil (pomegranate seed oil) by triggering multiple apoptotic signaling pathways and also reduce dose limiting single-drug toxicity. The quercetin loaded bio-SNEDDS were then characterized for the various physicochemical parameters. In light of potential data on quercetin for cancer treatment, we propose the use of bio-SNEDDS with natural bioactive oils (i.e., pomegranate seed oil (PSO)). This is an unmet need as there is currently very little or no research on quercetin SNEDDS with bioactive oils (PSO) for breast cancer; only the combination of quercetin SNEDDS alone or in combination with other bioactive compounds such as curcumin and genistein is under clinical study. And development of quercetin loaded bio-SNEDDS is an efficient and convenient method and has the great patient compliance. There are many techniques which are used to develop SNEDDS such as high pressure homogenization, ultrasonication method etc. and maintains the drug in dissolved state, in small droplets of oil all over its transit through GIT, prevents dissolution related problems which is rate-limiting factor and hence facilitates faster onset of action. Quercetin is a biflavonoid and natural bioactive compound used for its anti-oxidant, anticancer, anti-inflammatory activities. In different type of cancer such as breast cancer, lung cancer, ovarian cancer, and liver
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cancer it exhibit anti-apoptosis, anti- angiogenesis, anti-proliferative and cell cycle arrest, etc. but the drug has poor solubility, stability and oral bioavailability. The objective of our research was the development of bioactive self nanoemulsifying drug delivery system (SNEDDS) of Quercetin with synergistic effect using oil, surfactant and co surfactant that could improve its solubility, oral bioavailability and stability of formulation. The optimized formulation of Quercetin loaded bio-SNEDDS consists of Pomegranate seed oil and triacetin as oil, Tween 80 as surfactant and Transcutol HP as co-surfactant. Optimized liquid SNEDDS formulation showed 176.2 nm of droplet size with 0.358 PDI and 25.6 mV of zeta potential, respectively and had infinite dilution capability. The characterization parameters of the optimized formulation were exceedingly dependent on the independent variables. The optimum formulation showed better droplet size of 153.7 nm, 0.361 PDI and 24.8 mV zeta potential having infinite dilution capability. In vitro drug release of the optimized batch was found to be 86.64 % much higher than the pure Querctein drug solution. Thus, it was concluded from the research that the developed Q-bio-SNEDDS have better potential of efficacious treatments with enhanced solubility and dissolution by oral route with more cost-effective as well acceptable to patients because of convenience of application.
BRIEF DESCRIPTION OF FIGURES
These and other features, aspects, and advantages of the present invention will become better understood when the following detailed description is read with reference to the accompanying figures in which like characters represent like parts throughout the figures, wherein:
Figure 1, illustrates a view of the the optimized clear bio-SNEDDS formulation for the present invention.
Fig. 2: illustrates a view of Method of preparation of Q-bio-SNEDDS for the present invention.
Fig.3: illustrates a view of TEM photomicrograph of SNEDDS for the present invention. Fig.4: illustrates a view of the FTIR spectra of SNEDDS (a) Pure Drug (b) PSO
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(c)Triacetin (d) Tween 80 (e) Physical Mixture (f) Transcutol HP (g) SNEDDS for the present invention.
Fig: 5: illustrates a view of in vitro Drug Release Profile of Q-bio-SNEDDS and Pure Drug Solution for the present invention.
Further, skilled artisans will appreciate that elements in the figures are illustrated for simplicity and may not have been necessarily been drawn to scale. For example, the flowcharts illustrate the method in terms of the most prominent steps involved to help to improve understanding of aspects of the present invention. Furthermore, in terms of the construction of the device, one or more components of the device may have been represented in the figures by conventional symbols, and the figures may show only those specific details that are pertinent to understanding the embodiments of the present invention so as not to obscure the figures with details that will be readily apparent to those of ordinary skill in the art having benefit of the description herein.
DETAILED DESCRIPTION:
For the purpose of promoting an understanding of the principles of the invention, reference will now be made to the embodiment illustrated in the figures and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the invention is thereby intended, such alterations and further modifications in the illustrated system, and such further applications of the principles of the invention as illustrated therein being contemplated as would normally occur to one skilled in the art to which the invention relates.
It will be understood by those skilled in the art that the foregoing general description and the following detailed description are exemplary and explanatory of the invention and are not intended to be restrictive thereof.
Reference throughout this specification to “an aspect”, “another aspect” or similar language means that a particular feature, structure, or characteristic described in
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connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrase “in an embodiment”, “in another embodiment” and similar language throughout this specification may, but do not necessarily, all refer to the same embodiment.
