Description:A METHOD FOR SYNTHESIZING VORINOSTAT LOADED NANOPARTICLES COMPOSITION

FIELD OF THE INVENTION
[0001] This invention generally relates to a field of drug loaded nanoparticles, more particularly to a method for synthesizing vorinostat loaded nanoparticles composition.

BACKGROUND
[0002] The subject matter discussed in the background section should not be assumed to be prior art merely as a result of its mention in the background section. Similarly, a problem mentioned in the background section or associated with the subject matter of the background section should not be assumed to have been previously recognized in the prior art. The subject matter in the background section merely represents different approaches, which in and of themselves may also correspond to implementations of the claimed technology.
[0003] Suberoylanilide hydroxamic acid (SAHA) is also known as vorinostat that is a histone deacetylase (HDAC) inhibitor classified under the Biopharmaceutics Classification System (BCS) Class IV, indicating its low solubility and low permeability. The dual limitation presents challenges in its bioavailability and pharmacokinetics, with a biological half-life of approximately 2 hours. Other than these disadvantages, SAHA may show significant therapeutic potential, particularly in oncology. It is currently under investigation for the treatment of cutaneous T-cell lymphoma (CTCL) and is being explored in combination therapies to enhance its efficacy in glioblastoma multiforme and other cancers. The mechanism of action involves the inhibition of overexpressed HDAC enzymes, which play a critical role in cancer cell proliferation and survival. Other than oncology, SAHA may have potential benefits in treating psoriatic conditions by targeting HDAC enzymes in proliferative epidermal cells.
[0004] Conventionally known Vorinostat based nanoformulations utilizes large toxic quantities of organic solvents and surfactants which are not advised for human use and multiple preparation steps imposes problems during industrial scale-up. The drug is having low log P value of 1.44 and is slightly soluble in acetone and insoluble in dichloromethane that are commonly used organic solvents in the preparation of nanoparticles by nanoprecipitation and emulsion solvent evaporation methods respectively. In order to increase the solubility of drug in acetone, additional solvents such as ethanol, tetrahydrofuran, acetonitrile and dimethyl sulfoxide are used in combinations that are difficult to remove completely from the aqueous nanoparticle dispersion due to its higher boiling point, lower volatility and higher water miscibility.
[0005] According to a patent application “CN106821965A” titled “A kind of vitamin A acid multiple medicine delivers nanoparticle solution and its preparation and application together” discloses a nanoparticle solution and its application as anti-tumor medicine are delivered altogether the present invention relates to a kind of vitamin A acid multiple medicine. With vitamin A acid, histon deacetylase (HDAC) inhibitor, perifosine, emulsifying agent, matrix, cosolvent as raw material, nanoparticle is delivered by solvent dilution method one-step synthesis vitamin A acid multiple medicine altogether .Matrix, emulsifying agent, cosolvent in formula are auxiliary material, play a part of stabilization nanoparticle and regulation particle diameter;Histon deacetylase (HDAC) inhibitor, perifosine are used as ancillary drug, the tolerance for reversing tumor cell to retinoid medicine;Vitamin A acid is used as main ingredient, induced tumor cell differentiation and apoptosis. Each composition complements each other, and collaboration plays a role. Medicine collocation of the present invention is reasonable, and clearly, preparation process is simple, with tumour passive targeting, is expected to be applied to therapeutic field of tumor mechanism of action.
[0006] According to a patent application “WO2020077178A1” titled “Plga-peg/pei nanoparticles and methods of use” discloses a composition comprising a nanoparticle comprising poly (lactic acid-co-glycolic acid) (PLGA)-b-polyethylene glycol (PEG) (PLGA-PEG) copolymer formulated with polyethylenimine (PEI), and one or more cargo molecules (e.g., a nucleic acid molecule with or without a small molecule compound) associated with the nanoparticle. The nucleic acid molecule may be a plasmid or minicircle DNA expressing a gene or genes, CRISPR/Cas9 components, or an RNA molecule (e.g., small interfering RNA, miRNA, or lncRNA). Also provided are methods for delivering a cargo molecule to a cell in vitro and in vivo using the aforementioned composition.
[0007] However, existing vorinostat based nanoformulations utilizes some large toxic quantities of organic solvents and surfactants that are not advised for human use. Further, the drug is having low log P value of 1.44 and is slightly soluble in acetone and insoluble in dichloromethane, therefore there is a need of additional solvents such as ethanol, tetrahydrofuran, acetonitrile and dimethyl sulfoxide in order increase solubility of drug in acetone. Further, it is difficult to remove completely from the aqueous nanoparticle dispersion due to its higher boiling point, lower volatility and higher water miscibility.
OBJECTIVES OF THE INVENTION
[0008] The objective of present invention is to provide a method for synthesizing vorinostat loaded nanoparticles composition.
[0009] Further, the objective of present invention is to provide the method for synthesizing vorinostat loaded nanoparticles composition that utilizes low quantity of organic solvent and surfactant.
[0010] Furthermore, the objective of the present invention is to provide the method for synthesizing vorinostat loaded nanoparticles composition having higher drug loading, lower particle size and narrow polydispersity index.
[0011] Furthermore, the objective of the present invention is to provide the method for synthesizing vorinostat loaded nanoparticles composition in order to improve the efficacy of the drug by increasing the cellular uptake and reducing the rate of metabolism.
[0012] Furthermore, the objective of the present invention is to provide the method for synthesizing vorinostat loaded nanoparticles composition by using low quantity of acetone, thereby reducing the use of solvents such as ethanol, tetrahydrofuran, acetonitrile and dimethyl sulfoxide.

