AU2021104786A4 - Controlled Release of Docetaxel Within Functionalized Mesoporous Silica Nanoparticles for Cancer Therapy - Google Patents

Controlled Release of Docetaxel Within Functionalized Mesoporous Silica Nanoparticles for Cancer Therapy Download PDF

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AU2021104786A4
AU2021104786A4 AU2021104786A AU2021104786A AU2021104786A4 AU 2021104786 A4 AU2021104786 A4 AU 2021104786A4 AU 2021104786 A AU2021104786 A AU 2021104786A AU 2021104786 A AU2021104786 A AU 2021104786A AU 2021104786 A4 AU2021104786 A4 AU 2021104786A4
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formulation
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Sadaf Arfi
Satish Kumar Gupta
Satish Manchanda
Rishabh Maurya
K. Venkat Narapa Reddy
Partha ROY
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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/33Heterocyclic compounds
    • A61K31/335Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/337Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K9/00Medicinal preparations characterised by special physical form
    • A61K9/48Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51Nanocapsules; Nanoparticles
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    • A61K9/5115Inorganic compounds
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00Antineoplastic agents
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61JCONTAINERS SPECIALLY ADAPTED FOR MEDICAL OR PHARMACEUTICAL PURPOSES; DEVICES OR METHODS SPECIALLY ADAPTED FOR BRINGING PHARMACEUTICAL PRODUCTS INTO PARTICULAR PHYSICAL OR ADMINISTERING FORMS; DEVICES FOR ADMINISTERING FOOD OR MEDICINES ORALLY; BABY COMFORTERS; DEVICES FOR RECEIVING SPITTLE
    • A61J3/00Devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms

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Abstract

: In this present invention, mesoporous silica nanoparticle loaded with Antineoplastic drug had been prepared through emulsification followed with solvent evaporation method can be used via; different route of administration. Through this method mesoporous silica nanoparticle were prepared with optimized dependent and independent variables. Different formulations were prepared with varying ratio of drug and polymers which effects drug release and kinetics of drug release pattern. The entrapment of drug causes sustained drug release from mesoporous silica nanoparticle system. The research aims to study the drug release from mesoporous silica nanoparticle and its mechanism of release which ensures controlled and effective drug release. The formulation prepared were evaluated for drug entrapment, in-vitro drug release, drug release kinetics study, SEM (particle size determination), stability testing and Invitro MCF-7 cell line studies. The percentage drug entrapment for all the formulations was appropriate with formulation Fl giving the highest entrapment up to 66.43%. The formulation F2 and F3 62.95% and 54.72% drug entrapment respectively. The cumulative drug release for the formulation Fl, F2 and F3 was found to be 90.62%, 79.61% and 76.25% respectively, individually about a span 8 hours are required or controlled drug delivery model for the formulation. The maximum cumulative percentage drug release was shown by F1. The % tumor growth inhibition of formulation F1, F2 and F3 was found to be 32.64%, 28.42% and 21.24% respectively. The pure drug showed 18.2 % tumor growth inhibition. The formulation comprises the extreme surfactant proportion and the minor particle size (200nm-500nm) and it shows the maximum release initially. The formulations were found to be stable which gives controlled drug delivery for 6 hours. The formulation has nanoparticle size range around 200-500nm.