The terms "comprises", "comprising", or any other variations thereof, are intended to cover a non-exclusive inclusion, such that a process or method that comprises a list of steps does not include only those steps but may include other steps not expressly listed or inherent to such process or method. Similarly, one or more devices or systems or elements or structures or components proceeded by "comprises... a" does not, without more constraints, preclude the existence of other devices or other systems or other elements or other structures or other components or additional devices or additional systems or additional elements or additional structures or additional components.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The system, methods, and examples provided herein are illustrative only and not intended to be limiting.
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.
The terms “having”, “comprising”, “including”, and variations thereof signify the presence of a component.
Now the present invention will be described below in detail with reference to the following embodiment.
Example 1
Screening of Surfactant and Co-surfactant
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It is not necessary that the surfactant with good solubilizing potential also has a good affinity for oil. Thus, the screening of surfactant and co-surfactant is done based on their emulsification efficiency for oil, not only their ability to solubilize quercetin. So, the surfactants with HLB values in the range of 12–16 were screened on the basis of their emulsification efficiency and their solubilization of quercetin. Tween 80 and Cremophor EL were screened as they are non-ionic surfactants and considered safe and acceptable for oral ingestion as compared to ionic surfactants. They are also reported to provide better stability to emulsions over wide ranges of pH and ionic strength (Jain et al., 2013). Tween 80 showed the maximum oil solubilization capacity and also had a high HLB value of 15. So it was selected for further experimentation.
The screening of co-surfactants was done on the basis of their solubility with the drug and their ability to maximize the nanoemulsification area with the selected surfactant with the help of a pseudo-ternary phase diagram as well. Therefore, Transcutol HP has shown high solubilization potential with the drug, and then the pseudo-ternary diagram was constructed using different ratios of surfactant and co-surfactant. Transcutol HP showed the maximum emulsifying area as compared to PEG 400 due to its better compatibility with the chosen surfactant. The presence of co-surfactant enhanced the nanoemulsion area, and high emulsification efficiency was achieved. Hence, Trancutol HP was selected as the co-surfactant for the Q-bio-SNEDDS formulation.
Example 2
Construction of Pseudo Ternary Phase Diagram
A pseudo ternary diagram was constructed to select the optimum ratio of surfactant:co-surfactant and oil:Smix, consisting of Combination of PSO and triacetin as oily phase, Smix (Tween 80 and Transcutol HP) along with deionized water. These diagrams were prepared on the basis of the maximum isotropic nanoemulsion region, and it facilitated the self-nanoemulsifying efficiency of Smix, which is directly proportional to the size of the nanoemulsion region. The self-nanoemulsifying region is an area where clear and transparent formulations are obtained upon dilution. At first, the pomegranate oil was used as the oil phase and tested for the pseudo-ternary phase diagram with tween 80 as a
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surfactant and transcutol HP as a co-surfactant. But it was very difficult to construct the diagram with pomegranate oil as continuous phase separation from surfactant and instability was observed in the formation. So, the pseudo-ternary diagram was constructed using pomegranate oil and triacetin in combination as the oil phase, tween 80 as a surfactant, and transcutol HP as a co-surfactant. The self-nanoemulsifying area was first increased when we went from 2:1 to 3:1 with increasing concentrations of surfactant and co-surfactant. The maximum nanoemulasion was formed at 3:1 due to an increase in self-emulsification, decreased interfacial tension and rapid dispersion of oil in the aqueous phase. When the ratio was increased to 4:1, the decrease in nanoemulsion region was observed which showed that a further increment in the surfactant concentration did not increase the emulsification region. The maximum self-nanoemulsifying area was observed at a Smix ratio of 3:1. Different combinations in which the oil concentration varies from 20 to 35% were selected, the Smix was fixed at a ratio of 3:1, and then the bio-SNEDDS were prepared using this ratio.