SUMMARY
[0014] According to an aspect, the present embodiments a method for synthesizing vorinostat loaded nanoparticles composition. Further, the method comprises dissolving 25.5 mg of polylactic-co-glycolic acid (PLGA) in 500 µL of organic solvent to obtain polymer mixture. Further, the method comprises adding 4.5 mg of drug into the polymer mixture. Further, the drug particles are observed in polymer mixture due to low solubility of drug particles. Further, the method comprises adding 5 drops of 0.001M of aq. Solution of sodium hydroxide (NaOH) to polymer mixture to obtain organic phase. Further, the method comprises adding organic phase dropwise to 3 ml of 1% Tween 80 solution, followed by continuous stirring at 220 RPM using magnetic stirrer at room temperature. Further, the method comprises subjecting organic phase under reduced pressure by using rotary evaporator to obtain aqueous dispersion. Further, the method comprises subjecting aqueous dispersion to probe sonication for time interval of 30 seconds at 20% amplitude to obtain nanodispersion. Further, the method comprises centrifuging nanodispersion at 5000 rpm for 5 min to obtain supernatant. Further, the method comprises collecting synthesized nanoparticles composition from supernatant, followed by analysis for particle size, size distribution, and drug content.

BRIEF DESCRIPTION OF THE DRAWINGS
[0015] The accompanying drawings illustrate various embodiments of systems, methods, and embodiments of various other aspects of the invention. Any person with ordinary skills in the art will appreciate that the illustrated element boundaries (e.g. boxes, groups of boxes, or other shapes) in the figures represent one example of the boundaries. It may be that in some examples one element may be designed as multiple elements or that multiple elements may be designed as one element. In some examples, an element shown as an internal component of one element may be implemented as an external component in another, and vice versa. Furthermore, elements may not be drawn to scale. Non-limiting and non-exhaustive descriptions are described with reference to the following drawings. The components in the figures are not necessarily to scale, emphasis instead being placed upon illustrating principles.
[0016] FIG. 1 illustrates a flow chart of a method for synthesizing vorinostat loaded nanoparticles composition, according to an embodiment of the present invention; and
[0017] FIG. 2 illustrates a tabular representation of a value of particle size (nm) of the synthesized nanoparticles composition, Polydispersity index (PDI) of the synthesized nanoparticles composition, Zeta potential of the synthesized nanoparticles composition, and % Encapsulation efficiency of drug in the synthesized nanoparticles, according to an embodiment of the present invention.