Description

Title of Invention Controlled Release of Docetaxel Within Functionalized Mesoporous Silica Nanoparticles for Cancer Therapy
Background: Cancer tumor is usually described as a disease that is normally triggered by an out-of control department of irregular cells in a correct component of the human body. The word Cancer is originated from the Latin term i.e., 'crab' and the behavior are crab is that it adheres to correct component of the body in a stubborn manner due to its paws catching capacity. Therefore, the tumorous development can attack and abolish nearby pass on and buildings to isolated sites to trigger loss of life. Cancer tumor can have an effect on all existing cells in all age groups including children's old teenagers and they destroy and imbalance the physical as well as chemical characteristics of the body. The cancer is different at different site with several specific and non-specific factors. Since the price of the cancer treatment is much higher than the normal surgical treatment is mainly becomes a burden on females and their mental stress along with problem which related to their family and societies. Cancer is wide group of diseases which could start practically from any, part of the human body, when atypical cell grows ungovernably and yonder their partition to berate adjoining parts of the human body and outspread to other organs. This hindmost activity is called metastasizing and is the extensive cause of death from cancer. Although is this fast-growing world cancer is the 2nd largest paramount cause of death across the world, conceding for an approximate 9.6 million lives', which is one in six deaths, in 2018 lung, prostrate, colorectal, stomach, and liver cancer were the most regular types of cancer in males, though on other hand breast, colorectal, lung, cervical, and thyroid cancer were the most regular among females. However, Cancer could affect one an all young or old, rich or poor, men or women, even children's to are affected which constitutes an enormous burden for patient, families, and societies. Being one of the paramount causes of deaths worldwide. Though many in these deaths can be circumvented, between 30-50% of cancer are preventable aerobicized life style choices such as illusion of tobacco & by accepting public health measures like inoculation against cancer generating infection other can be spotted early, which could be served and rehabilitated. Even with later phase of cancer, the endurance of patients should be pleased with adequate palliative care. Cancer is the disease that transpire due to the variance in the cell cycle of the body, under normal condition the cell cycle is very highly regulated as the number of new cells formed by cell cycle is just adequate enough to replace the old cells which have undergone apoptosis or have died due to cell injury or some other cause. Cancer is the leading cause of death worldwide. Conventional chemotherapy is often characterized by clinical inefficiency and serious side-effects, mainly because of the leaking out of drugs during blood circulation and nonspecific cell/tissue distribution. The development of nanotechnology and nanomedicine in the past decades has facilitated the development of various nano vehicles for experimental and clinical application as drug delivery systems to solve these problems. To efficiently deliver therapeutics into cancer cells, a number of strategies have been recently investigated. The toxicity associated with the administration of chemotherapeutic drugs due to their random interactions throughout the body necessitates the development of drug encapsulating nano preparations that significantly mask, or reduce, the toxic side effects of the drugs. Simple passive nanocarrier delivery to the tumor site is unlikely to be enough to elicit a maximum therapeutic response as the drug-loaded carriers must reach the intracellular target sites, crossing the cell membrane barrier and reaching cytosol might still not be enough for achieving maximum therapeutic benefit, which necessitates the delivery of drugs directly to intracellular targets, such as bringing pro-apoptotic drugs to mitochondria, nucleic acid therapeutics to nuclei, and lysosomal enzymes to defective lysosomes. In this review, we discuss the strategies developed for tumor targeting, cytosolic delivery via cell membrane translocation, and finally organelle-specific targeting, which may be applied for developing highly efficacious, truly multifunctional, cancer-targeted nano preparations.
Besides organic and metallic nanoparticles, inorganic nanoparticles have attracted considerable attention as chemically inert and biocompatible drug carriers. The synthetic methods to generate many of these materials are simple and the building block relatively inexpensive. In addition, the morphology, size, and porosity of the nanoparticles can be controlled and tuned with relative ease. Metal-inorganic hybrid or organic-inorganic hybrid nanoparticles can be functionalized to generate materials with distinct drug delivery characteristics, which allows drug delivery and activation in a light-, pH-, or temperature- dependent manner. Current research primarily focuses on silica-based and calcium phosphate based nanoparticles. This research will be based on stimuli sensitive drug delivery systems based on MSN. Various materials can be used as gatekeepers to control drug release under acidic conditions. These materials have great potential for application in tumor therapy and for improving anti cancer drug efficiency and decreasing side effects. However, most work will focus on in-vitro and in-vivo studies. Thus, several challenges still need to be overcome for the further advancement of the biological and biomedical applications of MSNs. First, the differences in the physical condition between tumor microenvironment and normal tissues are minimal, making the manipulation of the stimuli-responsive drug delivery system in-vivo via stimuli-responsive. Second, the targeting effects of stimuli-responsive MSNs depending on EPR effects are low, thus causing nanoparticles accumulation in some organs, such as the heart, liver, and spleen. Upon accumulation in normal tissues, MSNs can be internalized into cells via endocytosis to trigger drug release, which may result in side effects. Third, the bio distribution, acute and chronic toxicities, changes in molecule level, long-term stability, and circulation properties of stimulus responsive drug delivery systems need to be further investigated before implementation in clinical practice. Docetaxel is used for the treatment of patients with locally advanced or metastatic breast cancer after failure of prior chemotherapy. It is also used as a single