Example 3
Quercetin loaded bio-SNEDDS
FTIR spectra of the drug, physical mixture and formulation were done to find out the possible interaction between the drug and excipients used in the formulation. The FTIR spectra of Quercetin peaks detected at C-H aromatic hydrocarbon, O-H stretching, O-H bending, C=O aryl ketone and C=C aromatic ring stretch at 1319 cm-1 3412.22 cm-1, 1381.09 cm-1, 1664.64 cm-1, and 1610.63, 1562.41 cm-1 respectively. Whereas the PSO shows N-H stretch at 3414.5 and –CH3 bending at 1377.23, and C-H band at 2928 cm-1and the peak of triacetin is C=O at 1726.36 and C-H band at 1298.15 cm-1alongwith the highest peak of Transcutol HP is detected at 3448.87 of OH band and C-H band at 2870 cm-1. In the physical mixture the C-H band shows a broad peak from 1300-1347 cm-1 and 2860-2924 cm-1 which means excipient shows no interaction with each other and is compatible. The spectra of SNEDDS shows the high intensity peak at 2924.21, 2854.77 and 1745.65 cm-1which are C=O stretching and C-H band. A medium peak was detected at 1163.13 which is amide and C-H bending can be observed at 723 alongwith –CH2 at
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1462.1 cm-1. The FTIR spectra show all the characteristic peaks of the groups in formulation without any interaction.
Example 7
In vitro drug release:
The in vitro release of drugs of Q-bio-SNEDDS and pure Quercetin were performed in 250 ml of phosphate buffer pH 6.8 separately. After 60 minutes, the percentage of drugs release of the SNEDDS was reached to be 21.4 ± 1.39, which further reached to 86.6 ± 1.22 after 24 hours and the percentage release of pure quercetin was 17.22 ± 0.34 and 59.62± 0.72 after 24 hrs which shows that the percentage release of Q-bio-SNEEDS was significantly higher (p ? 0.05) than the pure quercetin solution as shown in the table below. The enhanced release of drug from SNEDDS was due to the direct formation of nanoemulsion with nanometric droplet size range. Increased availability of Quercetin in the dissolved state could give the better absorption and improved bioavailability. Thedrug released rate were fitted in the different kinetics models includiing zero order, first order, Higuchi’s model, Korsmeyer Peppas and Hixon–Crowell. The finding of this study indicated the slow and sustained release of drugs from the Q-bio-SNEDDS Time (minutes) % CumulativeDrug Release (% CDR)* Q-bio-SNEDDS Drug Solution 0 0 0 5 12.21 ± 1.06 9.072 ± 0.72 10 13.84 ± 0.53 12.21 ± 1.41 15 15.47 ± 0.34 12.45 ± 1.22 30 16.52 ± 0.69 14.31 ± 1.32
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.
Table. 1; The percentage drug release of Q-bio-SNEDDS and Pure Quercetin
The NLC loaded with the drug was effectively prepared through a combination of hot shear homogenization and ultrasonication methods. The experimental design and optimization process employed response surface methodology utilizing the Central Composite Design approach. The objective of this study was to optimize the formulation variables and create a nanocarrier that is cost-effective, biodegradable, and stable, while also achieving higher drug entrapment and a sustained release profile.The optimized formulation exhibited a particle size of 302 nm, which falls within the acceptable range for oral drug delivery. It displayed a higher entrapment efficiency ranging from 42.3% to 76.5%, as well as an elevated drug release profile with a subsequent sustained release of 77.6%. The experimental findings revealed that Q-NLC improved the solubility and stability of Quercetin in aqueous solutions and facilitated its sustained release. This study represents a promising advancement in breast cancer prevention through the utilization of a novel, biodegradable, and biocompatible Q-NLC. It possesses enhanced anti-cancer properties while minimizing immunogenicity and side effects.
45 17.34 ± 0.87 15.82 ± 1.25 60 21.41 ± 1.39 17.22 ± 0.34 120 46.10 ± 1.72 18.85 ± 1.06 240 51.00 ± 1.45 21.41 ± 0.69 360 57.64 ± 1.79 40.51 ± 0.87 720 61.25 ± 1.39 53.91 ± 1.04 1440 86.64 ± 1.22 59.62 ± 0.72
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Variations and modifications of the foregoing are within the scope of the present invention. Accordingly, many variations of these embodiments are envisaged within the scope of the present invention.
The foregoing descriptions of specific embodiments of the present invention have been presented for purposes of description. They are not intended to be exhaustive or to limit the present invention to the precise forms disclosed, and obviously many modifications and variations are possible in light of the above teaching.
The embodiments were chosen and described in order to best explain the principles of the present invention and its practical application, and to thereby enable others skilled in the art to best utilize the present invention and various embodiments with various modifications as are suited to the particular use contemplated. It is understood that various omissions and substitutions of equivalents are contemplated as circumstances may suggest or render expedient, but such omissions and substitutions are intended to cover the application or implementation without departing from the spirit or scope of the present invention.