DETAILED DESCRIPTION
[0019] Some embodiments of this invention, illustrating all its features, will now be discussed in detail. The words “comprising,” “having,” “containing,” and “including,” and other forms thereof, are intended to be equivalent in meaning and be open ended in that an item or items following any one of these words is not meant to be an exhaustive listing of such item or items, or meant to be limited to only the listed item or items. It must also be noted that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise.
[0020] Although any systems and methods similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present invention, the preferred, systems and methods are now described. Embodiments of the present invention will be described more fully hereinafter with reference to the accompanying drawings in which like numerals represent like elements throughout the several figures, and in which example embodiments are shown. Embodiments of the claims may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. The examples set forth herein are non-limiting examples and are merely examples among other possible examples.
[0021] The present invention discloses a novel and effective method for synthesizing vorinostat loaded nanoparticles composition having higher drug loading, lower particle size and narrow polydispersity index and improve the efficacy of the drug by increasing the cellular uptake and reducing the rate of metabolism.
[0022] FIG. 1 illustrates a flow chart of a method (100) for synthesizing vorinostat loaded nanoparticles composition, according to an embodiment of the present invention.
[0023] The method (100) as depicted in flow chart may be described in a stepwise manner as follows. In this regard, each block may represent a module, segment, or portion of code, which comprises one or more executable instructions for implementing the specified logical function(s). It should also be noted that in some alternative implementations, the functions noted in the blocks may occur out of the order noted in the drawings. Any process descriptions or blocks in flowcharts should be understood as representing modules, segments, or portions of code that include one or more executable instructions for implementing specific logical functions or steps in the process, and alternate implementations are included within the scope of the example embodiments in which functions may be executed out of order from that shown or discussed, including substantially concurrently or in reverse order, depending on the functionality involved. The flowchart starts at the step (102) and proceeds till step (116).
[0024] In some embodiments, dissolving 25.5 mg of polylactic-co-glycolic acid (PLGA) in 500 µL of organic solvent to obtain polymer mixture, at step 102. 25.5 mg of polylactic-co-glycolic acid (PLGA) is dissolved in 500 µL of an organic solvent obtain polymer mixture. PLGA is a biodegradable and biocompatible polymer that may be dissolved in organic solvents. Further, the the organic solvent corresponds to acetone. The polymer mixture is configured to enable uniform distribution of PLGA to ensure consistent performance in its intended application.
[0025] Further, 500 µL of acetone is configured to eliminate the need for additional co-solvents like ethanol, tetrahydrofuran (THF), acetonitrile, and dimethyl sulfoxide (DMSO). The usage of 500 µL of acetone minimizes the risk of residual solvent contamination. The acetone is highly volatile and easily removed from aqueous systems through simple evaporation or vacuum techniques. Addition of acetone (i.e. organic solvent) in the synthesis process remained efficient while maintaining the safety, stability, and integrity of the nanoparticle dispersion, avoiding the complications posed by co-solvents with higher boiling points and water miscibility.
[0026] In some embodiments, adding 4.5 mg of drug into the polymer mixture. Further, the drug particles are observed in polymer mixture due to low solubility of drug particles, at step 104. The drug corresponds to Suberoylanilide hydroxamic acid (SAHA) or vorinostat that is classified under BCS Class IV. Further, 4.5 mg of the drug is added to the prepared polymer solution. Due to the low solubility of SAHA, the drug particles remain visible and undissolved in the polymer mixture, indicating incomplete dispersion. The phenomenon arises from the poor solubility of the drug in the chosen organic solvent that limits the ability of the drug particles to dissolve fully and uniformly integrate with the polymer. Such undissolved drug particles may affect the homogeneity and efficacy of the final composition.
[0027] In some embodiments, adding 5 drops of 0.001M of aq. Solution of sodium hydroxide (NaOH) to polymer mixture to obtain organic phase, at step 106. Further, the aq. solution of sodium hydroxide is configured to dissolve polymer and drug to obtain organic phase. Addition of the NaOH is configured to facilitate the formation of an organic phase. The aqueous NaOH acts as a mild base configured to promote ionization and partial dissolution of both the polymer and the drug (i.e., SAHA). Further, the interaction is configured to enhance the solubility of the drug and polymer within the organic phase by modifying the chemical environment, aiding in the dispersion or dissolution of the drug in the polymer.