agent in the treatment of patients with locally advanced or metastatic non-small cell lung cancer after failure of prior platinum-based chemotherapy. It is also used in combination with prednisone, in the treatment of patients with androgen independent (hormone refractory) metastatic prostate cancer. Furthermore, docetaxel has uses in the treatment of gastric adenocarcinoma and head and neck cancer. Docetaxel is an antineoplastic agent that acts by disrupting the microtubular network in cells that is essential for mitotic and interphase cellular functions. Docetaxel binds to free tubulin and promotes the assembly of tubulin into stable microtubules while simultaneously inhibiting their disassembly. This leads to the production of microtubule bundles without normal function and to the stabilization of microtubules, which results in the inhibition of mitosis in cells. Docetaxel binding to microtubules does not alter the number of protofilaments in the bound microtubules, a feature which differs from most spindle poisons currently in clinical use.
Summary: The major problem of conventional Docetaxel therapies possesses several drawbacks like low oral bioavailability, non-specific drug distribution in body, hypersensitivity reaction because of presence of Tween80 and ethanol and other related side effects. But the mesoporous silica nanoparticle formulation may be a better alternative for the Docetaxel therapy in the treatment of breast cancer. The developed system showed increase in bioavailability, cytotoxicity in cancerous cell, cellular uptake sustained drug release up to 8 hrs. and decrease size, hemolytic activity in RBCs, therefore, produce some benefit, such as hypersensitivity reaction, non specific drug distribution and dose related systemic side effects with improved patient compliance. All the formulations were prepared by emulsifying technique monitored with liquid vaporization technique with multiple ratios of polymer, fatty acid and surfactant. The percentage drug entrapment for all the formulations was appropriate with formulation Fl giving the highest entrapment up to 66.43%. The formulation F2 and F3 62.95% and 54.72% drug entrapment respectively. The cumulative drug release for the formulation Fl, F2 and F3 was found to be 90.62%, 79.61% and 76.25% respectively, individually about a span 8 hours are required or controlled drug delivery model for the formulation. The maximum cumulative percentage drug release was shown by F1. The % tumor growth inhibition of formulation Fl, F2 and F3 was found to be 32.64%, 28.42% and 21.24% respectively. The pure drug showed 18.2 % tumor growth inhibition. The formulation Fl shows maximum entrapment efficacy of 66.43% which shows that in the formulation there is a perfect blend of all the additives along with the drug i.e., Docetaxel which gives us optimum particle trapping efficacy in the MSNs and it also shows that increase in surfactant increase in drug entrapment. About a span 8 hours are required or controlled drug delivery model for the formulation. The maximum cumulative percentage drug release was shown by formulation Fl. The formulation F2 preparation obeys Korsmayer-Peppas discharge model that mainly explains that drug is released from the polymeric drug matrix. As from the release model kinetics data we can clearly see that that F1 follows zero order kinetics which proves that the drug is released through controlled way. Due to presence of low concentration of lipid it shows the highest and extreme drug release of the formulation. The "n" value of Korsmayer-Peppas, which indicates the mainly two different diffusion mechanism that is Fickian and Non-Fickian diffusion mechanism, from the data the preparations of Docetaxel F1, F2 and F3 all of them had "n" value which describes that when the value is above 0.45, they always follow non-Fickian diffusion pattern or mechanism. The low permeation of MSNs at high lipid loading may be attributed to the fact that low polymer and lipid swelling and high hydrophilic nature of drug entrapped in MSN. The release was fast at low lipid content and optimum polymer ratio because as dissolution media penetrates, as it tends to form gel layer which retards the release of hydrophilic drug and the thickness of this gel layer is greater in case of higher polymer and lipid content which shows greater resistance to drug release. The ICH guidelines with formulation F1, shows maximum stability i.e., 98.4% at 5 ±3'C ambient humidity conditions which is concluded through stability testing. All the studies result show that the formulations were can be well formulated in good condition and can be stored for long duration which further leads to a safer, efficient and completely homogenized for the drug delivery of the drug Docetaxel. The approach of increased therapeutic efficacy, controlled drug release and reduced toxicity show better applicability as compared to conventional anti-neoplastic dosage delivery methods due to enhanced bioavailability and patient compliance. Since all the physiochemical studies are successfully done it can be further forwarded to in- vivo or animal studies for the confirmation of its bio availability and several release aspects which are not yet discovered. All these results suggest that the mesoporous silica nanoparticle loaded with Antineoplastic system may be an appropriate vehicle for the IV delivery of hydrophobic anticancerous agent Docetaxel for treatment of breast cancer. Nevertheless, significant work still needs to be carried out to confirm these results i.e., in vivo investigations.
MODES FOR CARRYING OUT THE PREFERRED EMBODIMENTS The embodiments herein and the various features and advantageous details thereof are explained more fully with reference to the non-limiting embodiments that are illustrated in the accompanying drawings and detailed in the following description. Descriptions of well-known components and processing techniques are omitted so as to not unnecessarily obscure the embodiments herein. The examples used herein are intended merely to facilitate an understanding of ways in which the embodiments herein may be practiced and to further enable those of skill in the art to practice the embodiments herein. Accordingly, the examples should not be construed as limiting the scope of the embodiments herein. Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. As used herein, the singular forms "a", "an", "the" include plural referents unless the context clearly dictates otherwise. Further, the terms "like", "as such", "for example", "including" are meant to introduce examples which further clarify more general subject matter, and should be contemplated for the persons skilled in the art to understand the subject matter. Although this invention has been described in conjunction with the exemplary embodiments' below, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art. Accordingly, the exemplary embodiments of the invention as set forth above are intended to be illustrative and not limiting. Various changes may be made without departing from the spirit and scope of the invention.