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We claim,
1. A method for preparing quercetin loaded bioactive-self nanoemulsifying drug delivery system (SNEDDS) having anticancer effect against breast cancer cells comprising the steps of:
a) A calculated amount of drug is added to the oily phase of pomegranate oil and triacetin combination,
b) Mixed both the phases using vortex mixer.
c) Add calculated amount of surfactant and co-surfactant into clear drug solution,
d) Mixing of system was ensured using magnetic stirrer until homogeneous system is formed,
e) Final mixture was equilibrated for 24 hrs and observed for any sign of phase separation and turbidity.
2. A quercetin loaded bioactive-self nanoemulsifying formulation of Quercetin loaded bio-SNEDDS prepared by the method claimed in claim 1, comprises of Pomegranate seed oil and triacetin as oil, Tween 80 as surfactant and Transcutol HP as co-surfactant.
3. A quercetin loaded bioactive-self nanoemulsifying formulation of Quercetin loaded bio-SNEDDS prepared by the method claimed in claim 1, wherein liquid SNEDDS formulation showed 176.2 nm of droplet size with 0.358 PDI and 25.6 mV of zeta potential, respectively and had infinite dilution capability.
4. A quercetin loaded bioactive-self nanoemulsifying formulation of Quercetin loaded bio-SNEDDS prepared by the method claimed in claim 1, wherein in vitro drug release of the optimized batch was found to be 86.64 % much higher than the pure Querctein drug solution.
5. A quercetin loaded bioactive-self nanoemulsifying drug delivery system (SNEDDS) prepared by the method of claim 1, wherein said system enhances the bioavailability,
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increase the permeability through the membrane and reduced the first pass metabolism along with the synergistic effect with bioactive oil (pomegranate seed oil) by triggering multiple apoptotic signaling pathways and also reduce dose limiting single-drug toxicity.
Dated this 28/07/2023 G D Goenka University, Sohna Gurugram Road, Sohna, Haryana, India, 122103
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ABSTRACT
Quercetin loaded Bio-SNEDDS for the Breast Cancer
The present invention relates to develop quercetin loaded bioactive-self nanoemulsifying drug delivery system (SNEEDS) that offers anticancer effect against breast cancer cells. Solubility study of quercetin in different oils, surfactants and co-surfactants were performed.Pseudo ternary phase diagram was constructed for the selected mixture components to set the suitable range for each component. Then the experimental design approach was used to optimize the bio-SNEDDS that possess small globule size with enhanced emulsification and dissolution rates. Hence, developed quercetin loaded bioactive-SNEDDS which enhance the bioavailability, increase the permeability through the membrane and reduced the first pass metabolism along with the synergistic effect with bioactive oil (pomegranate seed oil) by triggering multiple apoptotic signaling pathways and also reduce dose limiting single-drug toxicity. In vitro drug release of the optimized batch was found to be 86.64 % much higher than the pure Querctein drug solution. , Claims:We claim,
1. A method for preparing quercetin loaded bioactive-self nanoemulsifying drug delivery system (SNEDDS) having anticancer effect against breast cancer cells comprising the steps of:
a) A calculated amount of drug is added to the oily phase of pomegranate oil and triacetin combination,
b) Mixed both the phases using vortex mixer.
c) Add calculated amount of surfactant and co-surfactant into clear drug solution,
d) Mixing of system was ensured using magnetic stirrer until homogeneous system is formed,
e) Final mixture was equilibrated for 24 hrs and observed for any sign of phase separation and turbidity.
2. A quercetin loaded bioactive-self nanoemulsifying formulation of Quercetin loaded bio-SNEDDS prepared by the method claimed in claim 1, comprises of Pomegranate seed oil and triacetin as oil, Tween 80 as surfactant and Transcutol HP as co-surfactant.
3. A quercetin loaded bioactive-self nanoemulsifying formulation of Quercetin loaded bio-SNEDDS prepared by the method claimed in claim 1, wherein liquid SNEDDS formulation showed 176.2 nm of droplet size with 0.358 PDI and 25.6 mV of zeta potential, respectively and had infinite dilution capability.
4. A quercetin loaded bioactive-self nanoemulsifying formulation of Quercetin loaded bio-SNEDDS prepared by the method claimed in claim 1, wherein in vitro drug release of the optimized batch was found to be 86.64 % much higher than the pure Querctein drug solution.
5. A quercetin loaded bioactive-self nanoemulsifying drug delivery system (SNEDDS) prepared by the method of claim 1, wherein said system enhances the bioavailability,
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increase the permeability through the membrane and reduced the first pass metabolism along with the synergistic effect with bioactive oil (pomegranate seed oil) by triggering multiple apoptotic signaling pathways and also reduce dose limiting single-drug toxicity.