[0028] In some embodiments, adding organic phase dropwise to 3 ml of 1% Tween 80 solution, followed by continuous stirring at 220 RPM using magnetic stirrer at room temperature, at step 108. The organic phase is added dropwise to 3 mL of a 1% Tween 80 solution, followed by continuous stirring at 220 RPM using a magnetic stirrer at room temperature. The continuous stirring may facilitate the formation of a stable emulsion by reducing the interfacial tension between the organic phase and the aqueous phase, aided by the surfactant properties of Tween 80. Further, the dropwise addition may ensure gradual mixing of the organic phase with Tween 80 in order to prevent phase separation and promoting uniform dispersion of the organic droplets within the aqueous medium. Further, the continuous stirring at an optimized speed may ensure consistent emulsification.
[0029] In some embodiments, subjecting organic phase under reduced pressure by using rotary evaporator to obtain aqueous dispersion, at step 110. Further, the organic solvent is removed under reduced pressure using the rotary evaporator. Further, the organic phase is subjected to reduced pressure using a rotary evaporator to obtain an aqueous dispersion. Further, the process may involve removal of organic solvent by evaporation under controlled vacuum and temperature conditions. The reduced pressure of rotary evaporator is configured to accelerate solvent removal without exposing the system to high temperatures in order to preserve integrity of temperature-sensitive components (i.e., drug and polymer). The dispersed polymer and drug form a stable aqueous dispersion on the evaporation of organic solvent. Further, the aqueous dispersion is free from residual organic solvents.
[0030] In some embodiments, subjecting aqueous dispersion to probe sonication for time interval of 30 seconds at 20% amplitude to obtain nanodispersion, at step 112. The aqueous dispersion is subjected to probe sonication for 30 seconds at 20% amplitude to convert it into a nanodispersion. Further, the probe sonication is configured to apply high-frequency ultrasonic waves to generate intense mechanical vibrations in order breakdown larger particles and aggregates into nanosized particles. The probe sonication process is configured to enhance the homogeneity and stability of the dispersion by reducing particle size to ensure uniform distribution of the polymer and drug. Further, the optimized parameters (i.e., short time interval and moderate amplitude) is configured to prevent excessive heat generation in order to obtain stable nanodispersion.
[0031] In some embodiments, centrifuging nanodispersion at 5000 rpm for 5 min to obtain supernatant, at step 114. Further, the centrifugation is performed to remove unentrapped drug and large particles from the nanodispersion. During the process of centrifugation, the centrifugal force is configured to drive heavier particles (i.e., aggregates and unencapsulated drug) to the bottom of the centrifuge tube as a pellet. While the lighter and uniform nanoparticles remain suspended in the supernatant. The process of centrifugation process is configured to remove impurities and oversized particles to provide purified nanodispersion. Further, the supernatant contains desired nanoparticles.
[0032] In some embodiments, collecting synthesized nanoparticles composition from supernatant, followed by analysis for particle size, size distribution, and drug content, at step 116. Further, particle size and size distribution of the synthesized nanoparticles composition is analysed by using Malvern Zetasizer. Further, the drug content in the synthesized nanoparticles composition are analysed by using High performance liquid chromatography (HPLC).
[0033] Further, the particle size and size distribution of the synthesized nanoparticle composition are analyzed using a Malvern Zetasizer that is configured to employ dynamic light scattering (DLS) to measure the hydrodynamic diameter of particles in the nanodispersion. Further, the analysis is configured to provide uniformity and stability of the nanoparticles. Additionally, the drug content within the synthesized nanoparticles is quantified using High-Performance Liquid Chromatography (HPLC). The technique configured to separate and measure the drug concentration with high precision, confirming the encapsulation efficiency and loading capacity of the nanoparticles.
[0034] FIG. 2 illustrates a tabular representation (200) of a value of particle size (nm) of the synthesized nanoparticles composition, Polydispersity index (PDI) of the synthesized nanoparticles composition, Zeta potential of the synthesized nanoparticles composition, and % Encapsulation efficiency of drug in the synthesized nanoparticles, according to an embodiment of the present invention.
[0035] In some embodiments, the average particle size of the synthesized nanoparticles composition is found to be 161.7 nm, with a small variation of ±1.32 nm, representing a uniform size distribution.
[0036] In some embodiments, the polydispersity index (PDI) measures the uniformity of particle size distribution having values below 0.2 indicating a highly homogeneous sample. Further, the PDI of 0.16±0.03 indicates that the synthesized nanoparticles composition is consistent and well-dispersed.
[0037] In some embodiments, zeta potential represents the surface charge of the nanoparticles composition. A value of -7.53 mV indicates moderate stability. Further, the negative charge is configured to provide electrostatic repulsion to prevent particle aggregation.
[0038] In some embodiments, the encapsulation efficiency represents the percentage of the drug encapsulated within the synthesized nanoparticles relative to the total drug used. An efficiency of 89.87% may represent the effective drug loading and high drug loading.