Working (Method of preparation): Drug-loaded MSNs were prepared by using a solvent evaporation technique A known amount of Docetaxel (100mg) was weighed and dissolved in 100ml of DMSO separately. 100ml de-ionized water was added to the Docetaxel-DMSO solution. The prepared solution was shaken through mechanical shaker for 72 hrs. at 370 C. After this the solution obtained was centrifuged at 12000 rpm for 45 mins. The pipette was taken and the topmost layer of the liquid was taken and solution of equal proportion of water and ethanol was used for washing the residual. Then filtered the solution and air dried the residue. The drug-loaded mesoporous nanoparticles were obtained.
Evaluation: Drug Entrapment The buffer was prepared freshly for the study. The weighed quantity of drug equivalent to 20 mg was taken and then it was taken in pestle and mortar for crushing followed by pouring it in the volumetric flask after filtration and likewise another formulation excluding drug i.e., blank formulation was prepared and kept separately. Both the dilutions were taken and analyzed under UV visible spectrophotometer for getting the data of drug trapping efficacy also called as drug entrapment. E.E % = API trapped / concentrated of drug taken x 100
SEM Studies
SEM is an essential tool for characterizing the particle size of the MSNs. They were first
disseminated in lesser quantity in the test tube containing acetone and was subjected to manual shaking in order to make particles without restrictions were spread. Then, the dispersed MSNs was released on the aluminum stumps and left for vacuity dehydrating in a vacuity desiccator for sometimes. After this, it was examined via SEM for the superficial inspection of MSNs.
X-Ray Diffraction Studies MSNs were examined for its diffraction patterns. This method had been opted for quantitative analysis, 3D dimensional structure, structure imperfections determination and crystal structure for crystalline material. 40mg of sample powder was taken and was scanned through 0-60 value. The XRD of drug, polymer and physical mixture was done separately in order to view the crystal lattice of the sample.
In-Vitro Cumulative Drug Release The amount of medicine loaded mesoporous silica nanoparticles equivalent to 50 mg of drug was dissolved in dissolution media 250 ml in conical flask and then it was placed in a mechanical
shaker. At regular intervals the aliquot was withdrawn around 5 ml and then it was substituted via recently set buffer solution. The sample was detected by the help of UV spectrophotometer at the
wavelength of 234 nm to a measure of the amount of drug present.
Kinetics of Drug Release For determining the drug discharge kinetics of the formulated batches, the all preparation were calculated into the given mathematical theory:
• Cumulative % Drug Release v/s Time considered as Zero Order Kinetics
• Log % Drug Retained v/s Time considered as First Order Kinetics
• Cumulative % Drug Release v/s Root Time considered as Higuchi Matrix • %Drug retained^1/3 v/sTime considered as Hixon-Crowell erosion equation
• Log Cumulative% Drug Release v/s Log Time named as Korsmeyer-Peppas model
Korsemayer Peppas model is used to interpret the the drug relase kinetics mechanism.The drug release mechanism model was understood by studying "n" as the dispersion exponent and accordingly the drug release pattern was interpreted. According to the diffusion exponent concept, if the significance of "n" is below 0.45 then it follows Fickinian meachanism for the release of drug; whereas if the value of "n" is in the range of 0.45-0.85 then it follows non-Fickinian model. Further it is seen that the release mechanism described as Case II transport or super case II transport if the value of n is 0.85 or above 0.85.
Stability Testing The method of estimating the capability to maintain the certain specific conditions and limits through its span of storing and use also term as shelf life and characteristics which it possesses during its preparations is called is stability testing of the formulation. The MSNs along with the drug was taken as same as 10 mg and was put in the Petri dish for 3 months in BOD. At different time intervals like 1, 2 and 3 months the samples were withdrawn and checked for the medicine content. As per ICHQ1A (General case) guidelines the three preparations were taken and it was exposed to stability testing:
> Long term (25± 2C/ 60 5% RH) > Intermediate (30 2°C / 65 5% RH) > Accelerated (40 2°C/ 75 5% RH)
> Refrigeration (5 3°C)
In-vitro cytotoxicity study of formulations: The in vitro cytotoxicity of the CPT formulations was performed on the human breast cancer cell line MCF-7. The concentration of drug was 10 pg/ml used for in vitro studied. Sensitivity of MCF-7 cells to formulations was determined individually by the MTT colorimetric assay. Cells were seeded in a flat-bottomed 96-well plate and incubated for 24 h at 37°C and in % C02. The cell line was exposed to all formulations mentioned above. The solvent DMSO treated cells served as control. Cells were then treated with MTT reagents (20[t/well) for 5 h at 37°C and then, DMSO (200[l) was added to each well to dissolve the formazan crystals. The optical density was recorded at 492 nm in a microplate reader. Percentage of residual cell viability was determined as (1-(OD of treated cells/OD of control cells)) x100. The drug show 18.2 % tumor growth inhibition and the formulation Fl showed 32.64% tumor growth inhibition, the formulation F1 has maximum% tumor growth inhibition. Formulation F2, and F3 showed 28.42 and 21.24 % tumor growth inhibition respectively. Formulation Fl showed maximum % tumor growth inhibition due to maximum drug release from the formulation.
Table 1: Percentage drug release of formulations
Time (Hrs) F1 F2 F3 0.5 19.73 14.46 12.45 1 31.62 25.12 21.93 1.5 40.55 35.38 27.63 2 48.62 45.82 41.83 2.5 57.99 53.24 49.66 3 60.72 56.11 51.26 3.5 67.71 58.33 54.26 4 72.94 62.38 58.11 4.5 74.33 64.91 61.35 5 76.83 66.67 64.91 5.5 78.34 67.41 65.18
6 80.39 68.63 66.82 6.5 82.84 70.38 68.35 7 85.37 72.83 70.22 7.5 87.35 75.88 73.64 8 90.62 79.61 76.25
Table 2: Stability studies of formulations
Storage Conditions F1 F2 F3 Long term 98.4% 97.3% 96.4%
(25°C ±2C/ 60% RH ±5% RH) Intermediate 97.7% 95.8% 94.5%
(30°C ±2C / 65% RH ± 5% RH) Accelerated 95.1% 92.3% 92.3% (40°C ±2C/ 75% RH ±5% RH)