[0039] FIG. 3 illustrates a graphical representation of the particle size of the synthesized nanoparticles composition, according to an embodiment of the present invention.
[0040] In some embodiments, the graphical representation shows the size distribution of particles based on intensity that is measured in nanometers (nm). Further, the x-axis represents particle size (d.nm) on a logarithmic scale and y-axis represents the intensity percentage. Further, the average particle size determined to be 161.7 nm and a small variation of ±1.32 nm. Further, the minimal variation may indicate the narrow size distribution representing uniformity in particle size. The sharp peak further confirms the homogeneous size distribution of the synthesized nanoparticles.
[0041] FIG. 4 illustrates a graphical representation of zeta potential of the synthesized nanoparticles composition, according to an embodiment of the present invention.
[0042] In some embodiments, the graphical representation illustrates the surface charge distribution of the synthesized nanoparticle composition. Further, the x-axis represents zeta potential distribution (mv) and y-axis represents the frequency percentage. Further, the measured value of -7.53 mV represents moderate stability of the nanoparticles in the suspension. The negative charge on the particle surface may facilitate electrostatic repulsion between particles, reducing aggregation. Further, the repulsion is responsible to maintain a dispersed and stable nanoparticle system.
[0043] In some embodiments, the in-vivo anti-psoriatic analysis is performed on IMQ induced psoriatic mouse model to determine the efficacy of synthesized nanoparticles. The in-vivo anti-psoriatic analysis of the synthesized nanoparticles is conducted using an Imiquimod (IMQ)-induced psoriatic mouse model to evaluate the therapeutic efficacy. In the in-vivo study, psoriasis is induced in mice by topical application of IMQ, which mimics the inflammatory and hyperproliferative conditions observed in human psoriasis. The synthesized nanoparticles composition is administered to assess the ability of the synthesized nanoparticles composition to alleviate psoriasis symptoms, such as erythema, scaling, and skin thickness.
[0044] In some embodiments, a swiss albino mice aged 10-14 weeks, weighing 35±3g are subjected to 5% of imiquimod cream. A dose of 62.5 mg of the cream was applied daily for five consecutive days on the shaved back and right ear skin of the mice. Further, the mice are divided into five groups to evaluate the therapeutic effects of various treatments: 1. Negative control group, 2. Positive control group, 3. Standard drug group (marketed drug), 4. Gel containing vorinostat group (0.05%), 5. Gel containing vorinostat nanoparticles group (0.05%). Further, the negative control group received no treatment to serve as a baseline. Further, the positive control group is subjected to imiquimod treatment for inducing psoriasis like skin inflammatory condition. Further, the standard drug group received the marketed drug to assess its effectiveness against the condition. Further, the mice in the gel containing vorinostat (0.05%) group are treated with the topical formulation of vorinostat, and those in the gel containing vorinostat nanoparticles (0.05%) group received the nanoparticle-based formulation.
[0045] In some embodiments, the efficacy of the treatment is evaluated by observing clinically accepted psoriasis area and severity index (PASI) scoring. In order to assess the extent of inflammation and psoriasis-like symptoms in the mice, a cumulative scoring system (0-12) is used based on erythema, thickness, and scaling. Further, the back skin thickness is measured using a digital vernier caliper. Further, the thickness of both the right and left ear skin is determined using a digital micrometer. An increase in the thickness of the back skin or right ear is indicative of the severity of psoriasis inflammation. Further, the back and ear skin samples of the animals are collected and examined histopathologically to evaluate tissue-level changes and confirm the effects of the treatments.
[0046] In some embodiments, the positive control group exhibited a progressive increase in cumulative scoring from day 1 to day 5, representing the successful induction of psoriasis-like inflammation. However, treatment groups receiving the standard drug (marketed product), gel containing vorinostat (0.05%), and gel containing vorinostat nanoparticles (0.05%) represented the significant therapeutic effects in reducing inflammation and psoriasis symptoms. Further, the gel containing vorinostat nanoparticles (0.05%) shows superior efficacy, outperforming both the standard drug and the gel with vorinostat alone.
[0047] In some embodiments, the histopathological analysis of back and ear skin further confirmed the enhanced effectiveness, revealing marked improvements in tissue structure and inflammation resolution with the nanoparticle-based gel. The potential of synthesized nanoparticle composition in improving drug delivery and therapeutic outcomes for psoriasis treatment.
[0048] It should be noted that the method (100) in any case could undergo numerous modifications and variants, all of which are covered by the same innovative concept; moreover, all of the details can be replaced by technically equivalent elements. In practice, the components used, as well as the numbers, shapes, and sizes of the components can be of any kind according to the technical requirements. The scope of protection of the invention is therefore defined by the attached claims.