Claims (8)

  1. Claims: 1. Herein we claim a Controlled Release Formulation of Docetaxel within finctionalized Mesoporous Silica Nanoparticles used for therapy of Cancer.
  2. 2. The Formulation claimed in 1 has sustained release drug delivery with high drug content.
  3. 3. The formulation claimed in 1 has low cost of therapy because no frequent administration of drug is required.
  4. 4. Formulation claimed in 1 is polysorbate-80 free, stable formulation to avoid hypersensitivity reactions.
  5. 5. Formulation claimed in 1 has enhanced cellular uptake by attaching targeting ligand.
  6. 6. Formulation claimed in 1 has no adverse effects to the normal cells.
  7. 7. Formulation claimed in 1 has better stability during storage.
  8. 8. We also claim the method of preparation, of the formulation claimed in 1, for which Drug-loaded MSNs were prepared by using a solvent evaporation technique.
    Fig. 1: FT-IR Spectra of Docetaxel
    Fig. 2: SEM image of drug loaded formulation
    70 66.43 62.95 60 54.72 % Drug entrapent
    50
    40
    30
    20
    10
    0 F1 F2 F3 Formulation
    Fig. 3: Percentage drug entrapment
    80
    70
    60 % Drug Release
    50
    40 F1 F2 F3 30 2021104786
    20
    10
    0 0 1 2 3 4 5 6 7 8 Time (Hrs.)
    Fig. 4: Percentage drug release of formulations
    35 32.64
    30 28.42
    25 21.24 20 18.2
    15
    10
    5
    0 Drug F1 F2 F3
    Fig 5. Percentage (%) Tumor growth inhibition
AU2021104786A 2021-08-01 2021-08-01 Controlled Release of Docetaxel Within Functionalized Mesoporous Silica Nanoparticles for Cancer Therapy Ceased AU2021104786A4 (en)

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