Dated this 28th Day of January, 2025
Ishita Rustagi (IN-PA/4097)
Agent for Applicant
, Claims:1. A method (100) for synthesizing vorinostat loaded nanoparticles composition, the method (100) comprises:
dissolving 25.5 mg of polylactic-co-glycolic acid (PLGA) in 500 µL of organic solvent to obtain polymer mixture, at step 102;
adding 4.5 mg of drug into the polymer mixture, wherein the drug particles are observed in polymer mixture due to low solubility of drug particles, at step 104;
adding 5 drops of 0.001M of aq. Solution of sodium hydroxide (NaOH) to polymer mixture to obtain organic phase, at step 106;
adding organic phase dropwise to 3 ml of 1% Tween 80 solution, followed by continuous stirring at 220 RPM using magnetic stirrer at room temperature, at step 108;
subjecting organic phase under reduced pressure by using rotary evaporator to obtain aqueous dispersion, at step 110;
subjecting aqueous dispersion to probe sonication for time interval of 30 seconds at 20% amplitude to obtain nanodispersion, at step 112;
centrifuging nanodispersion at 5000 rpm for 5 min to obtain supernatant, at step 114; and
collecting synthesized nanoparticles composition from supernatant, followed by analysis for particle size, size distribution, and drug content, at step 116.

2. The method (100) as claimed in claim 1, wherein the organic solvent corresponds to acetone.

3. The method (100) as claimed in claim 1, wherein the drug corresponds to Suberoylanilide hydroxamic acid (SAHA) or vorinostat that is classified under BCS Class IV.

4. The method (100) as claimed in claim 1, wherein aq. solution of sodium hydroxide is configured to dissolve polymer and drug to obtain organic phase.

5. The method (100) as claimed in claim 1, wherein the organic solvent was removed under reduced pressure using the rotary evaporator.

6. The method (100) as claimed in claim 1, wherein the centrifugation is performed to remove unentrapped drug and large particles from the nanodispersion.

7. The method (100) as claimed in claim 1, wherein the particle size and size distribution of the synthesized nanoparticles composition is analysed by using Malvern Zetasizer.

8. The method (100) as claimed in claim 1, wherein the drug content in the synthesized nanoparticles composition are analysed by using High performance liquid chromatography (HPLC).

9. The method (100) as claimed in claim 1, wherein the in-vivo anti-psoriatic analysis is performed on IMQ induced psoriatic mouse model to determine the efficacy of synthesized nanoparticles.

Dated this 28th Day of January, 2025
Ishita Rustagi (IN-PA/4097)
Agent for Applicant